Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript analyzed performance of immunotherapy using cytokine-activated autologous lymphocytes combination with tomotherapy in treating 63-year-old lung cancer patient. While the common used immuntherapy are TIL therapy using TIL isolated from tumor tissue, and CAR-T cells therapy, both of which are expanded and screened to identify tumor antigens, while the current study used the cytokine-activated PBMCs, which raise some concerns:

1. Theauthor should prove that the improved treatment effect is the result of the PBMCs.
2. In Figure 2, the percentage of CD3+CD16+/CD56+ should be labeled, and it seems that its percentage is not increased obviously, in addition, more phenotype and functional study of other cell types should be analyzed, for example, the in vivo cell killing test, and the effect/memory type of T cells, the cytokine production of T cells, NK cells and NKT cells.
3. A majorproblem of the manuscript is that the follow-up time is short, longer study of the case using CT and flow cytometry is needed to make the result more conceivable.
4. The discussion section should be refined, the value and novelty of the current studyshould be further emphasized here.
5. Fonts size in figures should be identical.

Author Response

Reviewer's comment: The manuscript analyzed performance of immunotherapy using cytokine-activated autologous lymphocytes combination with tomotherapy in treating 63-year-old lung cancer patient. While the common used immuntherapy are TIL therapy using TIL isolated from tumor tissue, and CAR-T cells therapy, both of which are expanded and screened to identify tumor antigens, while the current study used the cytokine-activated PBMCs, which raise some concerns:

Authors’ response: We thank for this critical point. To answer properly to this comment, we provide our step-by-step rebuttal to any raised concerns.

Although TIL and CAR T-cell therapy are currently the most widely studied forms of personalized cellular immunotherapy, the use of cytokine-activated lymphocytes (PBMC-based therapy, including CIK/LAK cells) is also a scientifically sound approach and continues to be clinically relevant for several reasons. First, PBMCs activated by cytokines (IL-7, IL-15, SCF, Flt3-L) are a heterogeneous population of T cells with enhanced cytotoxic function. This provides an advantage in MHC-independent tumor cell recognition, which in turn allows for the destruction of tumors with low HLA expression, which possess mechanisms for immune evasion. Second, compared to TILs and CAR T-cells, the simplicity, availability, and speed of obtaining activated cells are advantageous. PBMC therapy does not require surgical harvesting of tumor tissue. This eliminates the need for lengthy TIL cell expansion or complex antigen specificity verification. Unlike CAR-T, there is no genetic modification step. This makes the method of injecting activated lymphocytes more accessible. This also makes the method suitable for patients with stage 3-4 cancer or for those unable to obtain sufficient TILs. Unlike TILs and CAR-T, it is a good option specifically for elderly patients or patients with comorbidities for whom aggressive TIL or CAR-T protocols may be contraindicated. Third, this case and the case of other patients who received a similar therapy regimen for lung cancer demonstrated clinical efficacy: improved patient survival and demonstrated synergy with tomotherapy. The method of injecting activated lymphocytes provided clinical efficacy with low toxicity (which distinguishes it from some forms of CAR-T or intensive TIL protocols). In this case, the clinical effect was achieved by a combination of tomotherapy and cytokine-activated lymphocytes, which is particularly promising. This synergy of methods resulted in a clinical outcome because radiation therapy increases tumor immunogenicity, enhances the expression of stress-inducible molecules, and enhances immune cell infiltration. Fourth, activated lymphocytes have a high safety profile, a low risk of cytokine storm, minimal organ toxicity, and a good tolerability profile. Thus, TILs, CAR-t, and CIK/LAK cells are complementary, not mutually exclusive, immunotherapy methods. Each is effective in specific clinical settings.

 

Reviewer's comment: The author should prove that the improved treatment effect is the result of the PBMCs.

Authors’ response: We thank for this critical point. Cytokine activation of PBMCs resulted in the formation of a cell population with pronounced antitumor activity, CD8+ T lymphocytes, enhanced cytotoxicity, and the ability to recognize tumor cells independent of HLA expression, which is particularly important in lung cancer, which is characterized by frequent immune evasion mechanisms. The administration of such cells during tomotherapy has a biologically plausible synergism, as radiation induces tumor immunogenicity, increases the expression of stress-induced ligands, and enhances the susceptibility of tumor cells to immune destruction.

In this case, the patient was part of the main study group (10 patients), which received a combination of cell immunotherapy and tomotherapy. Treatment results for this group of patients demonstrated increased survival compared to the control group (10 patients without cell therapy, tomotherapy alone). In most cases, the main group demonstrated more pronounced decrease in tumor volume. The enhancement of the therapeutic effect demonstrates a clear temporal relationship with the administration of activated peripheral blood mononuclear cells. Despite limited tumor stabilization prior to cell therapy, the most pronounced regression or slowing of progression was observed after serial cycles of PBMC administration. Meanwhile, periods without cell therapy were characterized by less pronounced clinical dynamics. Patients also reported improved well-being during cell therapy. This pattern of observations forms a consistent causal pattern that meets key Bradford-Hill criteria, including temporal dependence, consistency, and biological plausibility, thereby confirming the significant contribution of PBMC to achieving the observed therapeutic effect.

The observed clinical dynamics exceed those typical of isolated radiation therapy in both the severity and sustainability of the effect, confirming the contribution of PBMC to the antitumor response. Thus, the authors attribute the enhanced therapeutic effect to the action of cytokine-activated peripheral blood mononuclear cells, as the combined clinical and biological data demonstrate a causal relationship between their administration and the observed improvement. The combination of biological plausibility, objective immunological data, a clear temporal relationship, and clinical observations provides compelling evidence that the patient's improvement is due to the action of the administered cytokine-activated peripheral blood mononuclear cells.

 

Reviewer's comment: In Figure 2, the percentage of CD3+CD16+/CD56+ should be labeled, and it seems that its percentage is not increased obviously, in addition, more phenotype and functional study of other cell types should be analyzed, for example, the in vivo cell killing test, and the effect/memory type of T cells, the cytokine production of T cells, NK cells and NKT cells.

Authors’ response: We modified Figure 2, and added percentages for the changes in the expression of all markers including CD3+CD16+/CD56+. Please note that the bars show actual measurements in the gate units, not percentages (proportion) of the cell phenotypes. The percentages shown above the bars show the change in “7 days culture” relatively to “before” measurement. We did not perform extended phenotypical analysis for cultured cells and did not measure cytokine production by the cells, we only evaluated the shifts in expression of specific types by testing various CD markers in flow cytometry.

 

Reviewer's comment: A major problem of the manuscript is that the follow-up time is short, longer study of the case using CT and flow cytometry is needed to make the result more conceivable.

Authors’ response: We thank for this critical comment and we agree that the longer follow-up period is needed to conclude about the effectiveness of the combined treatment. It is especially important to have repeated CT during the follow-up. However, we were unable to get the CT because the patient decided not to follow-up. On the other hand, early before the submission (August 2025) we have contacted to the patient directly by phone, he was alive and with a stable good condition.

 

Reviewer's comment: The discussion section should be refined, the value and novelty of the current study should be further emphasized here.

Authors’ response: We thank for this suggestion. We substantially modified the Discussion to reflect all raised issues.

 

Reviewer's comment: Fonts size in figures should be identical.

Authors’ response: We adjusted the font sizes where possible, but some of the figures were obtained in a raw format directly from the hardware and it is difficult to modify the image elements (like axis labels, etc.).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The result of lung biopsy is reported as “squamous cell carcinoma metastasis with keratinization of the lymph nodes”. Do the authors have the pathological report of the primary tumor site?
2. If possible, please reveal the driver mutations of this patient’s tumor.
3. What is the PD-L1 expression in this case?
4. When was the blood for lymphocyte isolation collected? Before or after radiotherapy?
5. The concentration of the activated lymphocyte was reported as ** per 10uL, but what’s the total volume or total cell number the patient received?
6. In Figure 2, the percentage of CD45+ cells are all below 60% in the isolated PBMC before or after activation. Why was the purity of immune cells so low?
7. It’s suggested that the authors add the percentage change of NK cells into Figure 2A.
8. It’s suggested that the authors show all the representative flow images of each type of immune cells.
9. Since the patient underwent simultaneous activated lymphocyte-based immunotherapy and radiotherapy, it is difficult to distinguish the relative contribution of each modality to clinical efficacy. The authors should discuss this limitation more thoroughly and interpret the treatment outcome with greater caution.

Author Response

Reviewer's comment: The result of lung biopsy is reported as “squamous cell carcinoma metastasis with keratinization of the lymph nodes”. Do the authors have the pathological report of the primary tumor site?

Authors’ response: Yes, the patient had the pathological report from the primary tumor site. The primary lung lesion was confirmed histologically as squamous cell carcinoma, consistent with the morphology described in the lymph-node biopsy showing metastatic keratinizing squamous cell carcinoma.

 

Reviewer's comment: If possible, please reveal the driver mutations of this patient’s tumor.

Authors’ response: Unfortunately, we did not perform the test for driver mutations.

 

Reviewer's comment: What is the PD-L1 expression in this case?

Authors’ response: We did not measure PD-L1 expression in this case.

 

Reviewer's comment: When was the blood for lymphocyte isolation collected? Before or after radiotherapy?

Authors’ response: The blood mononuclears were collected just before beginning of tomotherapy course.

 

Reviewer's comment: The concentration of the activated lymphocyte was reported as ** per 10uL, but what’s the total volume or total cell number the patient received?

Authors’ response: The values ​​of 1.86×10⁷, 7.36×10⁶, and 7.59×10⁶ cells per 10 µl refer to the initial cell suspension concentrations obtained before dilution. For clinical administration, the preparations were adjusted to a final volume of 200 ml; however, separate determination of the concentration and total cell count in the final infusate was not performed due to changes in suspension parameters during preparation. The amount of cell the patient received during each administration was approximately equal to the indicated values.

 

Reviewer's comment: In Figure 2, the percentage of CD45+ cells are all below 60% in the isolated PBMC before or after activation. Why was the purity of immune cells so low?

Authors’ response: We modified Figure 2, and added percentages for the changes in the expression of all markers including CD45+. Please note that the bars show actual measurements in the gate units, not percentages (proportion) of the cell phenotypes. The percentages shown above the bars show the change in “7 days culture” relatively to “before” measurement. The purity for lymphocytes in culture (in proportion to non-lymphocytic cells) was assessed at the level ~85% or above.

 

Reviewer's comment: It’s suggested that the authors add the percentage change of NK cells into Figure 2A.

Authors’ response: We modified Figure 2, and added percentages for the changes in the expression of all markers including NK cells.

 

Reviewer's comment: It’s suggested that the authors show all the representative flow images of each type of immune cells.

Authors’ response: We added the representative flow cytometry images for all types of CD markers to Figure2.

 

Reviewer's comment: Since the patient underwent simultaneous activated lymphocyte-based immunotherapy and radiotherapy, it is difficult to distinguish the relative contribution of each modality to clinical efficacy. The authors should discuss this limitation more thoroughly and interpret the treatment outcome with greater caution.

Authors’ response: We agree with this critical point. We extended our Discussion to cover this issue.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 The  interesting case report. The authors put a lot of work into developing the results and preparing the publication. The drawback of the article is the lack of assessment of the patient's geriatric condition (CGA or G8 or any geriatric scale). I also have doubts as to whether a 63-year-old patient should be considered geriatric. In the discussion section, I suggest expanding it to include a description of the most common complications of immunotherapy, especially in the geriatric patient group.

Author Response

Reviewer's comment: The interesting case report. The authors put a lot of work into developing the results and preparing the publication.

Authors’ response: We thank the reviewer for this positive remark on our manuscript.

 

Reviewer's comment: The drawback of the article is the lack of assessment of the patient's geriatric condition (CGA or G8 or any geriatric scale). I also have doubts as to whether a 63-year-old patient should be considered geriatric.

Authors’ response: We thank for this critical remark. Indeed, we did not perform assessment of geriatric state of the patient. Regarding the age of the patient – it is true that geriatric period is generally considered as 65 years and older. However, the clinical definition for geriatric age may be affected by the country/region according to the multiple factors like socioeconomical development and ecology. Kazakhstan is ranked as low as 108th place among other countries for the percentage of older population aged over 65 years (data for 2024), with only 9.6% of older people in total population. The average life expectancy for male people in Kazakhstan is about 70 years only. Also, despite Kazakhstan is considered now as an upper-middle-income country, the healthcare system is not developed well enough to cover all areas of early diagnostics for diseases; therefore, there is a risk for undiagnosed situation. The case of our patient is an example for this situation. Finally, the air, water, and soil pollution in Kazakhstan are measured at relatively high levels ranking this country at ~100^th place as well among all other countries. Altogether, these factors determine shorter life expectancy, especially for males, in Kazakhstan compared to many other countries with similar or higher incomes. Therefore, our patient may be considered as elderly (in Kazakhstan scale of ages) and therefore may be qualified as a person of nearly geriatric age. However, we realize that this issue is arguable.

 

Reviewer's comment: In the discussion section, I suggest expanding it to include a description of the most common complications of immunotherapy, especially in the geriatric patient group.

Authors’ response: We thank for this suggestion and we added the discussion concerning this issue.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made the corresponding revisions. Publication is recommended.

Author Response

We thank the Reviewer for evaluating our manuscript and suggesting corrections and improvements.