Lymph Node Follicles Research Articles

Bovine Gammaherpesvirus 6 Tropism in the Natural Host

Bovine gammaherpesvirus 6 (BoHV-6) is endemic in cattle in Europe, with a high prevalence. There is evidence that the virus is a commensal and not associated with disease processes. For other gammaherpesviruses, it is known that they have a rather specific target cell spectrum, generally including B cells and, at least in the early phase of infection, the epithelium of the respiratory tract. In a previous study we detected BoHV-6 by quantitative PCR for the gB gene sequence of BoHV-6 in lung, bronchial lymph nodes, spleen and tongue with variable loads, suggesting cells in these tissues as target cells. In the present study, formalin-fixed, paraffin embedded samples of the same tissues from 10 cattle, with high overall BoHV-6 copy numbers, were examined by RNA in situ hybridization for BoHV-6 ORF73. This revealed extremely limited viral ORF73 transcription. A signal was only detected in individual lymphocytes within lymphatic follicles in bronchial lymph nodes, and within very rare alveolar epithelial cells and interstitial cells in the lungs, without any evidence of pathological changes in the tissues. No signal was detected in the spleen or in the oral mucosa of the tongue. The results are consistent with previous findings with other gammaherpesviruses, murine herpesvirus-68, ovine herpesvirus-2 and/or Epstein–Barr virus. They provide further evidence that BoHV-6 is without any consequence to the host and can indeed represent a commensal in cattle.

Spatial technologies to evaluate the HIV-1 reservoir and its microenvironment in the lymph node.

The presence of the HIV-1 reservoir, a group of immune cells that contain intact, integrated, and replication-competent proviruses, is a major challenge to cure HIV-1. HIV-1 reservoir cells are largely unaffected by the cytopathic effects of viruses, antiviral immune responses, or antiretroviral therapy (ART). The HIV-1 reservoir is seeded early during HIV-1 infection and augmented during active viral replication. CD4+ T cells are the primary target for HIV-1 infection, and recent studies suggest that memory T follicular helper cells within the lymph node, more precisely in the B cell follicle, harbor integrated provirus, which contribute to viral rebound upon ART discontinuation. The B cell follicle, more specifically the germinal center, possesses a unique environment because of its distinct property of being partly immune privileged, potentially allowing HIV-1-infected cells within the lymph nodes to be protected from CD8+ T cells. This modified immune response in the germinal center of the follicle is potentially explained by the exclusion of CD8+ T cells and the presence of T regulatory cells at the junction of the follicle and extrafollicular region. The proviral makeup of HIV-1-infected cells is similar in lymph nodes and blood, suggesting trafficking between these compartments. Little is known about the cell-to-cell interactions, microenvironment of HIV-1-infected cells in the follicle, and trafficking between the lymph node follicle and other body compartments. Applying a spatiotemporal approach that integrates genomics, transcriptomics, and proteomics to investigate the HIV-1 reservoir and its neighboring cells in the lymph node has promising potential for informing HIV-1 cure efforts.

Abstract 3977: Neoadjuvant chemotherapy and radiation shape the local and regional adaptive antitumor immune response in lung squamous cell carcinomas

Abstract Chemotherapy (CT) and radiotherapy (RT) are known for their stimulating as well as inhibiting effects on the anti-tumor immune response. This study aimed to understand how CT and RT change the local immune microenvironment of the tumor and the regional immune response in draining lymph nodes. Evaluations were performed on lung squamous cell carcinomas (LUSC) with a special focus on tertiary lymphoid structures (TLS) and lymph follicles. A cohort of 21 untreated LUSC (naiveLUSC) and 21 matched neoadjuvantly treated LUSC (neoLUSC) was compiled. Digitized H&E whole slide images of the tumor and of two representative probes of a proximal and distal lymph node were assessed. A pathologist blinded to the groups assigned tumors to one of three immunophenotypes (inflamed, excluded, desert) by visual analysis. Tertiary lymphoid structures (TLS) in the tumor area and lymph follicles in the regional lymph nodes were counted and characterized according to their maturation status (with/without germinal center). RNAseq analyses were performed for a subset of tumors. NeoLUSC tended to more frequently have a desert immune phenotype (6/21 versus 1/21, p=0.0931). The mean number of TLS was significantly reduced in neoLUSC compared to naiveLUSC (4 versus 9, p=0.0179). Additionally, neoLUSC had a significantly lower likelihood for the presence of mature TLS with germinal centers (3/21 versus 9/21, p=0.0327). While the mean number of lymph follicles in regional lymph nodes was comparable between neoLUSC and naiveLUSC, neoLUSC had a significantly reduced number of mature lymph follicles (20% versus 41% of lymph follicles, p=0.0376) in distant lymph nodes and showed a similar, but less pronounced trend in proximal lymph nodes. In general, the amount of TLS correlated with the amount of lymph follicles in naiveLUSC patients (p=0.0154), but not in neoLUSC patients. Across the complete cohort, long-term survivors (>2 years) tended to have higher numbers of lymph follicles in lymph nodes than short term survivors (1.6 versus 1.1 lymph follicles/mm2, p=0.0805). RNAseq analysis revealed a significant upregulation of inflammatory gene sets and downregulation of cell cycle associated gene sets in neoLUSC as compared to naiveLUSC. This study demonstrates that CT and RT are potent, but multimodal influencers of the local tumor immune microenvironment as well as the regional immune response of the draining lymph nodes. While gene expression results underline the inflammation promoting effect of CT and RT, histological evaluations suggest that CT and RT negatively affect the adaptive anti-tumor immune response by reducing the development and maturation of TLS and lymph follicles in the lymph nodes. Next steps include an artificial intelligence based cell characterization and spatial evaluation of the tumor immune microenvironment to further unveil functional changes induced by CT and RT. Citation Format: Rosemarie Krupar, Justin Nieder, Johannes Braegelmann, Mareike Haarmann, Torsten Goldmann, Sharon Ruane, Timo Milbich, Cornelius Boehm, Lukas Ruff, Julika Ribbat-Idel, Christian Watermann, Sven Perner, Christiane Kuempers. Neoadjuvant chemotherapy and radiation shape the local and regional adaptive antitumor immune response in lung squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3977.

Assessment of CXCL13 plasma level in chronic lymphocytic leukemia and its relation to other prognostic markers

Abstract: BACKGROUND: Chronic lymphocytic leukemia is a mature B-cell malignancy where there is a progressive accumulation of leukemic cells with a distinctive immunophenotype as consequence to defective apoptosis and survival signals derived from the microenvironment. CXC chemokine ligand 13 (CXCL13) chemokines have recently emerged as crucial orchestrators for lymphocyte trafficking and activation. These secreted polypeptides exert their function by binding to specific cell surface receptors and can be divided into two categories: homeostatic and inflammatory. The CXCL13 is an efficacious attractant of naive B-cells in vitro and has been shown to be produced constitutively by stromal cells in lymphoid follicles of human lymph nodes. The CXCL13-CXCR5 axis has been previously shown to contribute to the progression of several malignancies and possibly CLL relapse. OBJECTIVE: The aim of this study to compare the plasma level of CXCL13 in a patient with CLL with healthy normal control and to correlate the plasma level of CXCL13 to beta-2 microglobulin (β2 M) and other hematological parameters in CLL. PATIENTS, MATERIALS AND METHODS: This cross-sectional study was conducted on 50 CLL patients who were newly diagnosed. A total of 30 healthy individuals were included in this study as a control group. Measurement of plasma CXCL13 and beta-2 microglobulin levels was done by the enzyme-linked immunosorbent assay. RESULTS: Fifty CLL patients were studied and compared with thirty control group of healthy individuals. The mean level of CXCL13 was 67.48 pg/ml in CLL patients while it was 69.2 pg/ml in control, so it is statistically not significant (P = 0.363). The mean level of β2 M was 50.89 ug/ml in CLL patients while it was 50.59 ug/ml in control, so it is statistically not significant (P = 0.702). The percentage of stage A of CLL patients was 22.44%, stage B was18.36%, and stage C was 10.20%. The percentages of lymphadenopathy, splenomegaly, and hepatomegaly were 66%, 32%, and 16%, respectively. The mean of malignant cell percentage in stage A was 55.18%; in stage B, it was 69.28%; and in stage C, it was 81%, so it is statistically significant (P < 0.001). CXCL13 shows no statistically significant between Cd38+ and CD38− and P value was 0.950. CONCLUSIONS: There was no correlation in level of CXCL13 between the CLL group and the control group. There was no correlation in level of CXCL13 and β2 M in the CLL group.

Extracellular matrix rigidity modulates physical properties of subcapsular sinus macrophage-B cell immune synapses

Subcapsular sinus macrophages (SSMs) play a key role in immune defense by forming immunological barriers that control the transport of antigens from lymph into lymph node follicles. SSMs participate in antibody responses by presenting antigens directly to naive B cells and by supplying antigens to follicular dendritic cells to propagate germinal center reactions. Despite the prominent roles that SSMs play during immune responses, little is known about their cell biology because they are technically challenging to isolate and study in vitro. Here, we used multicolor fluorescence microscopy to identify lymph node-derived SSMs in culture. We focused on the role of SSMs as antigen-presenting cells, and found that their actin cytoskeleton regulates the spatial organization and mobility of multivalent antigens (immune complexes [ICs]) displayed on the cell surface. Moreover, we determined that SSMs are mechanosensitive cells that respond to changes in extracellular matrix rigidity by altering the architecture of the actin cytoskeleton, leading to changes in cell morphology, membrane topography, and IC mobility. Changes to extracellular matrix rigidity also modulate actin remodeling by both SSMs and B cells when they form an immune synapse. This alters synapse duration but not IC internalization nor NF-κB activation in the B cell. Taken together, our data reveal that the mechanical microenvironment may influence B cell responses by modulating physical characteristics of antigen presentation by SSMs.

The extracellular matrix of lymph node reticular fibers modulates follicle border interactions and germinal center formation

Germinal center (GC) formation and antibody production in lymph node follicles require coordinated interactions between B-cells, T-cells and dendritic cells (DCs), orchestrated by the extracellular matrix-rich reticular fiber (RF) network. We describe a unique laminin 523-containing RF network around and between follicles that associates with PDGFrecβhighCCL19lowgp38low fibroblastic reticular cells (FRC). In the absence of FRC expression of laminin α5 (pdgfrb-cre:Lama5fl/fl), pre-Tfh-cells, B-cells and DCs are displaced from follicle borders, correlating with fewer Tfh-cells and GC B-cells. Total DCs are not altered in pdgfrb-cre:Lama5fl/fl mice, but cDC2s, which localize to laminin α5 in RFs at follicle borders, are reduced. In addition, PDGFrecβhighCCL19lowgp38low FRCs show lower Ch25h expression, required for 7α,25-dihydroxycholesterol synthesis that attracts pre-Tfh-cells, B-cells and DCs to follicle borders. We propose that RF basement membrane components represent a type of tissue memory that guides the localization and differentiation of both specialized FRC and DC populations, required for normal lymph node function.

Lymph Node Follicle-Targeting STING Agonist Nanoshells Enable Single-Shot M2e Vaccination for Broad and Durable Influenza Protection.

The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation-prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co-encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long-lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell-mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti-M2e humoral responses. STING agonist-triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1-biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single-shot nanovaccine further provides a translationally viable platform for pandemic preparedness.

Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimers as HIV-1 vaccine candidates

Uncleaved prefusion-optimized (UFO) design can stabilize diverse HIV-1 envelope glycoproteins (Envs). Single-component, self-assembling protein nanoparticles (1c-SApNP) can display 8 or 20 native-like Env trimers as vaccine candidates. We characterize the biophysical, structural, and antigenic properties of 1c-SApNPs that present the BG505 UFO trimer with wildtype and modified glycans. For 1c-SApNPs, glycan trimming improves recognition of the CD4 binding site without affecting broadly neutralizing antibodies (bNAbs) to major glycan epitopes. In mice, rabbits, and nonhuman primates, glycan trimming increases the frequency of vaccine responders (FVR) and steers antibody responses away from immunodominant glycan holes and glycan patches. The mechanism of vaccine-induced immunity is examined in mice. Compared with the UFO trimer, the multilayered E2p and I3-01v9 1c-SApNPs show 420 times longer retention in lymph node follicles, 20-32 times greater presentation on follicular dendritic cell dendrites, and up-to-4 times stronger germinal center reactions. These findings can inform future HIV-1 vaccine development.

Revisiting the Role of the CXCL13/CXCR5-Associated Immune Axis in Melanoma: Potential Implications for Anti-PD-1-Related Biomarker Research.

CXCL13 is a potent chemoattractant cytokine that promotes the migration of cells expressing its cognate receptor, CXCR5. Accordingly, T follicular helper cells and B cells migrate towards B cell follicles in lymph nodes, where the resulting spatial proximity promotes B cell/T cell interaction and antibody formation. Moreover, effector cells of the CXCL13/CXCR5-associated immune axis express PD-1, with corresponding circulating cells occurring in the blood. The formation of so-called ectopic or tertiary lymphoid structures, recently detected in different cancer types, represents an integral part of this axis, particularly in the context of its emerging role in anti-tumor defense. These aspects of the CXCL13/CXCR5-associated immune axis are highlighted in this review, which focuses on cutaneous malignant melanoma. Specifically, we elaborate on the role of this important immune axis as a possible ancillary target of immune checkpoint inhibition with anti-PD-1 antibodies in different therapeutic settings and as a potential source of predictive biomarkers regarding treatment efficacy.

Tingible body macrophages arise from lymph node-resident precursors and uptake B cells by dendrites.

Antibody affinity maturation depends on the formation of germinal centers (GCs) in lymph nodes. This process generates a massive number of apoptotic B cells, which are removed by a specialized subset of phagocytes, known as tingible body macrophages (TBMs). Although defects in these cells are associated with pathological conditions, the identity of their precursors and the dynamics of dying GC B cell disposal remained unknown. Here, we demonstrate that TBMs originate from pre-existing lymph node-resident precursors that enter the lymph node follicles in a GC-dependent manner. Intravital imaging shows that TBMs are stationary cells that selectively phagocytose GC B cells via highly dynamic protrusions and accommodate the final stages of B cell apoptosis. Cell-specific depletion and chimeric mouse models revealed that GC B cells drive TBM formation from bone marrow-derived precursors stationed within lymphoid organs prior to the immune challenge. Understanding TBM dynamics and function may explain the emergence of various antibody-mediated autoimmune conditions.

What can hide an enlarged lymph node of a patient with prostatic adenocarcinoma?

Introduction. Adenocarcinoma is the most common prostatic malignancy where clinical management, Gleason score and recent updates in prostate cancer staging play critical role. Mantle cell lymphoma originates from malignant transformation of B lymphocyte in the outer edge of lymph node follicle, with pathognomonic overexpression of cyclin D1. We present a rare case of two neoplasms occurred in the same individual person. Case report. In a 68-year-old patient planned for radical prostatectomy during the hospital preoperative examinations using multislice computed tomography tumor mass confined to the prostate but also excessive lymph node enlargement were revealed. Tissue specimens were analyzed after using hematoxylin and eosin staining, as well as immunohistochemical biomarker panel. Having performed a thorough histological examination, a diagnosis of prostatic adenocarcinoma, Gleason score 3+4=7, ISUP GG2, was given. Microscopic analysis of lymph node involvement showed unexpected, diffuse proliferation of small lymphoid cells, with irregular nuclei, wide mantle zone and hyalinized blood vessels. After using immunohistochemical staining for specific markers, another diagnosis was set, and it was non Hodgkin mantle cell lymphoma. Conclusion. A prostatic adenocarcinoma can extremely rarely be in a coexistence with undiagnosed lymphoproliferative disease, such as non Hodgkin mantle cell lymphoma in our case.

Spatial biology analysis reveals B cell follicles in secondary lymphoid structures may regulate anti-tumor responses at initial melanoma diagnosis.

IntroductionB cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients.MethodsFlow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs.ResultsB cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival.ConclusionThese data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Mannose-binding lectin and complement mediate follicular localization and enhanced immunogenicity of diverse protein nanoparticle immunogens.

SUMMARYNanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigate effects of MBL and complement on NP forms of HIV and other viral antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increases antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediate follicular trafficking and enhance GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determine that mannose patches at a minimal density of 2.1 × 10−3 mannose patches/nm2 are required to trigger follicular targeting, which increases with increasing glycan density up to at least ~8.2 × 10−3 patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants.

Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2GΔHR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with comparable potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2GΔHR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in the mouse model. Compared with the soluble spike, the I3-01v9 SApNP showed sixfold longer retention, fourfold greater presentation on follicular dendritic cell dendrites, and fivefold stronger germinal center reactions in lymph node follicles.

IL-21 enhances influenza vaccine responses in aged macaques with suppressed SIV infection.

Natural aging and HIV infection are associated with chronic low-grade systemic inflammation, immune senescence, and impaired antibody responses to vaccines such as the influenza (flu) vaccine. We investigated the role of IL-21, a CD4+ T follicular helper cell (Tfh) regulator, on flu vaccine antibody response in nonhuman primates (NHPs) in the context of age and controlled SIV mac239 infection. Three doses of the flu vaccine with or without IL-21–IgFc were administered at 3-month intervals in aged SIV+ NHPs following virus suppression with antiretroviral therapy. IL-21–treated animals demonstrated higher day 14–postboost antibody responses, which associated with expanded CD4+ T central memory cells and peripheral Tfh–expressing (pTfh–expressing) T cell immunoreceptor with Ig and ITIM domains (TIGIT), expanded activated memory B cells, and contracted CD11b+ monocytes. Draining lymph node (LN) tissue from IL-21–treated animals revealed direct association between LN follicle Tfh density and frequency of circulating TIGIT+ pTfh cells. We conclude that IL-21 enhances flu vaccine–induced antibody responses in SIV+ aged rhesus macaques (RMs), acting as an adjuvant modulating LN germinal center activity. A strategy to supplement IL-21 in aging could be a valuable addition in the toolbox for improving vaccine responses in an aging HIV+ population.

NK-B cell cross talk induces CXCR5 expression on natural killer cells

SummaryB cell follicles (BCFs) in lymph nodes (LNs) are generally exempt of CD8+ T and NK cells. African green monkeys (AGMs), a natural host of simian immunodeficiency virus (SIV), display NK cell-mediated viral control in BCF. NK cell migration into BCF in chronically SIVagm-infected AGM is associated with CXCR5+ NK cells. We aimed to identify the mechanism leading to CXCR5 expression on NK cells. We show that CXCR5+ NK cells in LN were induced following SIVagm infection. CXCR5+ NK cells accumulated preferentially in BCF with proliferating B cells. Autologous NK-B cell co-cultures in transwell chambers induced CXCR5+ NK cells. Transcriptome analysis of CXCR5+ NK cells revealed expression of bcl6 and IL6R. IL-6 induced CXCR5 on AGM and human NK cells. IL6 mRNA was detected in LN at higher levels during SIVagm than SIVmac infection and often produced by plasma cells. Our study reveals a mechanism of B cell-dependent NK cell regulation.

Mannose-Binding Lectin and Complement Mediate Follicular Localization and Enhanced Immunogenicity of Diverse Protein Nanoparticle Immunogens

Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigated effects of MBL and complement on NP forms of HIV and other clinically-relevant antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increased antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediated follicular trafficking and enhanced GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determined that mannose patches at a minimal density of 2.1x10-3 mannose patches/nm2 were required to trigger follicular targeting, which increased with increasing glycan density up to at least ~8.2×10-3 mannose patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-β. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Enhanced humoral immunity in breast cancer patients with high serum concentration of anti‐HER2 autoantibody

Humoral immunity plays a substantial role in the suppression of breast cancer. We have revealed that a high serum concentration of anti‐HER2 autoantibody (HER2‐AAb) is associated with favorable outcomes in patients with invasive breast cancer. Thus, we aimed to clarify the association between high serum concentration of HER2‐AAb and humoral immune response in the tumor microenvironment. Out of 500 consecutive patients with invasive breast cancer, we selected those whose HER2‐AAb values were high (n = 33) or low (n = 20) based on the distribution of HER2‐AAb values of 100 healthy individuals. Tumor and regional lymph node formalin‐fixed paraffin‐embedded samples prepared from the surgical specimens were subjected to immunohistochemistry. We confirmed that the recurrence‐free survival of the high HER2‐AAb group was significantly longer than that of the low HER2‐AAb group (p = 0.015). The numbers of tumor‐infiltrating CD20+ immune cells (ICs) (p < 0.001), IGKC+ICs (p = 0.023), and CXCL13+ ICs (p = 0.044) were significantly higher in the high HER2‐AAb group than in the low HER2‐AAb group. The number of CD4+ ICs in the B‐cell follicles of the regional lymph nodes was also significantly greater in the high HER2‐AAb group than in the low HER2‐AAb group (p = 0.026). Our findings indicate that a high level of HER2‐AAb is associated with enhanced humoral immunity against breast cancer and thus may provide a rationale for the association of HER2‐AAb with favorable prognosis.

Abstract 1604: Association of autoantibodies to HER2 with tumor microenvironments for humoral immunity and prognosis in patients with breast cancer

Abstract AIM: We have previously reported that the serum concentration of anti-HER2 autoantibodies (HER2-AAb) is associated with favorable outcomes in patients with invasive breast cancer (Tabuchi et al., Breast Cancer Res Treat 157:55-63, 2016). This study aimed to investigate the prognostic impact of HER2-AAb by examining the tumor microenvironments for humoral immunity. PATIENTS AND METHODS: From 500 consecutive patients with invasive breast cancer (Ibid.), we selected those whose log-transformed HER2-AAb values were above mean + 2SD (high HER2-AAb group, N = 33) or below mean − 2SD (low HER2-AAb group, N = 20). Tumor formalin-fixed paraffin-embedded (FFPE) samples and regional lymph node FFPE samples prepared from the patients’ surgical specimens were subjected to immunohistochemistry. Tumor-infiltrating immune cells (ICs) localized inside the tumor, in the adjacent stroma, and in the tumor stroma were separately counted. Expression of some proteins in tumor cells (TCs) was evaluated by the percentages of tumor cells showing presence of those proteins. RESULTS: We confirmed in the selected patients that the recurrence-free interval of the high HER2-AAb group was significantly longer than that of the low HER2-AAb group (log-rank P = 0.017; hazard ratio = 0.12). Tumor-infiltrating CD20-positive ICs (intratumoral + adjacent stromal ICs, P &amp;lt; 0.001), IGKC-positive ICs (intratumoral + adjacent stromal ICs, P = 0.023), and CXCL13-positive ICs (total ICs, P = 0.044) were significantly greater in the high HER2-AAb group than in the low HER2-AAb group. CD4-positive ICs in B-cell follicles of the regional lymph nodes were also significantly greater in the high HER2-AAb group than in the low HER2-AAb group (P = 0.026). Tumor-infiltrating PD-L1-positive ICs, CD8-positive ICs, FOXP3-positive ICs, as well as CD8/FOXP3 ratios, were not significantly different between the two groups. The expression of CXCL13, HLA-A/B/C, and PD-L1 in TCs was not significantly different between the two groups. The repertoire of B-cell receptors in ICs was not significantly different between the two groups but was considered to be skewed. HER2 expression and gene amplification and the existence of HER2 missense mutations were similar between the two groups. CONCLUSIONS: Increased HER2-AAb concentration was associated with enhanced humoral immunity in tumor microenvironments. Our findings indicate that a patient subpopulation with enhanced humoral immunity protecting against breast cancer recurrence exists. The recognition site of HER2-AAb may not be located in the HER2 polypeptide, but may be located in modifying factors, such as sugar chains. Citation Format: Masafumi Shimoda, Yasufumi Sato, Yoshiaki Sota, Tomohiro Miyake, Tomonori Tanei, Naofumi Kagara, Yasuto Naoi, Kenzo Shimazu, Seung Jin Kim, Shinzaburo Noguchi. Association of autoantibodies to HER2 with tumor microenvironments for humoral immunity and prognosis in patients with breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1604.