Anti-cancer immune reaction and lymph node macrophage; a review from human and animal studies

Lymph nodes are secondary lymphoid organs that appear as bean-like nodules usually <1 cm in size, and they are localized throughout the body. Many antigen-presenting cells such as dendritic cells and macrophages reside in lymph nodes, where they mediate host defense responses against pathogens such as viruses and bacteria. In cancers, antigenpresenting cells induce cytotoxic T lymphocytes (CTLs) to react to cancer cell–derived antigens. Macrophages located in the lymph node sinus are of particular interest in relation to anti-cancer immune responses because many studies using both human specimens and animal models have suggested that lymph node macrophages play a key role in activating anti-cancer CTLs. The regulation of lymph node macrophages therefore represents a potentially promising novel approach in anti-cancer therapy. Keyword: macrophage, lymph node, CD169, PD-L1, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 June 2021 doi:10.20944/preprints202106.0666.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. 2 The critical role of lymph nodes in anti-cancer immunotherapy Cancer cells are characterized by the accumulation of a variable number of genetic alterations that result in the production of neoantigens or cancer testis antigens. CD8 T cells recognize cancer cells via binding between the T-cell receptor and major histocompatibility complex class I/peptide complex [1]. Chen and Mellman (2013) suggested that the immune system is triggered to eliminate cancer cells via stimulation of the cancer-immunity cycle [2]. Immune checkpoint blockade therapy targeting cytotoxic T-lymphocyte (CTL)-associated antigen 4 (CTLA-4) or programmed death 1 (PD1)/programmed death ligand 1 (PD-L1) has become a promising anti-cancer immunotherapy approach [3]. Anti–PD-1 and anti–CTLA-4 therapy are reportedly effective for patients with several types of solid tumors, such as melanoma, non-smallcell lung cancer, renal cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma (SCC), esophageal SCC, gastric adenocarcinoma, triple-negative breast carcinoma, and microsatellite instability-high tumors [4]. CTLA-4 is expressed on T-lymphocytes and competitively inhibits the binding of CD28 to costimulatory molecules such as CD80 and CD86. PD-1 ligands are expressed on both cancer cells and immune cells. Among immune cells, antigen-presenting cells such as macrophages and dendritic cells (DCs) express high levels of PD-1 ligands [5]. Myeloid cells express PD-1 ligands in both the tumor microenvironment and lymph nodes [6]. Fransen et al. demonstrated that CD11b myeloid cells residing in lymph nodes express significantly higher levels of PD-L1 in tumor-bearing mice; lymph node resection in these mice abrogated the anti-tumor effect of anti–PD-1 therapy [7]. Fransen et al. also found that the anti-tumor effect of anti–PD-1 therapy was also suppressed by the S1P receptor inhibitor FTY720, which restricts T cells in lymphoid organs. Dammeijer et al. detected expression of PD-L1 in DCs and macrophages in tumor-draining lymph nodes; blocking PD-L1 on DCs (but not macrophages) induced an effective anti-tumor immune response [8]. These authors also found greater interaction between PD-1 and PD-L1 in the lymph nodes than tumors, which correlated with shorter relapse-free survival. These findings suggest that tumor-draining lymph nodes play a critical role in the anti-tumor immune responses induced by anti-PD1/PD-L1 therapy. Function of lymph node macrophages in mice Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 June 2021 doi:10.20944/preprints202106.0666.v1

The critical role of lymph nodes in anti-cancer immunotherapy Cancer cells are characterized by the accumulation of a variable number of genetic alterations that result in the production of neoantigens or cancer testis antigens. CD8 + T cells recognize cancer cells via binding between the T-cell receptor and major histocompatibility complex class I/peptide complex [1]. Chen and Mellman (2013) suggested that the immune system is triggered to eliminate cancer cells via stimulation of the cancer-immunity cycle [2]. Immune checkpoint blockade therapy targeting cytotoxic T-lymphocyte (CTL)-associated antigen 4 (CTLA-4) or programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) has become a promising anti-cancer immunotherapy approach [3]. Anti-PD-1 and anti-CTLA-4 therapy are reportedly effective for patients with several types of solid tumors, such as melanoma, non-smallcell lung cancer, renal cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma (SCC), esophageal SCC, gastric adenocarcinoma, triple-negative breast carcinoma, and microsatellite instability-high tumors [4].

CTLA-4 is expressed on T-lymphocytes and competitively inhibits the binding of
CD28 to costimulatory molecules such as CD80 and CD86. PD-1 ligands are expressed on both cancer cells and immune cells. Among immune cells, antigen-presenting cells such as macrophages and dendritic cells (DCs) express high levels of PD-1 ligands [5].
Myeloid cells express PD-1 ligands in both the tumor microenvironment and lymph nodes [6]. Fransen et al. demonstrated that CD11b + myeloid cells residing in lymph nodes express significantly higher levels of PD-L1 in tumor-bearing mice; lymph node resection in these mice abrogated the anti-tumor effect of anti-PD-1 therapy [7]. Fransen et al. also found that the anti-tumor effect of anti-PD-1 therapy was also suppressed by the S1P receptor inhibitor FTY720, which restricts T cells in lymphoid organs. Dammeijer et al. blocking PD-L1 on DCs (but not macrophages) induced an effective anti-tumor immune response [8]. These authors also found greater interaction between PD-1 and PD-L1 in the lymph nodes than tumors, which correlated with shorter relapse-free survival. These findings suggest that tumor-draining lymph nodes play a critical role in the anti-tumor immune responses induced by anti-PD1/PD-L1 therapy. Interest in the role of lymph node macrophages in the initiation of immune responses is increasing [9]. Research in this area has shown that lymph node sinus macrophages (SMs) express sialoadhesin (CD169), a 185-kDa type I lectin involved in phagocytosis of pathogens and cell-cell contact with lymphocytes via binding to CD43 (sialophorin). SMs are divided into two subtypes in mice: subcupsular SMs (SCSMs) and medullary SMs (MSM), which are characterized as CD11b + CD169 + F4/80 − and CD11b + CD169 + F4/80 + cells, respectively [10,11]. However, macrophages located in the medullary cord in both humans and rodents are CD169 − . CD169 expression is specifically restricted to macrophages, particularly resident macrophages in the spleen, liver, bone marrow, and intestines. CD169 is thought to play a role in the uptake of sialylated antigens and therefore considered a potentially useful target for antigen delivery in vaccine development in mice [12]. Targeting antigen delivery to CD169-expressing cells was

Function of lymph node macrophages in mice
shown to enhance immune responses in pigs [13]. anti-tumor immune response.
In the spleen, CD169 expression is restricted to marginal zone metallophilic macrophages [18]. Benhard et al. demonstrated that both DCs and CD169 + macrophages induce CTL responses in the spleen. They found that DCs induce CTLs that react to strongly binding epitopes, whereas macrophages induce CTLs that react to a broader range of epitopes [19]. Muraoka  Routine pathological analyses afford many opportunities for clinicians to observe the lymph nodes of cancer patients. The presence of metastasis to the lymph nodes is an important factor in determining disease stage, and the pathologist is responsible for making that decision. We examined CD169 expression in human lymph nodes immunohistochemically using paraffin-embedded sections. SMs were also positive for other macrophage markers such as CD163 and CD204, and Ki67-positive SMs were also observed ( Figure 2). The DC-related marker fascin was also expressed on SMs [34]. First, when we analyzed samples from cases of colorectal carcinoma, and interestingly, the rate of CD169 positivity of the SMs varied greatly among individual cancer-bearing patients and non-cancer controls [35]. This observation was inconsistent with CD169 expression in mouse lymph nodes. There were also no clear differences between SCSMs and MSMs, in contrast to mouse lymph nodes. Statistical analyses of postoperative survival and clinicopathological factors between patients with high and low CD169 expression in lymph nodes were also performed. In cases with colorectal carcinoma, high CD169 expression in SMs had significantly longer overall survival, smaller tumor size, and less lymph node metastasis [35]. Multivariate analysis identified CD169 positivity rate in SMs as an independent factor in determining overall survival in colorectal carcinoma patients.
The same analysis was performed for malignant melanoma, bladder cancer, endometrial carcinoma, gastric cancer, and esophageal cancer, and in cases with high CD169 expression, a significant prolongation of overall survival and cancer-specific survival was observed [36][37][38][39][40]. Multivariate analysis also indicated that a high number of CD169 + SMs is an independent prognostic factor in malignant melanoma, gastric cancer, and bladder cancer. Other researchers have reported similar results for prostate cancer and breast cancer [41,42]. Infiltrating CD8-positive T cells in tumor tissues, particularly in tumor nests, play a central role in the anti-tumor immune response in cancer patients. We hypothesized that CD169 + SMs activate CD8-positive T cells to promote anti-tumor immunity and therefore analyzed CD169 + SMs and the infiltration of CD8-positive T cells into tumor tissues. The number of CD8-positive T cells in tumor tissues was significantly higher in colorectal carcinoma, malignant melanoma, gastric cancer, breast cancer, and bladder cancer, which exhibited a high CD169 positivity rate in SMs [35,36,[38][39][40]43]. In endometrial carcinoma, the number of CD169 + SMs was correlated with infiltration of NK cells into the tumor [37]. The results of the present study demonstrate a significant correlation between CD169 + SMs in anti-tumor immune responses and a better clinical course. Other groups have also suggested that CD169 + SMs play a critical role in anti-tumor immune responses.
Gunnarsdottir et al. reported that the co-localization of CD169 + SMs and cancer cells in lymph node metastatic lesions was linked to improved recurrence-free survival in patients with breast cancer [42]. Those authors also examined PD-L1 expression in SMs; however, they found no significant association between PD-L1 expression on SMs and clinical course [42]. Using a rat prostate cancer model, Strömvall et al. identified several genes, including the gene encoding CD169, which are up-regulated in the pre-metastatic niche in tumor-draining lymph nodes [44]. In a subsequent study using the rat model, they found fewer CD169 + SMs in pre-metastatic tumor-draining lymph nodes, and a reduction in the number of CD169 + SMs was found to be closely associated with shortened relapse-free survival in prostate cancer patients [41]. Topf et al. reported that the metastatic spread of head and neck carcinoma to regional lymph nodes was associated with fewer CD169 + SMs in draining lymph nodes [45]. This reduction in the number of SMs was significant in cancer cases without human papilloma virus infection. Thus, accumulating evidence suggests that CD169 + SMs play a critical role in determining the clinical course of various types of cancer, but details regarding the relationship between SMs and cancer in humans remain to be determined.

Conclusions
Evidence indicating that CD169 + SMs play a significant role in anti-cancer immune responses is increasing, and many studies examining human samples and mouse models (C) Cell count and signal value data were evaluated using ImageJ software. Differences between two groups were evaluated using the Mann-Whitney U-test.