The Immune-Chemokine Axis in Alzheimer’s Disease: Roles of Adaptive Immune System in Neuroinflammation and Disease Progression

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provides an overview of the role of the immune system, particularly chemokines, in Alzheimer’s disease and discusses their potential relevance for neuroprotection and biomarker development. The manuscript is generally well written, clear, and easy to follow. The overall structure is logical, and the flow of the text is appropriate for a review article. The figures are of good quality and support the content. However, it is not clear whether they are original or adapted from other sources, and appropriate clarification or attribution should be provided. I recommend the manuscript for minor revisions.

However, several aspects of the manuscript would benefit from clarification, restructuring, and improved consistency.

1. “Implications for Neuroprotective Therapies and Biomarkers by Chemokines” part

Regarding the content, the part from the title “Implications for Neuroprotective Therapies and Biomarkers by Chemokines” is not sufficiently reflected in the main text. This aspect appears underdeveloped and should be expanded to match the scope suggested by the title.

2. Conclusion and Future Perspectives.

The main concern relates to the “Conclusion and Future Perspectives” section. In its current form, it does not fully function as a conclusion. It lacks a clear summary of therapeutic potential, including whether any of the discussed targets are being explored in ongoing or completed studies, and whether they represent realistic or promising strategies. Additionally, the sudden introduction of photobiomodulation (PBM) is confusing, as it is not discussed elsewhere in the manuscript. If the authors intend to include this topic, it should be properly introduced and described in a dedicated section within the main text. The conclusion should instead focus on synthesizing the presented information and providing the authors’ perspective on the most promising future directions.

3. References in Table 1.

Similarly, Table 1 would benefit from clearer integration with the reference list. If the studies listed in the table correspond to cited references, it would improve readability to include reference numbers directly in the table (e.g., in parentheses).

4. Underlined words (hyperlinked).

I have a few questions regarding formatting and editorial aspects. The presence of underlined words (hyperlinks) throughout the text is somewhat unclear. It would be helpful to clarify whether these are part of the journal’s standard formatting, added by the editorial system, or intentionally introduced by the authors. Additionally, it is not clear why only some references or terms are hyperlinked, while others are not.

5. Minor editorial issues.

There are also minor editorial issues that should be addressed. For example, in Figure 2 (“Microglial cells in Alzheimer disease brains”), “Microglial” should not contain a capital “I”. The list of abbreviations appears incomplete (e.g., PBM should be included), and the reference list requires careful revision, as some entries (e.g., 10, 16, 17, 26) differ in formatting style.

Author Response

 Dear reviewer

Thanks a lot for your valuable and constructive comments.

The manuscript provides an overview of the role of the immune system, particularly chemokines, in Alzheimer’s disease and discusses their potential relevance for neuroprotection and biomarker development. The manuscript is generally well written, clear, and easy to follow. The overall structure is logical, and the flow of the text is appropriate for a review article. The figures are of good quality and support the content. However, it is not clear whether they are original or adapted from other sources, and appropriate clarification or attribution should be provided. I recommend the manuscript for minor revisions.

However, several aspects of the manuscript would benefit from clarification, restructuring, and improved consistency.

1. “Implications for Neuroprotective Therapies and Biomarkers by Chemokines” part

Regarding the content, the part from the title “Implications for Neuroprotective Therapies and Biomarkers by Chemokines” is not sufficiently reflected in the main text. This aspect appears underdeveloped and should be expanded to match the scope suggested by the title.

The manuscript has been reorganized following new reviewer-2 suggestions again. However, this reviewer-2 requires shortened the conclusion you ask the opposite (increases the length of conclusion). Thus, we have extended a little bit the length of conclusion in this R2 version. Thanks for your valuable comments again¡

These aspects are included within the R2 version.

-The R2 manuscript has been improved compared with the previous R1 version, especially in the addition of chemokine-related discussions and schematic figures (two figures in this R2 version). In addition, the overall structure, figures and tables were improved following all reviewers recommendations, spetialy related suggestions of reviewer-2. Thus, the organization of this R2 version differ in lines as compared with the last one R1 version following reviewer-2 reorganization.

-In general, the overall structure of the R2 version is more clear with independent sections for innate immunity, including chemokines and chemokine signalling in AD. We believe it is necessary to include chemokines before inhate immune system because chemokines recruit monocytes in the brain of AD models. Thus, it is necessary to introduce before explaining adaptive immune system and T recruitment in the AD brain; the rest of reorganization followed your advice in this R2 version; thus, the point 5 about immune–chemokine crosstalk and T cells in AD after describing adaptive immune system responses by T cells in this R2 version

Additonally, we have added more information about crosstalk between chemokines and adaptive immune response in AD (point 5) and also we added information about chemokine signalling in AD

Thanks for your valuable comment again. The manuscript has been reorganized following new reviewer-2 suggestions again. However, this reviewer-2 requires shortened the conclusion you ask the opposite (increases the length of conclusion). Thus, we have extended a little bit the length of conclusion in this R2 version. Thanks for your valuable comments again¡

We have added this point 5. Chemokine signalling and T-cells in AD following your advice and also the point 6 indicate crosstalk between chemokines signalling (CXCR6 and CXCR3) in adaptive immune system in AD as follows in this R2 version:

1. Chemokine signalling and T-cells in AD

5.1. The CXCR4/SDF-1α axis influences microglia–T-cell crosstalk in AD models

 SDF1 alpha (also known CXCL12) correlated with an increase in brain-associated B-lineage cells in the AD brain, expressing its cognate CXCR4 receptor. In fact, CXCR4+ antibody-secreting cells are significantly reduced in the gut of 5XFAD AD mice. In contrast, CXCR4+ antibody-secreting cells (ASCs) are detected in the colon, while CXCR4+ B-cells and gut-specific IgA+ cells accumulate in the brain and dura mater. These findings suggest en-hanced SDF-1α–dependent recruitment of immune cells into the brain of 5XFAD mice. In this model, SDF-1α is released by astrocytes, and its levels correlated with the infiltration of CXCR4 + B-cells and gut-specific IgA + cells into the brain and dura mater. Additionally, these effects appear to be SDF-1α specific since CXCR4 blockade by AMD3100 (CXCR4 antagonist) abolished the migration of immune cells into the brain [69].

5.2. The CXCR3 and CXCR6 and microglia–T-cell recruitment in AD models

Chemokines increased the T-cell recruitment in a CXCL10-CXCR3 chemokine dependent manner since CXCR3 chemokine receptor enables recruitment of specific memory CD8+ T-cells to areas containing activated APCs that ex-press CXCL9/10/11 [139,140] through its CXCR3 chemokine receptor down-stream pathways in the cortex of 5xFAD AD transgenic mice [18].

CD8+ T-cells contribute to neurodegeneration through direct cytotoxicity and indirect glial-enhanced inflammatory responses. These CD8+ T-cells indirectly exacerbate neurodegeneration by IFN-γ-associated signalling in glial cells. CXCR3 is expressed in infiltrated CD8+ T within the hippocampus and cortex of 5xFAD mouse brain at 6- to 7-month-old [18]. Since these Aβ plaque-associated subset of CD8⁺ T-cells promote Type-I interferon signalling and recruit non-ISG T-cells through the CXCL10-CXCR3 CXCL10 axis, this type-I interferon responses in microglia cells near plaques could be target of drugs to prevent amyloid plaques accumulation in the human AD brain [141].

1. Conclusion and Future Perspectives.

The main concern relates to the “Conclusion and Future Perspectives” section. In its current form, it does not fully function as a conclusion. It lacks a clear summary of therapeutic potential, including whether any of the discussed targets are being explored in ongoing or completed studies, and whether they represent realistic or promising strategies. Additionally, the sudden introduction of photobiomodulation (PBM) is confusing, as it is not discussed elsewhere in the manuscript. If the authors intend to include this topic, it should be properly introduced and described in a dedicated section within the main text. The conclusion should instead focus on synthesizing the presented information and providing the authors’ perspective on the most promising future directions.

We have added a point 7 about therapeutic interventions in AD, including PBM withhmore detail and also improved the discussion.

1. References in Table 1.

Similarly, Table 1 would benefit from clearer integration with the reference list. If the studies listed in the table correspond to cited references, it would improve readability to include reference numbers directly in the table (e.g., in parentheses).

Done it¡. In addition, chemokines have been discussed by functional roles in the R2 version and tables-1 and 2 included all requirements of this reviwer-2. The table-1 includes major cell source, functional category, associated AD pathology, and cell type. The table-2 includes mean studies with T cells in AD (rodent and AD patients with emphasis on experimental model, immune cell subtype, key signalling pathways and protective vs detrimental effects in AD models and patients).

1. Underlined words (hyperlinked).

I have a few questions regarding formatting and editorial aspects. The presence of underlined words (hyperlinks) throughout the text is somewhat unclear. It would be helpful to clarify whether these are part of the journal’s standard formatting, added by the editorial system, or intentionally introduced by the authors. Additionally, it is not clear why only some references or terms are hyperlinked, while others are not.

We have improved these editorial errors.

1. Minor editorial issues.

There are also minor editorial issues that should be addressed. For example, in Figure 2 (“Microglial cells in Alzheimer disease brains”), “Microglial” should not contain a capital “I”. The list of abbreviations appears incomplete (e.g., PBM should be included), and the reference list requires careful revision, as some entries (e.g., 10, 16, 17, 26) differ in formatting style.

Done it¡. The list of abbreviations have been revised in this R2 version. Since the reviewer-2 ask more reorganization of this R2 manuscriopt, the lines are different as compared to the last one R1 version

Thanks again for all your valuable and constructive comments again.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review looks at how the immune system and chemokines are involved in Alzheimer’s disease, and the topic is definitely interesting and important. However, the paper mostly lists studies without clear structure or deeper discussion of key questions. It would benefit from better organization, clearer focus, and more critical analysis.

1. The title is not clear. It’s better to be revised to ‘The Immune–Chemokine Axis in Alzheimer’s Disease: Roles in Neuroinflammation and Disease Progression’
2. The Introduction would benefit from a clearer focus and logical flow. It is suggested to organize it into four paragraphs:

Paragraph 1 : brief intro to AD and neuroinflammation;

Paragraph 2 : the key challenges (like complex immune involvement);

Paragraph 3 : the main gaps in neuroinflammation area (especially chemokines), Paragraph 4 : the aim of this review.

3. For the main structure, it’s better to be shown in this way:

Section 1: Overview of immune-chemokine interactions in AD;

Section 2: Innate inmmunity in AD (Discuss about Micorglia, astrocytes);

Section 3: Adaptive immunity in AD (Discuss about T cells, B cells);

Section 4: Chemokine signaling in AD (Chemokine classification; functional roles, key chemokine axes. Don’t discuss by molecule, discuss by functional role.);

Section 5: Crosstalk between immune cells and chemokines (microglia and T cells crosstalk; CNS and peripheral immune system crosstalk; the bridge role of BBB)

Section 6: Controversies and knowledge gaps (The controversies of T cell functions; monocyte infiltration; different models;)

Section 7: Terapeutic implications (Chemokine targeting, immune modulation);

4. The Conclusion should be more concise and focused, highlighting key points and briefly addressing future directions.
5. There is no need to show the IHC staining figure in this review. Instead, the author should add some figure schematics to make the manuscript more easier to understand:

Figure 1. Immune–chemokine interactions in AD (mainly about how CNS and peripheral system interac with tau or Aβ);

Figure 2. Crosstalk between immune cells and chemokine signaling in AD (mainly summary how interaction between immune cells and chemokine signals);

Figure 3.  Potential therapeutic strategies targeting immune–chemokine pathways

6. The author should discuss the three questions in the manuscript deeply: 1) The dual role of immune cells in AD, under what conditions and at which disease stages do immune cells exert protective versus detrimental effects in AD? 2) What is the mechanistic role of chemokines in AD—are they drivers of pathology or secondary responses, and how do they regulate immune cell recruitment, BBB integrity, and neuroinflammation? 3) How does the peripheral immune system influence the brain in AD—do immune cells directly infiltrate across the BBB, or do they primarily exert indirect effects through CNS–periphery interactions?

Author Response

Reviewer-2 R2

Comments and Suggestions for Authors

This review discusses the role of immune cells and chemokine signaling in Alzheimer’s disease, and the topic is interesting and meaningful. The manuscript has been improved compared with the previous version, especially in the addition of chemokine-related discussions and schematic figures. However, the overall structure, figures and tables still needs to be improved.

Thanks for your valuable comments, which help us to improve this R2 version.

In the Introduction section, the overall logic is difficult to follow because of too many mechanistic details and molecule-specific examples, The author should simplify the Introduction and organize it more clearly. The author should also move detailed discussions of specific chemokines and signaling pathways to the later sections of the review.

The introduction has been shortened and simplify following your advice. These related details on chemokines in the last R1 version are removed in the introduction of this R2 version. The introduction in the R2 version is as follows:

 Immune system role in Alzheimer disease (AD)

AD is a progressive neurodegenerative disorder characterized by β-amyloid (Aβ) plaques and p-Tau hyperphosphorylated accumulation in the brain, together inflammation, microglia overactivation (the resident immune cells of the brain), astrogliosis and cognitive decline. Chemokines (chemotactic cytokines) are inflammatory mediators involved in the trafficking of immune cells into the brain parenchyma; additionally, chemokines act as modulators of the adaptive and immune responses by resident microglia cells. Chemokines are expressed by several cell types in the brain, including neurons, astrocytes, microglia and vascular cells. In general, synergic mechanisms as enhanced peripheral immune cells mobilization into the brain, metabolic and mitochondria alterations, breakdown of blood-brain barrier (BBB) integrity contribute to AD neuropathology [1-3]. Meanwhile, ApoE4 triggers inflammation in the brain, ultimately leading to the activation of microglia, astrocytes, and T cells activation in the brain of AD patients [4].

In general, chemokines exert neuroprotective effects but also contribute to neurodegeneration in AD rodent models [5,6,7]. Under controlled inflammatory conditions, microglial cells can promote neuroprotection. However, the recruitment of peripheral immune cells triggered (neutrophils, T Reg regulatory cells, T and B cells, natural killer -NK-) by chemokines contribute to AD pathology in rodent models and AD patients [16,17,6, 18]. These CD4+ and CD8+ T infiltrates into the brain can induce neuroprotective effects but overactivates microglia cells, leading to Aβ and tau deposition and, finally, cognitive dysfunction in AD models [18-21].

From a therapeutic viewpoint, new treatment with chemokine blockers, immune-modulators drugs that promotes T Reg-induced responses or complementary approach as photobiomodulation (PBM) are new strategies to treat AD in patients [22]

This review aims to summarize the roles of the innate (mainly microglial cells) and adaptive immune system (T and B cells), and infiltrated monocytes in AD pathology, with a particular focus on chemokines.

We also point out significant gaps in the field about drugs able to enhance amyloid-β plaques removal in AD patients. Other unsolved gap is of the lack of standarized protocols in AD clinical trials using chemokine antagonists; moreover, the study of confounding factors (age, infections, comorbidities) are not stimated in some clinical studies with AD patients. Furthermore, the long-term adverse effects of antigen-specific CD4+ T-cell based nano delivery therapies remain to be elucidated in AD patients. Finally, PBM treatment as complementary therapy requires more clinical evidence in AD patients.

1. In the main section organization, the overall structure of the manuscript is improved but still somewhat mixed and difficult to understand. The author should reorganize the review with clearer independent sections for innate immunity, adaptive immunity, chemokine signaling, and immune–chemokine crosstalk. The author should also discuss chemokines more by functional roles rather than listing individual molecules.

Thanks for your valuable comment again. We have followed your recommendation. The overall structure of the manuscript has been improved according to your suggestion. In general, the overall structure of the R2 version is more clear with independent sections for innate immunity, including chemokines and chemokine signalling in AD. We believe it is necessary to include chemokines before inhate immune system because chemokines recruit monocytes in the brain of AD models. Thus, it is necessary to introduce before explaining adaptive immune system and T recruitment in the AD brain; the rest of reorganization followed your advice in this R2 version; thus, the point 5 about immune–chemokine crosstalk and T cells in AD after describing adaptive immune system responses by T cells in this R2 version. Thus, points on chemokine signalling in Alzheimer and the crosstalk between chemokine -adaptive immune mediated responses are included at the end.

-We hope you are agree with this R2 structuration following reviewer-2 suggestions. However, this reviewer-2 requires shortened the conclusion while the reviewer-4 request us the opposite (increases the length of conclusion). Thus, we extend a litte bit the length of conclusion in this R2 version.

2. In Figure 1, why does the author not mention how the CNS and peripheral system interact with tau? The author should add a more comprehensive overview figure integrating Aβ/tau pathology, innate and adaptive immune responses, and chemokine-mediated crosstalk in AD.

Thanks again. The new R2 version includes two figures (fig-1 ; role of chemokines in BB) and the figure -2 integrates Aβ/tau pathology, innate and adaptive immune responses, and chemokine-mediated crosstalk in AD following your recommendation.

In a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells mediated neurodegeneration. The presence of activated T cells in the brains of transgenic mice with frontotemporal dementia (FTD)-like Tau protein pathology confirmed that infiltrates of T cells correlated with microglia overactivation and Tau deposition in the brain. The number of activated T cells (CD4+ and spetially CD8+ cells) are enriched the parenchyma in Tau mouse brains elevated in Tau pathology, leading to neurodegeneration. Interestingly, Tau transgenic mice on an APOE knockout background were protected against T cell infiltration, suggesting that T cell infiltration is associated with Tau pathology progression (Braak staging). The neuronal Tau aggregation triggers microglia activation, and results in recruitment, clonal expansion, and activation of T CD8 + cells in the brain. The inflamamtory cascade is enhanced by miicroglia MHC-II and CD11c cells, which activate T cells by IFNin the brain.

How are T cells activated in the brains of tauopathy mice?  It Is known that in microglia from tauopathy mice, genes associated with antigen representation, complement and interferon response, lysosomal pathways, and oxidative stress are upregulated. Furthermore, microglia had an antigen-presenting phenotype 2 characterized by the expression of major histocompatibility complex, MHC-II, proteins, and CD11c receptor, a disease-associated marker in TREM2+ microglia, which can provide a signal for T cell activation. Indeed, primary mouse microglia co-cultured with OT-1T cells (CD8+ T cells expressing TCRs that recognize ovalbumin as antigen) were able to stimulate OT-1T proliferation in the presence of their antigen, ovalbumin, which was exacerbated upon stimulation of microglia with the pro-inflammatory cytokine interferon-gamma, IFNγ. Inhibiting IFNγ signaling, or removing microglia by PLX3397 administration reduced p-Tau accumulation and brain atrophy in tauopathy mice. These results show that activated microglia have the capacity to trigger a T cell response that promotes Tau pathology and neurotoxicity. IFNγ secretion by immune cells could exacerbate neurodegeneration in tauopathies. Although, neurons signal such Tau alterations to microglia and induce T cell infiltration and activation remains to be clarified, it is known that depleting T cells reduced microglial MHC class II and CD11c expression, reduced p-Tau immunoreactivity and brain atrophy, and improved short-term memory. Blocking immunoregulation of T cells (PDCD1-PDL1 signaling) in TE4 mice through repeated anti-PD-1 antibody administration increased the percentage of immune suppressive regulatory T cells, but did not affect the percentage of effector T cells. However, at the age of 9.5 months, the anti-PD-1 treatment achieved a reduction in p-Tau and brain atrophy. In conclusion, targeting T cells and modulating immune response is a potential approach against Tau-mediated neurodegeneration (Askin, B., Wegmann, S. Interaction of microglia and T cells: a deadly duo behind Tau-mediated neurodegeneration. Sig Transduct Target Ther 8, 317 (2023). https://doi.org/10.1038/s41392-023-01563-9)

3. For able 1, it lacks mechanistic and translational information. The author should consider adding major cell source, functional category, associated AD pathology, and sample type.

Done it¡. Thanks again (please, read the R2 manuscript version that includes these aspects in tables 1 and 2). The figure 1 in the R2 version includes major cell source, functional category, associated AD pathology, and cell type.

1. For table 2, The author should reduce the title of the references and add: experimental model, immune cell subtype, protective vs detrimental effect, key chemokines/signaling pathways, main conclusion, and whether findings were from human or animal studies.

Done it¡. Moreover, the table-2 indicates mean studies with T cells in AD (rodent and AD patients with emphasis on experimental model, immune cell subtype, key signalling pathways and protective vs detrimental effects in AD models and patients. Thanks again

Comments and Suggestions for Authors

This review looks at how the immune system and chemokines are involved in Alzheimer’s disease, and the topic is definitely interesting and important. However, the paper mostly lists studies without clear structure or deeper discussion of key questions. It would benefit from better organization, clearer focus, and more critical analysis.

The title is not clear. It’s better to be revised to ‘The Immune–Chemokine Axis in Alzheimer’s Disease: Roles in Neuroinflammation and Disease Progression’

We include this title in this R1 and R2 version

The Introduction would benefit from a clearer focus and logical flow. It is suggested to organize it into four paragraphs:

Paragraph 1 : brief intro to AD and neuroinflammation;

Paragraph 2 : the key challenges (like complex immune involvement);

Paragraph 3 : the main gaps in neuroinflammation area (especially chemokines), Paragraph 4 : the aim of this review.

The introduction has been shortened and simplify, and these aspects were considered by us in the R2 version.

For the main structure, it’s better to be shown in this way:

Section 1: Overview of immune-chemokine interactions in AD;

Section 2: Innate inmmunity in AD (Discuss about Micorglia, astrocytes); Section 3: Adaptive immunity in AD (Discuss about T cells, B cells);

All  these requirements were answered in the last one R1 version. I also respond you in this R2 version

Section 4: Chemokine signaling in AD (Chemokine classification; functional roles, key chemokine

axes. Don’t discuss by molecule, discuss by functional role.);

Section 5: Crosstalk between immune cells and chemokines (microglia and T cells crosstalk; CNS and peripheral immune system crosstalk; the bridge role of BBB)

Section 6: Controversies and knowledge gaps (The controversies of T cell functions; monocyte infiltration; different models;)

Section 7: Terapeutic implications (Chemokine targeting, immune modulation);

The Conclusion should be more concise and focused, highlighting key points and briefly addressing future directions.

We extend the length of conclusion because other reviewer ask to add more details although the reviewer-2 request us to shortened this conclusion. Both aspects are opposite , and , we have extended a little bit the discussion following advice of reviewer-4.

There is no need to show the IHC staining figure in this review. Instead, the author should add some figure schematics to make the manuscript more easier to understand:

Figure 1. Immune–chemokine interactions in AD (mainly about how CNS and peripheral system

interac with tau or Aβ);

Figure 2. Crosstalk between immune cells and chemokine signaling in AD (mainly summary how interaction between immune cells and chemokine signals);

Figure 3. Potential therapeutic strategies targeting immune–chemokine pathways

The manuscript contains two figures, which grouped all these aspects in the R2 version. We have also added a spetial point 3 about chemokines signalling and AD and chemokines-inmune crosstalk in AD (point 5). in this R2 version (but without including a specific figure). The figure-2 includes all interactive effects between Abeta and tau accumulation in the brain of AD models

The figure 2 (A) indicates in a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells mediates neurodegeneration. Figure-2. Inflammatory mediators in the brain of Alzheimer`s patients

Figure-2. Inflammatory mediators in the brain of Alzheimer`s patients

(A) The progressive aggregation of amyloid-β in extracellular amyloid plaques and of Tau in neurofibrillary tangles are pathological hallmarks of AD. The neuroinflammation, and neuronal death synaptic loss, monocyte infiltration compromise the blood-brain barrier; these immune cells infiltrate the brain parenchyma and interact with glial cells and neurons; in a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells contribute to neurodegeneration in a CX3CR1-Fractalkine dependent manner. In fact, low soluble fractalkine release by vulnerable neurons impairs microglia functions in a CX3CR1 dependent manner, leading to p-Tau accumulation and amyloid beta deposition in the brain of AD and taupathy models [114]. The presence of activated T cells in the brains of transgenic mice with frontotemporal dementia (FTD)-like correlated with microglia overactivation and Tau deposition in the brain. In fact, enriched number of activated T cells (CD4+ and spetially CD8+ cells) are found in the parenchyma, leading to neurodegeneration. Interestingly, Tau transgenic mice on an APOE knockout background were protected against T cell infiltration, suggesting that T cell infiltration is associated with Tau pathology progression (Braak staging). The neuronal Tau aggregation triggers microglia activation, and results in recruitment, clonal expansion, and activation of T CD8 + cells in the brain. This inflammatory cascade is enhanced by microglia MHC-II and CD11c cells since IFN gamma released by clonal CD8 + cells activate T cells by in the brain. Collectivelley, these findings link T cell accumulation with p-tau and Amylid beta depostion, leading to neurotoxicity and neurodegeneration.

(B) Activated T cells can be found in postmortem brains of different neurological disorders, in AD, particularly in areas with neuronal loss and Tau pathology, i.e., the hippocampus and limbic structures. These infiltrated T cells in the brain parenquima activates astrocyte, which releases chemokines as CXCL10, CXCL9 and CC4. In fact, certain chemokines as CCR3 release by T infiltrated cells overactivates microglia, leading to neurotoxicity. This exacerbates local inflammation and enhances neuronal loss.

(C). CD4 and CD8+ T-cells infiltrates into the brain parenchyma promote detrimental effects, leading to Aβ plaque accumulation or microglia overactivation though IL-17 chemokine release by microglia cells. In fact, CD8+ clonal infiltates appear in the brain parenquima and these T cells activate microglia by IFN gamma release of activated CD8 + T cels. Thus, microglia overactivation release CXCL10 chemoines, which binds to astroglia carriers of CXCR3 chemokine receptor. Thus, CXCR3 contribute to neurodegeneration in conjunction with altered CXCR6-CXCL16 signalling by CD8+ T cells infiltratesin the brain. However, T Reg cells can also promote neuroprotective effects in AD models.

The author should discuss the three questions in the manuscript deeply: 1) The dual role of immune cells in AD, under what conditions and at which disease stages do immune cells exert protective versus detrimental effects in AD?

We response to these questions in the R1 submitted version. Anyway, we reply you in this R2 version. In fact, both situations are possible since chemokines aredrivers of AD pathology but can be also secondary given its capacity to regulate neuron-microglia interaction in AD since are pleiotphropic molecules.

Chemokines can regulate the BBB, which is often compromised in AD patients. Elevated levels of CCL2 and CCL5 can weaken BBB tight junctions, allowing peripheral immune cells (such as T-cells and monocytes) to infiltrate into the brain parenchyma.

2) What is the mechanistic role of chemokines in AD—are they drivers of pathology or secondary responses, and how do they regulate immune cell recruitment, BBB integrity, and neuroinflammation?

The role of chemokines in BBB disruption contribute to neuroinflammation in the brain. In this way, chemokines act as "homing" signals for microglia to play a dual role, functioning as either suppressors or inducers of pathogenic alterations though multiple signalling pathways in the AD brain. Certain chemokine ligands (ie: fractalkine) can regulate microglial chemotaxis via chemokine receptors (i.e:. CX3CR1), which sense these gradients and drive migration toward amyloid beta plaques in the brain [7].

The recruitment of inflammatory mediators in the brain is directed by chemokines in conjunction with cell adhesion molecules (selectins, VCAM-1, ICAM-1) and integrins (LFA-1, VLA-4, α4β1) [26]. Chemokines regulate the BBB integrity, which is often compromised in AD patients. The augmented levels of CCL2 and CCL5 can weaken BBB tight junctions, allowing peripheral immune cells (such as T-cells and monocytes) to infiltrate into the brain parenchyma. This exacerbates local neuroinflammation and increases neuronal loss. The binding of chemokine ligands to endothelial cells triggers signalling pathways and lead to the reorganization of tight junction proteins. In this way, BBB integrity depends on tight junctions (TJs), sealing the spaces between endothelial cells through interactions among zonula occludens-1 (ZO-1), claudins and occludins. Consequently, the “zipper-like" seal between cells loosens, allowing small molecules to reach the brain parenchyma. Chemokines induce matrix metalloproteinases (MMPs 2 and 9) that facilitate BBB breakdown by activating astrocytes, microglia, and endothelial cells. Thus, MPPs degrade the basement membrane and the extracellular matrix while chemokines stimulate the expression of adhesion molecules (i.e: ICAM-1 and VCAM-1) on the endothelial surface, which act as anchors for leukocytes, see figure-1 [27].

3) How does the peripheral immune system influence the brain in AD—do immune cells directly infiltrate across the BBB, or do they primarily exert indirect effects through CNS–periphery interactions?

These aspects have been included in the R2 version (see R2 pdf attached file within the Susy MDPI system). Briefly, we have added the point Thanks for your valuable comments. We have added this 3. Chemokine signaling in AD within the attached R2 version on line as follows for the point 5. Chemokine signalling and T-cells in AD as follows. This follows text was included within the new R2 attached version in MDPI system (see R2 file). The attached text is as follows:

5.Chemokine signalling and T-cells in AD

5.1. The CXCR4/SDF-1α axis influences microglia–T-cell crosstalk in AD models

 SDF1 alpha (also known CXCL12) correlated with an increase in brain-associated B-lineage cells in the AD brain, expressing its cognate CXCR4 receptor. In fact, CXCR4+ antibody-secreting cells are significantly reduced in the gut of 5XFAD AD mice. In contrast, CXCR4+ antibody-secreting cells (ASCs) are detected in the colon, while CXCR4+ B-cells and gut-specific IgA+ cells accumulate in the brain and dura mater. These findings suggest enhanced SDF-1α–dependent recruitment of immune cells into the brain of 5XFAD mice. In this model, SDF-1α is released by astrocytes, and its levels correlated with the infiltration of CXCR4 + B-cells and gut-specific IgA + cells into the brain and dura mater. Additionally, these effects appear to be SDF-1α specific since CXCR4 blockade by AMD3100 (CXCR4 antagonist) abolished the migration of immune cells into the brain [69].

5.2. The CXCR3 and CXCR6 and microglia–T-cell recruitment in AD models

Chemokines increased the T-cell recruitment in a CXCL10-CXCR3 chemokine dependent manner since CXCR3 chemokine receptor enables recruitment of specific memory CD8+ T-cells to areas containing activated APCs that ex-press CXCL9/10/11 [139,140] through its CXCR3 chemokine receptor down-stream pathways in the cortex of 5xFAD AD transgenic mice [18].

CD8+ T-cells contribute to neurodegeneration through direct cytotoxicity and indirect glial-enhanced inflammatory responses. These CD8+ T-cells indirectly exacerbate neurodegeneration by IFN-γ-associated signalling in glial cells. CXCR3 is expressed in infiltrated CD8+ T within the hippocampus and cortex of 5xFAD mouse brain at 6- to 7-month-old [18]. Since these Aβ plaque-associated subset of CD8⁺ T-cells promote Type-I interferon signalling and recruit non-ISG T-cells through the CXCL10-CXCR3 CXCL10 axis, this type-I interferon responses in microglia cells near plaques could be target of drugs to prevent am-yloid plaques accumulation in the human AD brain 141]. Moreover, certain chemokines as CCR2/CCL2 axis that recruit leukocytes in the brain and pro-mote the differentiation of naïve T-cells [40]. Other chemokines as CXCL8, can activate neutrophils and T-cells since it is a marker of T-cell effector function in human newborns and inhibits CD8+ T-cell infiltration [96].

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, the authors summarize the immune system in Alzheimer´s disease cognitive disfunction, and the neuroprotective therapies and biomarkers by chemokines was reviewed. However, some revisions should be made to show the significance of this study.

1. I can see the part of “1. Introduction” and “3. Role of chemokines in Alzheimer neuropathology”. However, the part of “2. XXX” was not found in the manuscript.
2. “Figure 1 a,b. Hypertrophic astroglial cells and plaque amyloid surrounding microglial cells” and “Figure-1c. MIcroglial cells close to Amyloid beta plaques in the brain of Alzheimer disease” were provided in the part of Original Images. However, these two figures were not found in the manuscript. Where is the figure 1? The figures related to this review should be provided and checked carefully.
3. In the part of “3. Role of chemokines in Alzheimer neuropathology”, the authors only provide a brief description on the role of chemokines in Alzheimer neuropathology, but it lacks a deep mechanism summary on the role of chemokines in Alzheimer neuropathology.
4. In the Table 1. Relevance of immune system in Alzheimer disease, the full reference has been provided. The format of these references should be revised according to the requirement of the journal. 

Comments on the Quality of English Language

English is OK.

Author Response

Reviewer-3

Comments and Suggestions for Authors

In this review, the authors summarize the immune system in Alzheimer´s disease cognitive disfunction, and the neuroprotective therapies and biomarkers by chemokines was reviewed. However, some revisions should be made to show the significance of this study.

1. I can see the part of “1. Introduction” and “3. Role of chemokines in Alzheimer neuropathology”. However, the part of “2. XXX” was not found in the manuscript.

Thanks a lot for your comments, which help us to improve the quality of this R1 manuscript. We have merged  these parts of the introduction in the new R1 version. The R1 version have been reorganized following all suggestions of four reviewers, which lead to a different organization as compare to the original manuscript.

Please, take into account that the manuscript has been totally reorganized following reviwer                adviced of 4 revieiwers. So, many parts of different and we added a image, therapeutic approachs and gaps following other reviewers comments.

1. “Figure 1 a,b. Hypertrophic astroglial cells and plaque amyloid surrounding microglial cells” and “Figure-1c. MIcroglial cells close to Amyloid beta plaques in the brain of Alzheimer disease” were provided in the part of Original Images. However, these two figures were not found in the manuscript. Where is the figure 1? The figures related to this review should be provided and checked carefully.

We have to removed these figures since Biomolecules indicated us is not allowed the publication of original photographies in reviewers, which are own original and not published figures anywhere. We have added a scheme (figure) and tables 1 and 2 in this R1 version because the reviewer- 2 request us this figure.

1. In the part of “3. Role of chemokines in Alzheimer neuropathology”, the authors only provide a brief description on the role of chemokines in Alzheimer neuropathology, but it lacks a deep mechanism summary on the role of chemokines in Alzheimer neuropathology.

The role of “Role of chemokines in Alzheimer neuropathology” has been extenden and integrated with the role of microglia and T cells in AD but the aim of this review is not proved deep knoledment about chemokines in AD. In fact, there are previous reviews about this topic. We have tried to include more deep mechanism summary on the role of chemokines in Alzheimer neuropathology. However, in our opinion is more actual describe the interaction between T cells, chemokines and AD.

1. In the Table 1. Relevance of immune system in Alzheimer disease, the full reference has been provided. The format of these references should be revised according to the requirement of the journal. 

The format of these references have been revised according to the requirement of the journal and number included. Pleas e, take into account the R1 version contains new added information and numbers could be different as compare to the original submission.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

• Organisation defficiencies:
• The overall section numbering is inconsistent. Introduction subsections are numbered 1.1, 1.2, 1.3, and 1.4, yet section 2 (Chemokines in AD) begins a new top-level heading, and section 3 (Role of chemokines in AD neuropathology) partially overlaps with section 2. These must be merged and restructured logically.
• Sections 1.2 (“Contribution of adaptive immune responses”) and 1.3 (“Adaptative (?) immune responses” are mostly overlapping. please merge. 
• The title mentions “cognitive dysfunction” but the manuscript body does not discuss cognitive topics explicitly in relation to the immune mechanisms. Title needs revision or add a dedicated section on cognition.
• The subsection on monocyte infiltration (3.a) and neutrophil migration (3.a — duplicate label) appear under “Chemokines and innate immunity” but they do not concentrate on the chemokine. Either make them separate sections or integrate them within chemokine signalling.

language and editing errors

• Line 51: “ and orchest neuroinflammatory responses” — “orchest” appears to be a truncated word (likely “orchesters” or “orchestrates”). 
• Line 53: “protective BBB dysfunction” is contradictory — BBB dysfunction is consistently described as detrimental. 
• Line 130: “brain inabilit to orchestrate” — typo and incomplete sentence. Revise.
• Line 189: Citation “Choi20203” appears to be a formatting error (possibly Choi 2020 or 2023). Verify and correct.
• Line 175: Citation “Su2023” is not good.
• Lines 288–289: Two numbered list items (100, 101) appear in the text.
• Line 264: “Reduces NLRP3 inflammasome-driven IL-1β/IL-18 hypersecretion” what induces? treatment of pathophysiology? 

Data are thrown together without a critical integration:

• A lot of experimental findings listed but rarely synthesised or critically evaluated. 
• The role of chemokines is introduced as the central focus, yet the chemokine sections (2 and 3) are comparatively brief and descriptive relative to the T-cell content. Either expand the chemokine coverage to justify the title, or change the title.
• In the end appears the photobiomodulation (PBM) as a future perspective but it is dropped from the sky, without being mentioned anywhere in the manuscript. PBM should be introduced in the main text with the mechanism or taken out.

• Writing is sloppy: please revise and use a text checker...
• many grammatical errors, incomplete sentences, and non-standard terminology throughout. 
• Specific examples (non-exhaustive): line 45 (“Immnometabolic” — typo); line 46 “and can, shifting the microglia” (grammatically incorrect); line 130 (“brain inabilit”); line 166 (“there the less immunosuppressive”); line 392 (“inmunometabolic” — likely a Spanish-English mix); line 390 (“re removal depuration” — redundant and unclear).
• The term “adaptative” (section 1.3 heading and line 194) is not standard English; the correct term is “adaptive.”
• Abbreviations (e.g., ASCs, DAMP, TEMRA, PBM) should be defined on first use, and a complete abbreviation list should be provided.
• References 44 and 37 (Meibers et al., 2023)  duplicate entries for the same publication? 
• References 114 and 115 (Makhijani et al., 2025) appear to be the same study cited twice. Confirm and consolidate.
• Reference 46 in the bibliography (Wang et al., 202) contains an incomplete publication year (“202”). Correct to the full year.

Comments on the Quality of English Language

• Writing is sloppy: please revise and use a text checker...
• many grammatical errors, incomplete sentences, and non-standard terminology throughout. 
• Specific examples (non-exhaustive): line 45 (“Immnometabolic” — typo); line 46 “and can, shifting the microglia” (grammatically incorrect); line 130 (“brain inabilit”); line 166 (“there the less immunosuppressive”); line 392 (“inmunometabolic” — likely a Spanish-English mix); line 390 (“re removal depuration” — redundant and unclear).
• The term “adaptative” (section 1.3 heading and line 194) is not standard English; the correct term is “adaptive.”
• Abbreviations (e.g., ASCs, DAMP, TEMRA, PBM) should be defined on first use, and a complete abbreviation list should be provided.
• References 44 and 37 (Meibers et al., 2023)  duplicate entries for the same publication? 
• References 114 and 115 (Makhijani et al., 2025) appear to be the same study cited twice. Confirm and consolidate.
• Reference 46 in the bibliography (Wang et al., 202) contains an incomplete publication year (“202”). Correct to the full year.

Author Response

Comments and Suggestions for Authors

• Organisation defficiencies:
• The overall section numbering is inconsistent. Introduction subsections are numbered 1.1, 1.2, 1.3, and 1.4, yet section 2 (Chemokines in AD) begins a new top-level heading, and section 3 (Role of chemokines in AD neuropathology) partially overlaps with section 2. These must be merged and restructured logically.

 

Dear reviewer. Thanks for your valuable comments, which help us to improve the quality of this R1 version.

We have corrected numbers and also reorganized the R1 version following all four reviewer`s comment. Thus, lines and points are different in this R1 version as compare to the original manuscript.

 

• Sections 1.2 (“Contribution of adaptive immune responses”) and 1.3 (“Adaptative (?) immune responses” are mostly overlapping. please merge. 

 

We have merged these parts but other reviewer require to separe both of them. We hope to solve this inconvenience in the new R1 version.

 

• The title mentions “cognitive dysfunction” but the manuscript body does not discuss cognitive topics explicitly in relation to the immune mechanisms. Title needs revision or add a dedicated section on cognition.

 

You are right.The new title removes “cognitive dysfunction” and the new R1 manuscript body does not discuss cognitive topics explicitly and it is more focused about the role of T cell and pheripheral infiltration in the brain of AD and Ad patients. relation to the immune mechanisms. Thus, the title has been update as follows an , thus,  we have not added a dedicated section on cognition by this reason. Thanks again¡

 

• The subsection on monocyte infiltration (3.a) and neutrophil migration (3.a — duplicate label) appear under “Chemokines and innate immunity” but they do not concentrate on the chemokine. Either make them separate sections or integrate them within chemokine signalling.

We have divided the role of chemokines depending of their pleitrophic fiunctions but we have not include specific chemokine axis because other reviewer required include the role of chemokines by function but not by specific chemokine pairs (CXCR4/SDF1 alpha, etc9. Please, see the R1 manuscript (lines 290-394)

 

language and editing errors

 

Since the requirements of 4 reviewers were include in this R1 version, these lines are not the same but your requirements and corrections have been taking into account by us in this R1 verison although these numbers are different in the R1 version.

 

• Line 51: “ and orchest neuroinflammatory responses” — “orchest” appears to be a truncated word (likely “orchesters” or “orchestrates”). 
• Line 53: “protective BBB dysfunction” is contradictory — BBB dysfunction is consistently described as detrimental. 

    Thanks. We have correct4edin this R1 version. We are right

 

• Line 130: “brain inabilit to orchestrate” — typo and incomplete sentence. Revise.
• Line 189: Citation “Choi20203” appears to be a formatting error (possibly Choi 2020 or 2023). Verify and correct.

This citation is as follows: Choi, S.J.; Koh, J.Y.; Rha, M. S.; Seo, I.H., Lee, H.; Jeong, S.; Park, S.H.; Shin, E.C. KIR⁺CD8⁺ and NKG2A⁺CD8⁺ T cells are distinct innate-like populations in humans. Cell reports 2023, 42, 112236.

 

• Line 175: Citation “Su2023” is not good.

This citation has been replaced by other more appropriate. Thanks again (see R1 PDDF version)

• Lines 288–289: Two numbered list items (100, 101) appear in the text.

Now, these citations are not repeated in the R1 version

• Line 264: “Reduces NLRP3 inflammasome-driven IL-1β/IL-18 hypersecretion” what induces? treatment of pathophysiology? 

This sentence has been removed in the new R1 version. NLRP3 inflammasome contribute to AD pathology (in general)

 

Data are thrown together without a critical integration:

• A lot of experimental findings listed but rarely synthesised or critically evaluated. 

We have tried to include concepts with a more critically view of point in this R1 version

 

• The role of chemokines is introduced as the central focus, yet the chemokine sections (2 and 3) are comparatively brief and descriptive relative to the T-cell content. Either expand the chemokine coverage to justify the title, or change the title.

 

Yes, the coverage of chemokine has bene extended and the title has been changed as follows ¨The Immune-Chemokine Axis in Alzheimer´s Disease: Roles of adaptive immune system in neuroinflammation and Disease Progression¨, followong your advice in this R1 version

 

• In the end appears the photobiomodulation (PBM) as a future perspective but it is dropped from the sky, without being mentioned anywhere in the manuscript. PBM should be introduced in the main text with the mechanism or taken out.

 

Following your advice, the photobiomodulation (PBM) appears in the introduction and we have extended the information about PBM as alternative therapy at the end of the manuscript together other treatments following the advice of other review.  Thus, the manuscript has been reorganized following the advice of all four reviewers. This is the reason by which the organization of the manuscript is totally different in this R1 version.

 

• Writing is sloppy: please revise and use a text checker...
• many grammatical errors, incomplete sentences, and non-standard terminology throughout. 
• Specific examples (non-exhaustive): line 45 (“Immnometabolic” — typo); line 46 “and can, shifting the microglia” (grammatically incorrect); line 130 (“brain inabilit”); line 166 (“there the less immunosuppressive”); line 392 (“inmunometabolic” — likely a Spanish-All these grammatical errors, incomplete sentences hae been corrected but some of tyhese typo were removed in this R1 version as (“Immnometabolic” — typo), etc

 

• The term “adaptative” (section 1.3 heading and line 194) is not standard English; the correct term is “adaptive.”

          We have corrected in all the R1 version. Thanks¡

• Abbreviations (e.g., ASCs, DAMP, TEMRA, PBM) should be defined on first use, and a complete abbreviation list should be provided.
• References 44 and 37 (Meibers et al., 2023)  duplicate entries for the same publication? 

Now, Meibers et al., 2023 does not appear twice inthis R1 version

• References 114 and 115 (Makhijani et al., 2025) appear to be the same study cited twice. Confirm and consolidate.

Now, (Makhijani et al., 2025) does not appear twice inthis R1 version. However, the lines of both manuscript are different in the R1 version given all reviewer´s responses (4 reviwers) included in this R1 version.

 

• Reference 46 in the bibliography (Wang et al., 202) contains an incomplete publication year (“202”). Correct to the full year.

We have completed Wang et al., 2025 in tis R1 version

 

Comments on the Quality of English Language

• Writing is sloppy: please revise and use a text checker...
• many grammatical errors, incomplete sentences, and non-standard terminology throughout. 
• Specific examples (non-exhaustive): line 45 (“Immnometabolic” — typo); line 46 “and can, shifting the microglia” (grammatically incorrect); line 130 (“brain inabilit”); line 166 (“there the less immunosuppressive”); line 392 (“inmunometabolic” — likely a Spanish-English mix); line 390 (“re removal depuration” — redundant and unclear).
• The term “adaptative” (section 1.3 heading and line 194) is not standard English; the correct term is “adaptive.”
• Abbreviations (e.g., ASCs, DAMP, TEMRA, PBM) should be defined on first use, and a complete abbreviation list should be provided.
• References 44 and 37 (Meibers et al., 2023)  duplicate entries for the same publication? 
• References 114 and 115 (Makhijani et al., 2025) appear to be the same study cited twice. Confirm and consolidate.
• Reference 46 in the bibliography (Wang et al., 202) contains an incomplete publication year (“202”). Correct to the full year.

Your requirements were reply in the last parragrah. Thanks a lot.

Submission Date

11 March 2026

Date of this review

12 Apr 2026 08:48:39

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This review discusses the role of immune cells and chemokine signaling in Alzheimer’s disease, and the topic is interesting and meaningful. The manuscript has been improved compared with the previous version, especially in the addition of chemokine-related discussions and schematic figures. However, the overall structure, figures and tables still needs to be improved.

 

1. In the Introduction section, the overall logic is difficult to follow because of too many mechanistic details and molecule-specific examples, The author should simplify the Introduction and organize it more clearly. The author should also move detailed discussions of specific chemokines and signaling pathways to the later sections of the review.
2. In the main section organization, the overall structure of the manuscript is improved but still somewhat mixed and difficult to understand. The author should reorganize the review with clearer independent sections for innate immunity, adaptive immunity, chemokine signaling, and immune–chemokine crosstalk. The author should also discuss chemokines more by functional roles rather than listing individual molecules.
3. In Figure 1, why does the author not mention how the CNS and peripheral system interact with tau? The author should add a more comprehensive overview figure integrating Aβ/tau pathology, innate and adaptive immune responses, and chemokine-mediated crosstalk in AD.
4. For table 1, it lacks mechanistic and translational information. The author should consider adding major cell source, functional category, associated AD pathology, and sample type.
5. For table 2, The author should reduce the title of the references and add: experimental model, immune cell subtype, protective vs detrimental effect, key chemokines/signaling pathways, main conclusion, and whether findings were from human or animal studies.

Author Response

Thanks for your valuable comments, which help us to improve this R2 version.

1.In the Introduction section, the overall logic is difficult to follow because of too many mechanistic details and molecule-specific examples, The author should simplify the Introduction and organize it more clearly. The author should also move detailed discussions of specific chemokines and signaling pathways to the later sections of the review.

The introduction has been shortened and simplify following your advice. These related details on chemokines in the last R1 version are removed in the introduction of this R2 version. The introduction in the R2 version is as follows:

 Immune system role in Alzheimer disease (AD)

AD is a progressive neurodegenerative disorder characterized by β-amyloid (Aβ) plaques and p-Tau hyperphosphorylated accumulation in the brain, together inflammation, microglia overactivation (the resident immune cells of the brain), astrogliosis and cognitive decline. Chemokines (chemotactic cytokines) are inflammatory mediators involved in the trafficking of immune cells into the brain parenchyma; additionally, chemokines act as modulators of the adaptive and immune responses by resident microglia cells. Chemokines are expressed by several cell types in the brain, including neurons, astrocytes, microglia and vascular cells. In general, synergic mechanisms as enhanced peripheral immune cells mobilization into the brain, metabolic and mitochondria alterations, breakdown of blood-brain barrier (BBB) integrity contribute to AD neuropathology [1-3]. Meanwhile, ApoE4 triggers inflammation in the brain, ultimately leading to the activation of microglia, astrocytes, and T cells activation in the brain of AD patients [4].

In general, chemokines exert neuroprotective effects but also contribute to neurodegeneration in AD rodent models [5,6,7]. Under controlled inflammatory conditions, microglial cells can promote neuroprotection. However, the recruitment of peripheral immune cells triggered (neutrophils, T Reg regulatory cells, T and B cells, natural killer -NK-) by chemokines contribute to AD pathology in rodent models and AD patients [16,17,6, 18]. These CD4+ and CD8+ T infiltrates into the brain can induce neuroprotective effects but overactivates microglia cells, leading to Aβ and tau deposition and, finally, cognitive dysfunction in AD models [18-21].

From a therapeutic viewpoint, new treatment with chemokine blockers, immune-modulators drugs that promotes T Reg-induced responses or complementary approach as photobiomodulation (PBM) are new strategies to treat AD in patients [22]

This review aims to summarize the roles of the innate (mainly microglial cells) and adaptive immune system (T and B cells), and infiltrated monocytes in AD pathology, with a particular focus on chemokines.

We also point out significant gaps in the field about drugs able to enhance amyloid-β plaques removal in AD patients. Other unsolved gap is of the lack of standarized protocols in AD clinical trials using chemokine antagonists; moreover, the study of confounding factors (age, infections, comorbidities) are not stimated in some clinical studies with AD patients. Furthermore, the long-term adverse effects of antigen-specific CD4+ T-cell based nano delivery therapies remain to be elucidated in AD patients. Finally, PBM treatment as complementary therapy requires more clinical evidence in AD patients.

1. In the main section organization, the overall structure of the manuscript is improved but still somewhat mixed and difficult to understand. The author should reorganize the review with clearer independent sections for innate immunity, adaptive immunity, chemokine signaling, and immune–chemokine crosstalk. The author should also discuss chemokines more by functional roles rather than listing individual molecules.

Thanks for your valuable comment again. We have followed your recommendation. The overall structure of the manuscript has been improved according to your suggestion. In general, the overall structure of the R2 version is more clear with independent sections for innate immunity, including chemokines and chemokine signalling in AD. We believe it is necessary to include chemokines before inhate immune system because chemokines recruit monocytes in the brain of AD models. Thus, it is necessary to introduce before explaining adaptive immune system and T recruitment in the AD brain; the rest of reorganization followed your advice in this R2 version; thus, the point 5 about immune–chemokine crosstalk and T cells in AD after describing adaptive immune system responses by T cells in this R2 version. Thus, points on chemokine signalling in Alzheimer and the crosstalk between chemokine -adaptive immune mediated responses are included at the end.

-We hope you are agree with this R2 structuration following reviewer-2 suggestions. However, this reviewer-2 requires shortened the conclusion while the reviewer-4 request us the opposite (increases the length of conclusion). Thus, we extend a litte bit the length of conclusion in this R2 version.

2. In Figure 1, why does the author not mention how the CNS and peripheral system interact with tau? The author should add a more comprehensive overview figure integrating Aβ/tau pathology, innate and adaptive immune responses, and chemokine-mediated crosstalk in AD.

Thanks again. The new R2 version includes two figures (fig-1 ; role of chemokines in BB) and the figure -2 integrates Aβ/tau pathology, innate and adaptive immune responses, and chemokine-mediated crosstalk in AD following your recommendation.

In a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells mediated neurodegeneration. The presence of activated T cells in the brains of transgenic mice with frontotemporal dementia (FTD)-like Tau protein pathology confirmed that infiltrates of T cells correlated with microglia overactivation and Tau deposition in the brain. The number of activated T cells (CD4+ and spetially CD8+ cells) are enriched the parenchyma in Tau mouse brains elevated in Tau pathology, leading to neurodegeneration. Interestingly, Tau transgenic mice on an APOE knockout background were protected against T cell infiltration, suggesting that T cell infiltration is associated with Tau pathology progression (Braak staging). The neuronal Tau aggregation triggers microglia activation, and results in recruitment, clonal expansion, and activation of T CD8 + cells in the brain. The inflamamtory cascade is enhanced by miicroglia MHC-II and CD11c cells, which activate T cells by IFNin the brain.

How are T cells activated in the brains of tauopathy mice?  It Is known that in microglia from tauopathy mice, genes associated with antigen representation, complement and interferon response, lysosomal pathways, and oxidative stress are upregulated. Furthermore, microglia had an antigen-presenting phenotype 2 characterized by the expression of major histocompatibility complex, MHC-II, proteins, and CD11c receptor, a disease-associated marker in TREM2+ microglia, which can provide a signal for T cell activation. Indeed, primary mouse microglia co-cultured with OT-1T cells (CD8+ T cells expressing TCRs that recognize ovalbumin as antigen) were able to stimulate OT-1T proliferation in the presence of their antigen, ovalbumin, which was exacerbated upon stimulation of microglia with the pro-inflammatory cytokine interferon-gamma, IFNγ. Inhibiting IFNγ signaling, or removing microglia by PLX3397 administration reduced p-Tau accumulation and brain atrophy in tauopathy mice. These results show that activated microglia have the capacity to trigger a T cell response that promotes Tau pathology and neurotoxicity. IFNγ secretion by immune cells could exacerbate neurodegeneration in tauopathies. Although, neurons signal such Tau alterations to microglia and induce T cell infiltration and activation remains to be clarified, it is known that depleting T cells reduced microglial MHC class II and CD11c expression, reduced p-Tau immunoreactivity and brain atrophy, and improved short-term memory. Blocking immunoregulation of T cells (PDCD1-PDL1 signaling) in TE4 mice through repeated anti-PD-1 antibody administration increased the percentage of immune suppressive regulatory T cells, but did not affect the percentage of effector T cells. However, at the age of 9.5 months, the anti-PD-1 treatment achieved a reduction in p-Tau and brain atrophy. In conclusion, targeting T cells and modulating immune response is a potential approach against Tau-mediated neurodegeneration (Askin, B., Wegmann, S. Interaction of microglia and T cells: a deadly duo behind Tau-mediated neurodegeneration. Sig Transduct Target Ther 8, 317 (2023). https://doi.org/10.1038/s41392-023-01563-9)

3. For table 1, it lacks mechanistic and translational information. The author should consider adding major cell source, functional category, associated AD pathology, and sample type.

Done it¡. Thanks again (please, read the R2 manuscript version that includes these aspects in tables 1 and 2). The figure 1 in the R2 version includes major cell source, functional category, associated AD pathology, and cell type.

1. For table 2, The author should reduce the title of the references and add: experimental model, immune cell subtype, protective vs detrimental effect, key chemokines/signaling pathways, main conclusion, and whether findings were from human or animal studies.

Done it¡. Moreover, the table-2 indicates mean studies with T cells in AD (rodent and AD patients with emphasis on experimental model, immune cell subtype, key signalling pathways and protective vs detrimental effects in AD models and patients. Thanks again

The manuscript contains two figures, which grouped all these aspects in the R2 version. We have also added a spetial point 3 about chemokines signalling and AD and chemokines-inmune crosstalk in AD (point 5). in this R2 version (but without including a specific figure). The figure-2 includes all interactive effects between Abeta and tau accumulation in the brain of AD models

The figure 2 (A) indicates in a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells mediates neurodegeneration. Figure-2. Inflammatory mediators in the brain of Alzheimer`s patients

Figure-2. Inflammatory mediators in the brain of Alzheimer`s patients

(A) The progressive aggregation of amyloid-β in extracellular amyloid plaques and of Tau in neurofibrillary tangles are pathological hallmarks of AD. The neuroinflammation, and neuronal death synaptic loss, monocyte infiltration compromise the blood-brain barrier; these immune cells infiltrate the brain parenchyma and interact with glial cells and neurons; in a rodent model of taupathy, neuroinflammatory response by activated microglia and effector-type T cells contribute to neurodegeneration in a CX3CR1-Fractalkine dependent manner. In fact, low soluble fractalkine release by vulnerable neurons impairs microglia functions in a CX3CR1 dependent manner, leading to p-Tau accumulation and amyloid beta deposition in the brain of AD and taupathy models [114]. The presence of activated T cells in the brains of transgenic mice with frontotemporal dementia (FTD)-like correlated with microglia overactivation and Tau deposition in the brain. In fact, enriched number of activated T cells (CD4+ and spetially CD8+ cells) are found in the parenchyma, leading to neurodegeneration. Interestingly, Tau transgenic mice on an APOE knockout background were protected against T cell infiltration, suggesting that T cell infiltration is associated with Tau pathology progression (Braak staging). The neuronal Tau aggregation triggers microglia activation, and results in recruitment, clonal expansion, and activation of T CD8 + cells in the brain. This inflammatory cascade is enhanced by microglia MHC-II and CD11c cells since IFN gamma released by clonal CD8 + cells activate T cells by in the brain. Collectivelley, these findings link T cell accumulation with p-tau and Amylid beta depostion, leading to neurotoxicity and neurodegeneration.

(B) Activated T cells can be found in postmortem brains of different neurological disorders, in AD, particularly in areas with neuronal loss and Tau pathology, i.e., the hippocampus and limbic structures. These infiltrated T cells in the brain parenquima activates astrocyte, which releases chemokines as CXCL10, CXCL9 and CC4. In fact, certain chemokines as CCR3 release by T infiltrated cells overactivates microglia, leading to neurotoxicity. This exacerbates local inflammation and enhances neuronal loss.

(C). CD4 and CD8+ T-cells infiltrates into the brain parenchyma promote detrimental effects, leading to Aβ plaque accumulation or microglia overactivation though IL-17 chemokine release by microglia cells. In fact, CD8+ clonal infiltates appear in the brain parenquima and these T cells activate microglia by IFN gamma release of activated CD8 + T cels. Thus, microglia overactivation release CXCL10 chemoines, which binds to astroglia carriers of CXCR3 chemokine receptor. Thus, CXCR3 contribute to neurodegeneration in conjunction with altered CXCR6-CXCL16 signalling by CD8+ T cells infiltratesin the brain. However, T Reg cells can also promote neuroprotective effects in AD models.

The author should discuss the three questions in the manuscript deeply: 1) The dual role of immune cells in AD, under what conditions and at which disease stages do immune cells exert protective versus detrimental effects in AD?

We response to these questions in the R1 submitted version. Anyway, we reply you in this R2 version. In fact, both situations are possible since chemokines aredrivers of AD pathology but can be also secondary given its capacity to regulate neuron-microglia interaction in AD since are pleiotphropic molecules.

Chemokines can regulate the BBB, which is often compromised in AD patients. Elevated levels of CCL2 and CCL5 can weaken BBB tight junctions, allowing peripheral immune cells (such as T-cells and monocytes) to infiltrate into the brain parenchyma.

2) What is the mechanistic role of chemokines in AD—are they drivers of pathology or secondary responses, and how do they regulate immune cell recruitment, BBB integrity, and neuroinflammation?

The role of chemokines in BBB disruption contribute to neuroinflammation in the brain. In this way, chemokines act as "homing" signals for microglia to play a dual role, functioning as either suppressors or inducers of pathogenic alterations though multiple signalling pathways in the AD brain. Certain chemokine ligands (ie: fractalkine) can regulate microglial chemotaxis via chemokine receptors (i.e:. CX3CR1), which sense these gradients and drive migration toward amyloid beta plaques in the brain [7].

The recruitment of inflammatory mediators in the brain is directed by chemokines in conjunction with cell adhesion molecules (selectins, VCAM-1, ICAM-1) and integrins (LFA-1, VLA-4, α4β1) [26]. Chemokines regulate the BBB integrity, which is often compromised in AD patients. The augmented levels of CCL2 and CCL5 can weaken BBB tight junctions, allowing peripheral immune cells (such as T-cells and monocytes) to infiltrate into the brain parenchyma. This exacerbates local neuroinflammation and increases neuronal loss. The binding of chemokine ligands to endothelial cells triggers signalling pathways and lead to the reorganization of tight junction proteins. In this way, BBB integrity depends on tight junctions (TJs), sealing the spaces between endothelial cells through interactions among zonula occludens-1 (ZO-1), claudins and occludins. Consequently, the “zipper-like" seal between cells loosens, allowing small molecules to reach the brain parenchyma. Chemokines induce matrix metalloproteinases (MMPs 2 and 9) that facilitate BBB breakdown by activating astrocytes, microglia, and endothelial cells. Thus, MPPs degrade the basement membrane and the extracellular matrix while chemokines stimulate the expression of adhesion molecules (i.e: ICAM-1 and VCAM-1) on the endothelial surface, which act as anchors for leukocytes, see figure-1 [27].

3) How does the peripheral immune system influence the brain in AD—do immune cells directly infiltrate across the BBB, or do they primarily exert indirect effects through CNS–periphery interactions?

 

These aspects have been included in the R2 version (see R2 pdf attached file within the Susy MDPI system). Briefly, we have added the point Thanks for your valuable comments. We have added this 3. Chemokine signaling in AD within the attached R2 version on line as follows for the point 5. Chemokine signalling and T-cells in AD as follows. This follows text was included within the new R2 attached version in MDPI system (see R2 file). The attached text is as follows:

 

5.Chemokine signalling and T-cells in AD

 

5.1. The CXCR4/SDF-1α axis influences microglia–T-cell crosstalk in AD models

 SDF1 alpha (also known CXCL12) correlated with an increase in brain-associated B-lineage cells in the AD brain, expressing its cognate CXCR4 receptor. In fact, CXCR4+ antibody-secreting cells are significantly reduced in the gut of 5XFAD AD mice. In contrast, CXCR4+ antibody-secreting cells (ASCs) are detected in the colon, while CXCR4+ B-cells and gut-specific IgA+ cells accumulate in the brain and dura mater. These findings suggest enhanced SDF-1α–dependent recruitment of immune cells into the brain of 5XFAD mice. In this model, SDF-1α is released by astrocytes, and its levels correlated with the infiltration of CXCR4 + B-cells and gut-specific IgA + cells into the brain and dura mater. Additionally, these effects appear to be SDF-1α specific since CXCR4 blockade by AMD3100 (CXCR4 antagonist) abolished the migration of immune cells into the brain [69].

 

5.2. The CXCR3 and CXCR6 and microglia–T-cell recruitment in AD models

Chemokines increased the T-cell recruitment in a CXCL10-CXCR3 chemokine dependent manner since CXCR3 chemokine receptor enables recruitment of specific memory CD8+ T-cells to areas containing activated APCs that ex-press CXCL9/10/11 [139,140] through its CXCR3 chemokine receptor down-stream pathways in the cortex of 5xFAD AD transgenic mice [18].

CD8+ T-cells contribute to neurodegeneration through direct cytotoxicity and indirect glial-enhanced inflammatory responses. These CD8+ T-cells indirectly exacerbate neurodegeneration by IFN-γ-associated signalling in glial cells. CXCR3 is expressed in infiltrated CD8+ T within the hippocampus and cortex of 5xFAD mouse brain at 6- to 7-month-old [18]. Since these Aβ plaque-associated subset of CD8⁺ T-cells promote Type-I interferon signalling and recruit non-ISG T-cells through the CXCL10-CXCR3 CXCL10 axis, this type-I interferon responses in microglia cells near plaques could be target of drugs to prevent am-yloid plaques accumulation in the human AD brain 141]. Moreover, certain chemokines as CCR2/CCL2 axis that recruit leukocytes in the brain and pro-mote the differentiation of naïve T-cells [40]. Other chemokines as CXCL8, can activate neutrophils and T-cells since it is a marker of T-cell effector function in human newborns and inhibits CD8+ T-cell infiltration [96].

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The signal pathways related on the roles of adaptive immune system in neuroinflammation and disease progression should be added.

Comments on the Quality of English Language

English is OK.

Author Response

Dear reviewer

Thanks for your valuable comment again. The manuscript has been reorganized following new reviewer-2 suggestions again. However, this reviewer-2 requires shortened the conclusion you ask the opposite (increases the length of conclusion). Thus, we have extended a little bit the length of conclusion in this R2 version. Thanks for your valuable comments again¡

We have added this point 5. Chemokine signalling and T-cells in AD following your advice and also the point 6 indicate crosstalk between chemokines signalling (CXCR6 and CXCR3) in adaptive immune system in AD as follows in this R2 version:

1. Chemokine signalling and T-cells in AD

5.1. The CXCR4/SDF-1α axis influences microglia–T-cell crosstalk in AD models

 SDF1 alpha (also known CXCL12) correlated with an increase in brain-associated B-lineage cells in the AD brain, expressing its cognate CXCR4 receptor. In fact, CXCR4+ antibody-secreting cells are significantly reduced in the gut of 5XFAD AD mice. In contrast, CXCR4+ antibody-secreting cells (ASCs) are detected in the colon, while CXCR4+ B-cells and gut-specific IgA+ cells accumulate in the brain and dura mater. These findings suggest en-hanced SDF-1α–dependent recruitment of immune cells into the brain of 5XFAD mice. In this model, SDF-1α is released by astrocytes, and its levels correlated with the infiltration of CXCR4 + B-cells and gut-specific IgA + cells into the brain and dura mater. Additionally, these effects appear to be SDF-1α specific since CXCR4 blockade by AMD3100 (CXCR4 antagonist) abolished the migration of immune cells into the brain [69].

5.2. The CXCR3 and CXCR6 and microglia–T-cell recruitment in AD models

Chemokines increased the T-cell recruitment in a CXCL10-CXCR3 chemokine dependent manner since CXCR3 chemokine receptor enables recruitment of specific memory CD8+ T-cells to areas containing activated APCs that ex-press CXCL9/10/11 [139,140] through its CXCR3 chemokine receptor down-stream pathways in the cortex of 5xFAD AD transgenic mice [18].

CD8+ T-cells contribute to neurodegeneration through direct cytotoxicity and indirect glial-enhanced inflammatory responses. These CD8+ T-cells indirectly exacerbate neurodegeneration by IFN-γ-associated signalling in glial cells. CXCR3 is expressed in infiltrated CD8+ T within the hippocampus and cortex of 5xFAD mouse brain at 6- to 7-month-old [18]. Since these Aβ plaque-associated subset of CD8⁺ T-cells promote Type-I interferon signalling and recruit non-ISG T-cells through the CXCL10-CXCR3 CXCL10 axis, this type-I interferon responses in microglia cells near plaques could be target of drugs to prevent amyloid plaques accumulation in the human AD brain 141].

Moreover, certain chemokines as CCR2/CCL2 axis that recruit leukocytes in the brain and pro-mote the differentiation of naïve T-cells [40]. Other chemokines as CXCL8, can activate neutrophils and T-cells since it is a marker of T-cell effector function in human newborns and inhibits CD8+ T-cell infiltration [96].

On the other hand, the Cxcr6 deficiency or CD8+ T depletion decreased the clonal expansion of brain PD-1+ CD8+ T-cells and increased proinflammatory cytokines released in microglia cells. In this study, the high number of CD3+TCRβ+ T-cells is attributable to the accumulation of CD8+ T-cells [113]. In addition, the CXCR6 chemokine plays a role in memory formation and its deficiency disrupts the spatial localization of CD8+ T-cells near Aβ plaque-associated microglia, leading to memory deficits in AD transgenic mice. Once recruited, T-cells engage in reciprocal interactions with microglia to r-inforce their activation and neurodegeneration [113]. These findings suggest a key role of CXCR6 chemokine in AD. In fact, the analysis of single-cell RNA sequencing datasets of brain-derived CD8⁺ T-cells confirmed a CXCR6-related immunosuppressive cluster with stem-like features in Cxcr6-deficient AD mice. Since CXCR6-related CD8⁺ T-cells decrease Aβ pathology, a neuroprotective role of these CD8 cells into the brain [80] (see table-2). The analysis of single-cell RNA sequencing samples from CSF in AD patients, as compared with 45 cognitively normal subjects (aged 54-82 years), revealed a role for CXCR6 in CD8 T-cells recruitment. In this study, defective CD8 T cytokine signalling is associated with decreased expression of lipid transport genes in monocytes. Collectively, these findings suggest that CXCL16-CXCR6 signalling axis promotes antigen-specific T-cell recruitment into the brain via CD8+ T effector memory T-cells [142]. Other study in the CSF of cognitively AD impaired patients, re-ported increased expression of CXCR6 in CD8+ memory T-cells in conjunction with CXCL16 augmented levels in microglia and monocytes, which suggest that this chemokine axis regulated the brain-resident T-cells recruitment into dam-ages areas of the central nervous system [76, 143].

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The evolution is clear, the changes are very good and comprehensive and the paper can be published, after the elimination of a few problems:

The conclusion is still too short (only three sentences) but it's not synthetic and does not cover all that we have learned. I suggest about 200 word and a clear expansion on a nice proposal of a research agenda.

Minor changes:

reference 140 is in Spanish 

The sentence about Tregs "rejuvenating" the brain’s immune response appears twice in close proximity (approximately lines 452–455 and 460–461 of Version 2). One variant should be removed

Line 285 of Version 2 contains the string "[photobiomodulation41]" — an artefact to be removed.

Version 2 adds PBM and TEMRA to the abbreviations list, which is a positive step. However, many abbreviations used frequently in the text are not defined in the list, including: NLRP3, MCP-1, DAM, NK, APC, MHC, TCR, LTP, ROS, ATP, ICAM-1, VCAM-1, MMP. Please add them and re-alphabetize

 

Author Response

Dear reviewer

Thanks a lot for your valuable comments, which help us to improve this R2 version. The manuscript has been reorganized following new reviewer-2 suggestions again. However, this reviewer-2 requires shortened the conclusion you ask the opposite (increases the length of conclusion). Thus, we have extended a little bit the length of conclusion in this R2 version. Thanks for your valuable comments again¡ So, we hope you are agree with this new R2 conclusion since the other request the opposite (increase the length of discussion)

We have added this point 5. Chemokine signalling and T-cells in AD following your advice and also the point 6 indicate crosstalk between chemokines signalling (CXCR6 and CXCR3) in adaptive immune system in AD as follows in this R2 version:

Minor changes:

reference 140 is in Spanish

Please, notice that lines are totally different in this R2 version because reviewer-2 request us a new organization, which we follows in this R2 version.

This reference was added in English.

Thanks again¡

Author Response File: Author Response.pdf