CD4 Helper Research Articles

Abstract 4689: Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer

Abstract Introduction: High levels of tumor infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in patients with HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC) . Recently, we demonstrated that 60% of BC TIL are organized in tertiary lymphoid structures (TLS) located in the stroma. We further identified a CXCL13-producing CD4+ T follicular helper cell (Tfh) subpopulation and demonstrated that this chemokine, important for TLS formation, is associated with positive clinical outcomes in BC. The aim of the present study was to investigate how conventional CD4+ Tfh cells, expressing the CXCL13 receptor CXCR5, contribute to immune function and regulation in BC-associated TLS. Methods: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to prepare tumor supernatant and TIL for flow cytometric analysis and sorting. Matching formalin-fixed paraffin-embedded (FFPE) were used for subsequent spatial analysis by dual IHC and confocal microscopy. Results: Flow cytometric analyses show that ~15% CD4+, ~13% CD8+ and >95% B cell TIL express CXCR5 while confocal microscopy reveals that these CXCR5+ TIL subpopulations co-localize in BC TLS and their presence is tightly correlated. BC Tfh TIL (compared to activated tonsillar Tfh) have a memory phenotype and express ICOS and PD-1 suggesting they are activated, but BCL6 is undetectable and CXCL13 positive cells are rare in only some tumors. RNA analysis detected high expression of IL-21, IL-10 and CXCL13 in Tfh TIL. Investigation into the role of Tfh cells in TLS functionality was accomplished via an in vitro assay where Tfh TIL were activated with allogeneic splenic B cells. These experiments reveal that only ICOS+PD-1+Tfh TIL from TN/HER2+ BC are capable of inducing IgM and IgG secretion by B cells. A comparison of ICOS+PD-1+ with ICOS-PD-1- Tfh TIL confirms an activated, functional nature for the former characterized by high levels of IL-21, IL-10 and CXCL13 mRNA expression. We also found specialized follicular regulatory T cells (Tfr), expressing CXCR5, CD25, demethylated Foxp3 and GARP, localized in TLS. To understand the regulation of Tfh function(s) by Tfr we quantified immunoglobulins in the primary tumor supernatant, finding a correlation between the Tfh/Tfr ratio (fresh tissues) and IgG and IgM production (supernatant) in TN/HER2+ BC. These data suggest that the balance between effector and regulatory Tfh influences B cell differentiation in TLS. Conclusions: We show that it is possible to isolate functional Tfh TIL from fresh BC tissues, demonstrate their activation and functional state and link a Tfr presence with negative regulation of TLS function. These data begin to shed light on anti-tumor immune responses occurring in TLS at the tumor site, whose functional activities may have important treatment implications, particularly for immunotherapy in BC. Citation Format: Mireille Langouo, Gregory Noël, Gert Van den Eynden, Alexandre De Wind, Soizic Garaud, Pushpamali De Silva, Cinzia Solinas, Anais Boisson, Celine Naveaux, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo. Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4689.

Abstract 3785: Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis

Abstract Vacuolar ATPase (V-ATPase) is expressed intracellularly on vesicular membranes, and controls intracellular pH and acidification of various intracellular vesicles. Most cancer cells express V-ATPase on their cell surface where they appear to acidify the extracellular environment and help cancer cells to metastasize. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells plays a key role during breast tumor progression and metastasis. However, the role of a2V of hematopoietic stem cells during breast tumor pathogenesis is not known. Therefore, to understand the role of a2V in hematopoietic stem cells during breast tumor progression, we conditionally knocked out a2V (cKO) from the hematopoietic stem cells of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the periphery and spleen of the a2V cKO mice compared to control mice. We hypothesized that reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster-growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that deletion of a2V led to larger and faster-growing breast tumors compared to control mice. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of tumor microenvironment demonstrated that out of 499 genes that were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and proapoptotic genes. Collectively, these results demonstrate that deletion of a2V from the hematopoietic stem cells leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster-growing and larger breast tumor. Further study is needed to understand the relative contribution of CD4+ or CD8+ T cells during breast tumor pathogenesis. Citation Format: Manoranjan Sahoo, Gajendra K. Katara, Mahamood Y. Bilal, Sara Fleetwood, Arpita Kulshrestha, Safaa A. Ibrahim, Kenneth D. Beaman. Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3785.

Immune Responses in Renal Transplant Recipients at Risk of BK-Virus Associated Nephropathy – Impact of Three Immunosuppressive Regimens

Introduction Early detection of patients at risk of BK virus-associated graft nephropathy (BKAN) would allow prevention by early adjusting immunosuppressive treatment. Materials and Methods We analyzed intracellular cytokine responses, CD4+ T helper function and T-cell dependent and independent B cell responses up to 24 months post-transplant in a prospective study of 105 renal transplant recipients, who were randomized to a CsA/MMF (n=35), Tacr/MMF (n=37) and Tacr/ERL (n=33) regimen, respectively. 18 drop-outs were excluded from analysis. BK virus (BKV) screening was performed by real time PCR with hybridization probes in serum and urine specimens. Results Risk of BK viremia was lowest in Tacr/ERL patients (4/30 (13%), CsA/MMF; 14/36 (39%), Tacr/MMF; 1/21 (5%), Tacr/ERL; p=0.005). BKAN was histologically proven in 6 patients (n=1, CsA/MMF; n=4, Tacr/MMF; n=1, Tacr/ERL). Tacr/ERL patients showed increased T-dependent B cell responses (p=0.004, 4 months; p=0.019, 1 year), increased CD4 cell IL4 (PMA; p=0.032, 1 year), CD14 IL6 (LPS; p=0.044, 4 months) and lowest CD4, CD19 and CD14 cell IL10 responses (CD4: PMA; p=0.009, 1year; CD19: PMA; p=0.002, 4 months; CD14: LPS; p=0.048, 2 years) "compared to CsA/MMF and Tacr/MMF patients". Intracellular CD4 and CD8 cell IL-2, IL-4 and IFN-γ production as well as B cell responses did not differ between patients with and without BK viremia. Pretransplant, increased IL-10R expression was detected in patients developing BK viremia (CD4+ T cells, p=0.005; CD8+ T cells, p=0.004; CD14+ monocytes, p=0.004). Post-transplant, we found an increased CD4 helper activity (p=0.008, 4 months), a decreased IL1β response of CD14+ monocytes (LPS; p=0.008, 4 months) and increased IL10 responses of CD4+ and CD8+ T cells (PMA; CD4: p=0.003, 1 year; p=0.038, 2 years; CD8: p=0.013, 1 year) in these patients. Conclusion Our data demonstrate a significantly lower risk of BK viremia on Tacr/ERL compared to CsA/MMF and Tacr/MMF treatment. The previously reported increased risk of donor-specific antibody (DSA) formation on everolimus treatment is supported by our finding of significantly increased T-dependent B cell responses but not the low CD4, CD8 and CD14 cell IL-10 production. Whereas pretransplant increased IL-10R expression may predict an increased risk of developing BK viremia, the increased post-transplant Th2 responses (CD4 helper activity and IL10 responses) found in these patients may increase the risk of DSA formation.

Abstract 3753: Indoximod modulates AhR-driven transcription of genes that control immune function

Abstract The IDO pathway mediates immunosuppressive effects by metabolizing tryptophan (Trp) into kynurenine (Kyn). The depletion of Trp stimulates downstream signaling through nutrient sensors GCN2 and mTOR, while the production of Kyn stimulates signaling through the aryl-hydrocarbon receptor (AHR) transcription factor. The activation of these signaling pathways has pleiotropic effects on immune cells, including influencing the differentiation of dendritic cells (DCs), helper T cells, and regulatory T cells (Treg) as well as enhancing the proliferation of effector T cells and Treg. Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creating an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. We hypothesized that indoximod's activity could also include the disruption of AHR activation by Kyn and other Trp catabolites, causing differential AHR signaling and transcriptional activity, resulting in an augmented antitumor immune response. We observed that indoximod activates AHR-dependent transcriptional activity in HepG2 cells as evidenced by increased AHR-driven luciferase and endogenous CYP1A1 activity. AHR activation by indoximod was also observed in primary human T cells, as measured by the induction of CYP1A1 mRNA. In a Kyn-driven Treg differentiation assay, indoximod altered transcription of genes associated with T helper and Treg phenotypes. Indoximod induced upregulation of Rorc expression, a TH17-associated transcription factor, while concurrently downregulating transcription of Foxp3, the master transcription factor of Treg cells. These effects were reverted by an AHR inhibitor. Consistent with the gene expression profiles, indoximod shifted the cellular phenotype from Foxp3+ Treg toward Th17-producing CD4 helper T cells. Transcription of Ido1 is controlled by IFNγ and AHR-response elements in its promoter. Consistent with the role of indoximod in blocking the IDO pathway, we observed that indoximod downregulated the expression and function of IDO in in vitro derived plasmacytoid DC that normally would express IDO under the same differentiation conditions. When these in vitro differentiated human pDCs were used in an MLR culture, the indoximod-induced downregulation of IDO resulted in decreased Kyn production and increased T cell proliferation. Moreover, indoximod treatment of tumor-bearing mice decreased expression of IDO in the pDC in tumor-draining LN. Together, these data suggest that indoximod modulates AHR signaling to exert multiple immunomodulatory effects, including a shift from suppressive Foxp3+ Treg toward TH17 helper T cells as well as the downregulation of IDO expression in pDC, contributing to enhanced antitumor immunity. Citation Format: Erik L. Brincks, James Adams, Michael Essmann, Benjamin A. Turner, Lifu Wang, Jiyuan Ke, Agnieszka Marcinowicz, Nicholas Vahanian, Charles J. Link, Mario R. Mautino. Indoximod modulates AhR-driven transcription of genes that control immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3753.

Abstract 3145: Characterization of novel immune checkpoint receptors within the breast cancer tumor microenvironment

Abstract Introduction. The interruption of immune checkpoint receptor (ICR)/ligand interaction within the tumor microenvironment has become an important immunotherapy strategy. In breast cancer (BC), neither the ICR phenotype of immune cells of each BC subtype nor the associated immune response is fully characterized. We hypothesize that there are differences in ICR phenotypes between BC subtypes. Methods. To analyze the expression of ICRs (i.e. PDCD1 (PD-1), TIGIT, HAVCR2 (Tim-3), and LAG3 in the tumor microenvironment (TME) of BC, the 956- patient BC cohort of The Cancer Genome Atlas (TCGA) gene expression data was analyzed and normalized against CD3E, a T-cell specific transcript. As validation, we examined various ICR by flow cytometry on 14 matched dissociated breast tumor and peripheral blood mononuclear cells (PBMC) and compared differences across BC subtypes with t-test and ANOVA. In addition, differential gene expression and multispectral analysis were investigated using FFPE specimens and the NanoString and Vectra platforms. Results. TCGA data revealed a higher expression of TIGIT, LAG3 and PD-1 in triple-negative breast cancer (TNBC) when compared to other subtypes (p <0.001, Table 1). PD-L1 (CD274) expression was higher in TNBC than ER/PR+/Her2- cancer (p=-0.008). On flow cytometry, there was a higher co-expression of TIGIT and Tim-3 on CD4 T cells in TNBC than all other subtypes (p=0.003) while ER/PR+Her2- cancers had the highest CD4 expression of TIGIT alone (p=0.04). Compared to ER/PR+Her2- cancers, TNBC had increased expression of transcripts associated with myeloid derived suppressor cells, and decreased expression of transcription factors that regulate lineage specification for CD4 helper T cells. Conclusions. In addition to PD-1, we demonstrated that other ICRs may play significant roles in TME of Her2+ and TNBC BC subtypes. Our results support the development of novel anti-ICR therapy which may synergize with current treatment strategies against these unfavorable BC subtypes. Citation Format: Austin D. Williams, Jean S. Campbell, Lauri D. Aicher, Kyle K. Payne, Jose R. Conejo-Garcia, Robert H. Pierce, Julia Tchou. Characterization of novel immune checkpoint receptors within the breast cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3145.

Follicular CD4 T Helper Cells As a Major HIV Reservoir Compartment: A Molecular Perspective.

Effective antiretroviral therapy (ART) has prevented the progression to AIDS and reduced HIV-related morbidities and mortality for the majority of infected individuals. However, a lifelong administration of ART is necessary, placing an inordinate burden on individuals and public health systems. Therefore, discovering therapeutic regimens able to eradicate or functionally cure HIV infection is of great importance. ART interruption leads to viral rebound highlighting the establishment and maintenance of a latent viral reservoir compartment even under long-term treatment. Follicular helper CD4 T cells (TFH) have been reported as a major cell compartment contributing to viral persistence, consequent to their susceptibility to infection and ability to release replication-competent new virions. Here, we discuss the molecular profiles and potential mechanisms that support the role of TFH cells as one of the major HIV reservoirs.

Cutting Edge: The Histone Methyltransferase G9a Is Required for Silencing of Helper T Lineage-Associated Genes in Proliferating CD8 T Cells.

Helper versus cytotoxic T lineage decision in the thymus has been studied as a model for silencing of alternative lineage genes. Although the transcription factor RUNX3 is required for the initiation of Cd4 silencing in developing CD8 T cells, it is unknown how silencing of Cd4 and other helper T lineage genes is maintained. We show that the histone methyltransferase G9a is necessary for silencing helper T lineage genes in proliferating mouse CD8 T cells. Despite normal initial Cd4 downregulation, G9a-deficient CD8 T cells derepress Cd4 and other helper lineage genes during repeated division in lymphopenia or in response to tumor Ag. However, G9a was dispensable for continued silencing of those genes in CD8 T cells that respond to infection by Listeria monocytogenes These results demonstrate that G9a facilitates maintenance of cellular identity of CD8 T cells during cell division, which is further reinforced by inflammatory signals.

Helper T-cell responses and pulmonary fungal infections.

The mucosal surface of the respiratory tract encounters microbes, such as fungal particles, with every inhaled breath. When pathogenic fungi breach the physical barrier and innate immune system within the lung to establish an infection, adaptive immunity is engaged, often in the form of helper CD4 T-cell responses. Type 1 responses, characterized by interferon-γ production from CD4 cells, promote clearance of Histoplasma capsulatum and Cryptococcus neoformans infection. Likewise, interleukin-17A (IL-17A) production from Th17 cells promotes immunity to Blastomyces dermatitidis and Coccidioides species infection by recruiting neutrophils. In contrast the development of T helper type 2 responses, characterized by IL-5 production from T cells and eosinophil influx into the lungs, drives allergic bronchopulmonary aspergillosis and poor outcomes during C. neoformans infection. Experimental vaccines against several endemic mycoses, including Histoplasma capsulatum, Coccidioides, Cryptococcus and Blastomyces dermatitidis, induce protective T-cell responses and foreshadow the development of vaccines against pulmonary fungal infections for use in humans. Additionally, recent work using antifungal T cells as immunotherapy to protect immune-compromised patients from opportunist fungal infections also shows great promise. This review covers the role of T-cell responses in driving protection and pathology in response to pulmonary fungal infections, and highlights promising therapeutic applications of antifungal T cells.

Tfh1 Cells in Germinal Centers During Chronic HIV/SIV Infection.

T follicular helper CD4 cells (Tfh) are essential for the development and maintenance of germinal center (GC) reactions, a critical process that promotes the generation of long-lived high affinity humoral immunity. It is becoming increasingly evident that GC-Tfh cells are heterogeneous in nature with some cellular characteristics associated with a Th1, Th2, and Th17 phenotype. Emerging studies suggest that GC-Tfh cells are directed to differentiate into distinct phenotypes during chronic HIV/SIV infection and these changes in GC-Tfh cells can greatly impact the B cell response and subclass of antibodies generated. Studies in HIV-infected humans have shown that certain Tfh phenotypes are associated with the generation of broadly neutralizing antibody responses. Moreover, the susceptibility of particular GC-Tfh subsets to HIV infection within the secondary lymphoid sites can also impact GC-Tfh/B cell interactions. In this review, we discuss the recent advances that show Tfh heterogeneity during chronic HIV/SIV infection. In particular, we will discuss the dynamics of GC-Tfh cells, their altered differentiation state and function, and their impact on B cell responses during HIV/SIV infection. In addition, we will also discuss the potential role of a recently described novel subset of follicular homing CXCR5+ CD8 T cells (Tfc) and their importance in contributing to control of chronic HIV/SIV infection. A better understanding of the mechanistic role of follicular homing CD4 and CD8 T cells during HIV/SIV infection will aid in the design of vaccines and therapeutic strategies to prevent and treat HIV/AIDS.

Vaccine Designs Utilizing Invariant NKT-Licensed Antigen-Presenting Cells Provide NKT or T Cell Help for B Cell Responses.

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4+ T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8+ T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.

A natural killer T-cell subset that protects against airway hyperreactivity

Infection of suckling mice with influenza virus expands a CD4-CD8- double-negative (DN) natural killer T(NKT) cell subpopulation that protects the mice as adults against allergen-induced airway hyperreactivity (AHR). However, this NKT cell subset has not been characterized, and the underlying mechanisms of protection remain unknown. We characterized this specific NKT cell subpopulation that developed during influenza infection in neonatal mice and that suppressed the subsequent development of AHR. A cell-surface marker was identified by comparing the mRNA expression profile of wild-type CD4+ NKT cells with that of suppressive Vα14 DN NKT cells. The marker-enriched NKT cell subset was then analyzed for its cytokine profile and its suppressive invitro and invivo abilities. We showed that DN NKT cells with high CD38 expression produced IFN-γ, but not IL-17, IL-4, or IL-13, and inhibited development of AHR through contact-dependent suppression of helper CD4 T-cell proliferation. The NKT subset expanded in the lungs of neonatal mice after infection with influenza and also after treatment of neonatal mice with Nu-α-GalCer, which effectively increased DN CD38hi NKT cell numbers. These results suggest that early/neonatal exposure to infection or antigen challenge affects subsequent lung immunity by altering the cellular composition of cells in the lung and that some subsets of NKT cells suppress AHR. These results provide a possible mechanism by which prior infections can protect against the development of allergic asthma and might be further explored as a protective measure for young children.

The Kinase Complex mTOR Complex 2 Promotes the Follicular Migration and Functional Maturation of Differentiated Follicular Helper CD4+ T Cells During Viral Infection.

Follicular helper CD4+ T (TFH) cells are critical for optimal B-cell-mediated humoral immunity by initiating, fueling, and sustaining germinal center reactions. The differentiation of TFH cells relies on multiple intrinsic and extrinsic factors; however, the details by which these factors are integrated to coordinate TFH differentiation are largely unknown. In this study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) viral infection, we demonstrate that mTOR complex 2 (mTORC2) kinase integrates TCR signaling and ICOS-mediated co-stimulation to promote late differentiation and functional maturation of virus-specific TFH cells. Specifically, mTORC2 functions to maintain TFH lineage specifications, including phenotypes, migratory characteristics, and functional properties. Thus, our results highlight the importance of mTORC2 in guarding TFH phenotypic and functional maturation.

Investigating the role of CX3CR1 in a GITRL- dependent signal 4 checkpoint during LCMV clone 13 infection

Abstract T cell-APC interactions early during chronic viral infection are critical for determining viral set point and disease outcome. Our recent work (Chang et al. Immunity. 2017) revealed a division of labour whereby monocyte-derived APCs (infAPCs), with low MHC II and CD80/86 expression compared to classical DCs (cDCs), preferentially up-regulate TNF receptor superfamily ligands, GITRL, 4-1BBL, OX40L and CD70, in response to type I interferon (signal 3) to provide a post-priming checkpoint (signal 4) for CD4 T cell responses. While cDCs contribute to signal 1 & 2 during priming in response to LCMV clone 13, GITRL on infAPCs binding to GITR on CD4 T cells results in increased surface expression of pro-survival receptors, including CD25, CD127 and OX40 to sustain Th1 helper cell accumulation, help for virus-specific CD8 T cell responses and viral control. Interestingly, CX3CR1, the receptor for fractalkine, was also upregulated on CD4 T cells by GITR signalling during clone 13 infection. CX3CR1 was recently shown to contribute to tumor-infiltration of CD4-helped CD8 CTLs (Ahrends et al. Immunity. 2017). In the chronic LCMV model, we observed that CX3CR1high virus-specific CD4 T cells in the spleen exhibit a markedly increased effector profile when compared to their CX3CR1int-low counterparts, with both higher frequency and per cell expression of Tbet and IFNg production. To investigate how CX3CR1 on CD4 T cells contributes to CD4 T cell help for the CD8 T cell response, we have crossed CX3CR1 deficient mice with TCR transgenic SMARTA mice, expressing an MHC II-restricted TCR specific for LCMV gp61–80. Overall, our data so far indicate that upregulation of CX3CR1 marks the most activated splenic CD4 Th1 effectors during chronic LCMV infection.

Absence of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis

Abstract Vacuolar ATPase (V-ATPase) is expressed on surface of most cancer cells where it acidifies the extracellular milieu and promotes metastasis. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells, plays a key role during breast tumor progression and metastasis. However, the role of a2V in hematopoietic stem cells (HSC) during breast tumor progression is not known. Therefore, to understand the role of a2V in HSC during breast tumor progression, we conditionally knocked out a2V (cKO) from the HSC of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the spleen of the a2V cKO mice compared to control mice. We hypothesized that the reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that lack of a2V leads to larger and faster growing breast tumors compared to controls. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of the tumor microenvironment demonstrated that out of 499 genes those were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and pro-apoptotic genes. Collectively, these results demonstrate that deletion of a2V from the HSC leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster growing and larger breast tumor.

Group 3 innate lymhoid cells are absent in DOCK8-defective HIES patients

Abstract Innate lymphoid cell (ILC) subsets ILC1, ILC2 and ILC3 mirror image the functions helper CD4+ T cell subsets, Th1, Th2 and Th17, respectively; rely on similar transcription factors for their development, and produce similar effector cytokines. Thus, they have recently been shown to be crucial for protective immunity in mice. More importantly, in several chronic inflammatory diseases activated or increased frequency of ILCs have been reported in the circulation or affected tissues of patients. Our previous work with Dock8-defective Dock8pri/pri mice revealed that Dock8 is required for the development/function and survival of murine ILC3s. Thus Dock8pri/pri mice lacked ILC3s and was susceptible to Citrobacter rodentium infections. However to date, whether DOCK8 regulates ILC3 development or functions in humans has not been addressed. In the current study, using 11 DOCK8 mutant patients from across Turkey, we show, for the first time, that humans with DOKC8 deficiency lack peripheral ILC3s. Reduction in blood ILC3 could be verified by flow cytometry. Additionally, sorted total blood ILC population obtained from HIES patients with DOCK8 mutations shows diminished ILC3-specific Gm-csf, AhR and il23r gene expression in line with ILC3 loss. The defect is dramatic in ILC3s and less so in ILC2s in circulation. Flow cytometric examination of 11 patients revealed full restroration of ILC3s following hematopoietic stem cell transplantation. We also show that sorted peripheral ILC precursor population obtained from DOCK8 mutant patients has impaired capacity to proliferate in ILC1-ILC2-ILC3 polarizing conditions. This is the first report in the literature to show a selective deletion of ILC3s in HIES patients with DOCK8 mutations.

Programmed death-1 restrains the germinal center reaction in type 1 diabetes

Abstract Programmed death-1 (PD-1) is a T cell inhibitory receptor important for tolerance maintenance. PD-1 deficiency or blockade accelerates autoimmune diabetes in non-obese diabetic (NOD) mice, but the mechanism remains unclear. Autoantibody production is a hallmark of autoimmunity, and has also been reported in patients treated with PD-1 blockade, suggesting that PD-1 might regulate this process. Autoantibody production results from B cell:CD4 T cell interactions in the germinal center of the lymph node. The dynamics and regulation of the germinal center response in spontaneous autoimmunity and in the face of PD-1 deprivation are not well understood, primarily due to an inability to track self-specific lymphocytes. To bridge this knowledge gap, we used tetramers to phenotype islet-specific CD4 T cells and B cells in mice. PD-1-deficient mice, and NOD mice treated with anti-PD-1, had increased insulin autoantibodies, as well as increased insulin-specific germinal center T follicular helper CD4 T cells and germinal center B cells compared to controls. This work provides a mechanistic explanation for autoantibody onset following PD-1 blockade in the clinic, and has important implications for cancer immunotherapy and autoimmunity.

Antigen targeting of Fc-receptors induces strong T cell responses in vivo independent of ITAM signaling but dependent on dendritic cell subsets

Abstract Dendritic cells (DCs) are important antigen presenting cells (APCs) and responsible for the induction of immune responses and preserving peripheral tolerance. We showed that targeting of antigens via C-type lectin receptors to different specialized DC subpopulations induced either CD4+ or CD8+ T cell responses in vivo. Fc receptors are also highly active in endocytosis enabling APCs to take up antigens in form of immune complexes. As they are expressed on various APCs, we aimed to identify responsible APCs for primary and secondary immune responses. Therefore, we assessed their expression in various organs and delivered antigens by specific recombinant antibodies. The targeting of the Fc receptors induced CD4+ and CD8+ T cell responses in a transgenic as well as a naïve system. Moreover, especially antigen delivery to the activating FcγRIV was superior in inducing CD4+ and CD8+ T cell responses at the same time, which could not be observed by classical targeting of the C-type lectin receptors DEC205 or DCIR2. Additionally, targeting of antigens to FcγRIV induced a pronounced CD4 helper response in naïve mice, whereas targeting to DCIR2 was inefficient. As FcγRIV is expressed on both major splenic DC subsets, we used this receptor to verify the subset intrinsic preference of DCs for CD4+ or CD8+ T cell responses. This was clearly shown by the induction of CD4+ T cell responses by splenic CD8− DCs, whereas splenic CD8+ DCs induced a CD8+ T cell response. The induced naïve CD8+ T cell responses were functional relevant, as we could demonstrate efficient dose-dependent killing of target cells in vivo. Therefore, we suggest this strategy as useful tool for the induction of de novo as well as the modulation of immune responses for therapeutic applications.

Immunophenotypic characterization of CSF B cells in virus-associated neuroinflammatory diseases.

Intrathecal antibody synthesis is a well-documented phenomenon in infectious neurological diseases as well as in demyelinating diseases, but little is known about the role of B cells in the central nervous systems. We examined B cell and T cell immunophenotypes in CSF of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) compared to healthy normal donors and subjects with the other chronic virus infection and/or neuroinflammatory diseases including HIV infection, multiple sclerosis (MS) and progressive multifocal leukoencephalopathy. Antibody secreting B cells (ASCs) were elevated in HAM/TSP patients, which was significantly correlated with intrathecal HTLV-1-specific antibody responses. High frequency of ASCs was also detected in patients with relapsing-remitting multiple sclerosis (RRMS). While RRMS patients showed significant correlations between ASCs and memory follicular helper CD4+ T cells, CD4+CD25+ T cells were elevated in HAM/TSP patients, which were significantly correlated with ASCs and HTLV-1 proviral load. These results highlight the importance of the B cell compartment and the associated inflammatory milieu in HAM/TSP patients where virus-specific antibody production may be required to control viral persistence and/or may be associated with disease development.

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4+ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca2+ signals, mainly mediated by the store operated Ca2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4+ T cell subsets show differential Ca2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4+ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca2+ release activated Ca2+ currents (ICRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca2+ profiles of helper CD4+ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4+ T cells.

A-103 Combined immune-based strategies targeting viral persistence in ART-suppressed SIV-infected rhesus macaques

Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals, but does not eliminate an extremely durable reservoir of latently infected cells that is established early after infection. Understanding the phenotype and location of latently infected cells represents a critical challenge in designing a cure for HIV; furthermore, many HIV-infected individuals given ART exhibit residual inflammation, which may contribute to virus persistence. This presentation will discuss immune based strategies targeting HIV persistence developed over the past several years using the model of SIV infection in rhesus macaques. Recent work identified PD-1+ follicular helper CD4+ T-cells as an important cellular compartment for viral persistence. We have described that CTLA-4+PD-1-memory CD4+ T-cells, which share phenotypic markers with regulatory T-cells and localize outside the B-cell follicle of the lymph nodes, are significantly enriched in SIV-DNA; contain robust levels of replication-competent virus; and increase their contribution to the SIV reservoir with prolonged ART. Finally, we showed that Interleukin-21 and IFN-alpha administration in ART-treated, SIV-infected RMs reduces residual immune activation during ART and temporally delay viral rebound after ART treatment interruption. These recent advancements highlight the complexity and diversity of the mechanisms and T-cell populations that can contribute to the residual reservoirs of virally infected cells. Developing a range of different interventions to target individual components of viral reservoirs represent both a formidable challenge and an exciting opportunity for the years to come.