Cells In Secondary Lymphoid Organs Research Articles

Abstract 5609: Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that shows striking resistance to standard therapies. Immunotherapy is an exciting new treatment strategy for PDAC, but has yet to reproducibly demonstrate clinical efficacy. This finding may reflect poor effector T cell infiltration into PDAC or mechanisms of peripheral T cell tolerance. We hypothesize that successful immunotherapy in PDAC will require strategies that reverse elements of innate immunity that regulate T cell tolerance. This hypothesis is based on our prior studies in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model of PDAC where macrophages residing outside of the tumor microenvironment were found to regulate the efficacy of a vaccine strategy involving the combination of chemotherapy and a CD40 agonist (FGK45). Specifically, we found that depletion of peripheral phagocytes using clodronate-encapsulated liposomes (CEL) was necessary for gemcitabine chemotherapy and a CD40 agonist to induce productive anti-tumor T cell immunity against spontaneously arising PDAC tumors in the KPC model. We now investigate the mechanism by which CEL-depleted phagocytes regulate T cell tolerance in PDAC using the KPC mouse model and an orthotopic implantation model. We found that CEL depletes macrophages in the spleen and abdominal lymph nodes, thereby reversing the pathologic accumulation of macrophages in these secondary lymphoid organs that is induced by PDAC tumors. CEL targeted two macrophage subsets in the lymph node, CD11b+CD169+F4/80neg subcapsular sinus macrophages (SSM) and CD11b+CD169+F4/80+ medullary sinus macrophages (MSM). These macrophage populations may be exposed to tumor-derived antigens and apoptotic cell vesicles entering the lymph node, and have been previously identified as central orchestrators of tolerance in autoimmune models. We found that both fluorescently-labeled liposomes and OVA-FITC peptide injected into the peritoneal cavity were specifically targeted to SSM and MSM with little uptake by dendritic cells (DC). In contrast, depletion of these first-line phagocytes by CEL shifted the cellular uptake of these model antigens increasing uptake by CD11b+CD11c+ DC which are more efficient in antigen presentation and show increased expression of co-stimulatory molecules after FGK45 treatment. Using an orthotopic model of PDAC, we then determined that CEL can enhance the T cell stimulatory capacity of gemcitabine chemotherapy in combination with FGK45 as seen by a significant increase in CD38, CD44, CD69 and PD-1 expression on CD4 and CD8 T cells in secondary lymphoid organs. Together, our findings suggest that tumor-derived antigens may be sequestered by tolerogenic macrophages in peritumoral lymph nodes that act to impair optimal induction of T cell responses. Thus, strategies aimed at disrupting the tolerogenic functions of these macrophages may offer a novel approach for improving immunotherapy in PDAC. Citation Format: Fee Bengsch, Anni Liu, Kathleen Graham, Gregory L. Beatty. Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5609. doi:10.1158/1538-7445.AM2017-5609

Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets.

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4+ T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4+ T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3+ CCR5+ CD4+ T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3+ T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14+ CD16+ monocytes and MAC387+ macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387+ macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages.IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4+ T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3+ CCR5+ CD4+ T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3+ cells, in CD14+ CD16+ monocytes and MAC387+ macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes.

An Antigen-Presenting and Apoptosis-Inducing Polymer Microparticle Prolongs Alloskin Graft Survival by Selectively and Markedly Depleting Alloreactive CD8+ T Cells.

Selectively depleting the pathogenic T cells is a fundamental strategy for the treatment of allograft rejection and autoimmune disease since it retains the overall immune function of host. The concept of killer artificial antigen-presenting cells (KaAPCs) has been developed by co-coupling peptide–major histocompatibility complex (pMHC) multimer and anti-Fas monoclonal antibody (mAb) onto the polymeric microparticles (MPs) to induce the apoptosis of antigen-specific T cells. But little information is available about its in vivo therapeutic potential and mechanism. In this study, polyethylenimine (PEI)-coated poly lactic-co-glycolic acid microparticle (PLGA MP) was fabricated as a cell-sized scaffold to covalently co-couple H-2Kb-Ig dimer and anti-Fas mAb for the generation of alloantigen-presenting and apoptosis-inducing MPs. Intravenous infusions of the biodegradable KaAPCs prolonged the alloskin graft survival for 43 days in a single MHC-mismatched murine model, depleted the most of H-2Kb-alloreactive CD8+ T cells in peripheral blood, spleen, and alloskin graft in an antigen-specific manner and anti-Fas-dependent fashion. The cell-sized KaAPCs circulated throughout vasculature into liver, kidney, spleen, lymph nodes, lung, and heart, but few ones into local allograft at early stage, with a retention time up to 36 h in vivo. They colocalized with CD8+ T cells in secondary lymphoid organs while few ones contacted with CD4+ T cells, B cells, macrophage, and dendritic cells, or internalized by phagocytes. Importantly, the KaAPC treatment did not significantly impair the native T cell repertoire or non-pathogenic immune cells, did not obviously suppress the overall immune function of host, and did not lead to visible organ toxicity. Our results strongly document the high potential of PLGA MP-based KaAPCs as a novel antigen-specific immunotherapy for allograft rejection and autoimmune disorder. The in vivo mechanism of alloinhibition, tissue distribution, and biosafety were also initially characterized, which will facilitate its translational studies from bench to bedside.

Regulatory T cells exhibit distinct lymph node homing and trafficking kinetics as compared to conventional CD4+ T cells

Abstract Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. We have previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal significant differences in Treg versus Tconv homing and transit through peripheral and mesenteric LNs. We find that Tregs with an effector phenotype are preferentially retained in LNs, and complementary transwell assays demonstrate that Tregs have blunted response to CCL21 compared to Tconvs. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs, taking advantage of a novel gut SLO stabilization device allowing up to 10 hours of continuous imaging. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in LNs.

Abstract 561: GITR Activation Promotes Regulatory CD4+ T-cell Responses and Stimulates Leukocyte Recruitment in Atherosclerotic Plaques

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) - a costimulatory molecule - is expressed on CD4(+) effector memory T cells and regulatory T cells as well as antigen-presenting cells and mast cells; while its ligand (GITRL) is mainly found on antigen-presenting cells and endothelial cells. However, the definitive role of GITR in atherosclerosis is not fully understood. Our hypothesis is that signaling through GITR plays a vital role in atherosclerosis progression. Low-density lipoprotein receptor-deficient mice (Ldlr -/- ) with B-cell-restricted overexpression of GITRL ( Gitrl tg ) fed a high-cholesterol diet showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs in comparison to wild-type controls. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased GITRL and interleukin-2 transcript levels. Atherosclerotic lesions of Ldlr -/- Gitrl tg mice contained more total CD3 + T cells as well as Foxp3 + regulatory T cells overall, leading to significantly less severe atherosclerosis. Conversely, atherosclerosis was found to be less severe in mice deficient in apolipoprotein E and GITR (ApoE -/- GITR -/- ). Atherosclerotic lesions in these mice were found to contain less macrophages and CD3-positive T-cells. Perfusion assays using two-photon excitation microscopy revealed less wild type leukocyte adhesion on GITR-deficient endothelium, with a further reduction in adhesion by GITR-deficient leukocytes to both wild type and GITR-deficient endothelia. Finally, expression of GITR expression in human plaque tissue was significantly increased in ruptured plaques. In conclusion, these data indicate that continuous GITR stimulation through B cell GITRL acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells, while GITR activation on endothelial cells promotes atherogenesis by stimulating leukocyte recruitment into the plaque.

Graft-Versus-Host Disease Following Liver Transplantation: Development of a High-Incidence Rat Model and a Selective Prevention Method.

Graft-versus-host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT-GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti-CD8α mAb treatment, which invariably resulted in acute LT-GvHD. Compared with those in the peripheral counterpart, graft CD4+ CD25- passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4+ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti-TCRαβ mAb. T cell-depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT-GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT-GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high-risk donor-recipient combinations and for analyzing immunoregulatory mechanisms of the liver.

Abstract A069: Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells

Abstract Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. Our laboratory has previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal surprising differences in Treg versus Tconv trafficking in mesenteric versus other peripheral LNs. We find that Treg LN transit time is heavily dependent on recognition of self-pMHC in peripheral but not mesenteric LN. Furthermore, we describe a novel imaging platform which enables high resolution intravital imaging of gut SLOs, especially mesenteric LN, for up to 10 hours. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in peripheral LNs. Citation Format: Alexander Tong. Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A069.

Abstract B129: Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion

Abstract Immune checkpoint blockade has revolutionized cancer immunotherapy, and combination treatment with antibodies against multiple inhibitory receptors promises to improve efficacy substantially. However, the spatiotemporal and combinatorial expression profile of various checkpoint receptors on effector T cells and the effect of combined blockade on immune function remain poorly defined. In this study we set out to determine the co-expression kinetics and functional impact of multiple checkpoint receptors (PD-1, LAG-3, TIM-3, and BTLA) on tumor antigen-specific effector T cells in controlled in vitro culture systems and in the tumor environment. To elicit tumor antigen-specific responses we immunized mice with irradiated MC38 tumor cells engineered to express chicken ovalbumin (OVA; MC38.OVA) and used pentamer reagents to identify OVA257-264-specific CD8 T cells. Repeated stimulation with irradiated MC38.OVA cells or irradiated OVA-pulsed splenocytes ex vivo resulted in robust proliferation and activation of antigen-specific T cells as determined by BrdU incorporation and production of IFN-γ. Most OVA-specific cells expressed PD-1 ex vivo and maintained PD-1 expression during repeated cycles of in vitro stimulation. In contrast, antigen-specific T cells did not express TIM-3 or LAG-3 ex vivo, yet both receptors were induced on the majority of PD1 positive, but not PD-1 negative, cells upon repeated stimulation ex vivo, resulting in double and triple positive cells. The frequency of BTLA expressing cells remained low under all conditions. The addition of PD-1 and TIM-3 blocking antibodies during ex vivo restimulation enhanced T cell activation, demonstrating that these receptors mediate inhibitory function. Consistent with our in vitro data demonstrating that repeated antigen stimulation promotes the coordinated expression of checkpoint receptors, we found that the vast majority of MC38.OVA or Colon26 tumor infiltrating T cells (TILs) express PD-1, and most PD-1 positive cells also co-express TIM-3 and LAG-3, whereas these receptors are largely absent from T cells in secondary lymphoid organs. Importantly, combination treatment of tumor-bearing mice with anti-PD-1 and anti-TIM-3, or anti-PD-1 and anti-LAG-3 antibodies enhanced the immune control of tumor growth in both models as compared to the respective monotherapies. Finally, in vitro activation of human PBMC-derived T cells resulted in a similar sequential upregulation of checkpoint inhibitors ultimately resulting in their co-expression. Here, we have established a culture system to delineate the hierarchical expression of inhibitor checkpoint receptors on tumor antigen-specific murine T cells during repeated antigen stimulation. Defining the inhibitory checkpoint receptor landscape will guide the process of identifying the most promising combinations of checkpoint blockers in cancer immunotherapy. Citation Format: Robert J. Durso, Jerry Pei, Michelle Russell, Pratha Budhani, Elena Burova, Chandrika Taduriyasas, Ella Ioffe, Markus Mohrs, Gavin Thurston, Jie Dai. Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B129.

CollagenVI-Cre mice: A new tool to target stromal cells in secondary lymphoid organs.

Stromal cells in secondary lymphoid organs (SLOs) are non-hematopoietic cells involved in the regulation of adaptive immune responses. Three major stromal populations have been identified in adult SLOs: fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs) and marginal reticular cells (MRCs). The properties of these individual populations are not clearly defined, mainly due to the lack of appropriate genetic tools, especially for MRCs. Here, we analyzed stromal cell targeting in SLOs from a transgenic mouse strain that expresses Cre recombinase under the CollagenVI promoter, using lineage tracing approaches. We show that these mice target specifically MRCs and FDCs, but not FRCs in Peyer’s patches and isolated lymphoid follicles in the intestine. In contrast, stromal cells in lymph nodes and the spleen do not express the transgene, which renders ColVI-cre mice ideal for the specific targeting of stromal cells in the gut-associated lymphoid tissue (GALT). This funding further supports the hypothesis of organ-specific stromal precursors in SLOs. Interestingly, in all tissues analyzed, there was also high specificity for perivascular cells, which have been proposed to act as FDC precursors. Taken together, ColVI-Cre mice are a useful new tool for the dissection of MRC- and FDC-specific functions and plasticity in the GALT.

Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4 + T-Cell Responses—Brief Report

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4(+) effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. Low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) were lethally irradiated and reconstituted with either bone marrow from B-cell-restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr(-/-) Gitrl(tg) mice showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr(-/-) Gitrl(tg) chimeras contained more total CD3(+) T cells as well as Foxp3(+) regulatory T cells overall, leading to significantly less severe atherosclerosis. These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells.

Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells

Abstract Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. Our laboratory has previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal surprising differences in Treg versus Tconv trafficking in mesenteric versus other peripheral LNs. Furthermore, we find that Treg LN transit time is heavily dependent on recognition of self-pMHC. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in peripheral LNs.

Innate lymphoid cells in secondary lymphoid organs.

The family of innate lymphoid cells (ILCs) has attracted attention in recent years as its members are important regulators of immunity, while they can also cause pathology. In both mouse and man, ILCs were initially discovered in developing lymph nodes as lymphoid tissue inducer (LTi) cells. These cells form the prototypic members of the ILC family and play a central role in the formation of secondary lymphoid organs (SLOs). In the absence of LTi cells, lymph nodes (LN) and Peyer's Patches (PP) fail to form in mice, although the splenic white pulp can develop normally. Besides LTi cells, the ILC family encompasses helper-like ILCs with functional distinctions as seen by T-helper cells, as well as cytotoxic natural killer (NK) cells. ILCs are still present in adult SLOs where they have been shown to play a role in lymphoid tissue regeneration. Furthermore, ILCs were implicated to interact with adaptive lymphocytes and influence the adaptive immune response. Here, we review the recent literature on the role of ILCs in secondary lymphoid tissue from the formation of SLOs to mature SLOs in adults, during homeostasis and pathology.

Intermediate Filament (IF) Protein Vimentin Regulates T Cell Mediated Immune Response in Gvhd

Graft-versus-host-disease (GVHD) is an alloimmune response complicating allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T lymphocytes and recipient antigen presenting cells (APCs) are the primarily mediators of GVHD, the molecular and cellular basis are not well understood. Recent published studies investigated T cell migration and homing in GVHD, especially effects of adhesion molecules including chemokines and integrins. Intermediate filaments (IFs) are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support. As the most abundant IF protein and the only known IF protein in leukocytes, vimentin plays an important role in stabilizing intracellular architecture, maintaining cellular integrity and providing resistance against stress. Vimentin-deficient (Vim-/-) mice are viable with impaired wound healing, and defects in PMN and lymphocyte adhesion to endothelial cells. In addition, Vimentin has been reported to be involved in colitis, Crohn's disease and allograft rejection. Herein, we report that vimentin regulates the organization of proteins in cell adhesion, migration and signaling, all of which are important for T cell activation in GVHD.Using homotypic aggregation assay, we found there was significantly reduced aggregation of primary T cells isolated from Vim-/- mice compared to WT control. However, the expression of both LFA-1 and ICAM-1 are similar between WT and Vim-/- T cells, thus the reduced T cell adhesion is not attributed to LFA-1 expression. We further investigated whether vimentin regulates mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and demonstrated there was a significant reduction of both CD4+ and CD8+ T cell proliferation in the absence of vimentin (Vim-/-) compared to WT control. Moreover, the frequency of IFN-γ (Th1) and IL-17 (Th17) producing CD4+ cells was significantly reduced in T cells isolated from Vim-/- mice compared to WT control. To investigate the role of vimentin in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells, and found vimentin was enriched proximal to the membrane and associated with the prominent CD3/LFA-1 cluster upon TCR stimulation. In the control without any TCR stimulation, the predominant pattern was different with vimentin evenly distributed beneath the cell membrane. In the absence of vimentin (Vim-/-), immune synapse failed to form in mouse CD8+ T cells upon TCR stimulation. The data demonstrate that vimentin can regulate immunological synapse function and signal transduction in T cell activation and proliferation.To determine whether vimentin expression in T cells plays a role in GVHD, the MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d) transplantation, was used to establish GVHD. T cells from WT or Vim-/- (C57BL/6 background) mice were used as donors and Balb/c mice as recipients. Irradiated BALB/c mice received 5x106 T cell-depleted bone marrow cells (WT) and 10×106 T cells (WT or Vim-/-). In comparison to WT control, mice received Vim-/- T cells showed a lower mortality rate. Within 8 weeks post-transplantation, about 65% of mice received Vim-/- T cells survived, compared with only less than 10% of control mice received WT T cells (P=0.036; n=15 in each group). Control recipients had severe GVHD in the skin, intestine and liver. Mice received Vim-/- T cells exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores. There were significantly reduced donor-derived CD4+ and CD8+ T cells in secondary lymphoid organs. Thus, the reduced homing and proliferation of Vim-/- T cells in vivo led to the reduced mortality and morbidity associated with GVHD.In summary, we have shown that there are significantly reduced T cell adhesion, proliferation and Th1/17 polarization in Vim-/- T cells, and vimentin participates in TCR clustering and immunological synapse formation in CD8+ T cells. Furthermore, vimentin plays an important role in GVHD through regulating donor T cell adhesion, proliferation and activation. Our data will not only significantly advance our knowledge of GVHD, but also define a new function of vimentin and IF proteins in health and diseases, and thus provide a rationale for using vimentin inhibitors as potential novel therapeutic interventions for GVHD. DisclosuresNo relevant conflicts of interest to declare.

Modulation of CNS autoimmune responses by CD8+ T cells coincides with their oligoclonal expansion

MS is a highly prevalent neuroinflammatory disease of presumed autoimmune origin. Clinical observations and animal studies suggest that CD8+ T cells play an important role in MS but their exact mechanisms are ill defined. When we actively induced EAE in CD8 knock-out DA rats, or adoptively transferred encephalitogenic CD4+ T cells into CD8 knock-out DA rats, the disease course was indistinguishable from controls. Since our previous findings had revealed that the absence of CD8+ T cells in Lewis rats ameliorated EAE, we compared antigen-induced T cell differentiation in both strains. Disease onset and the composition of the draining lymph nodes were similar but T cell activation in DA rats was much weaker. Moreover, oligoclonal expansion of CD8+ T cells was exclusively observed in Lewis but not in DA rats. This suggests that myelin-specific CD8+ T cells are involved in the differentiation of encephalitogenic CD4+ T cells in Lewis rats, whilst they do not impact CD4+ T cell priming in DA rats. Hence, clonal expansion of CD8+ T cells in secondary lymphoid organs appears to be linked to their ability to modulate CNS autoimmune responses.

Suppression of Autoimmunity and Renal Disease in Pristane-Induced Lupus by Myeloperoxidase.

Myeloperoxidase (MPO) locally contributes to organ damage in various chronic inflammatory conditions by generating reactive intermediates. The contribution of MPO in the development of experimental lupus is unknown. The aim of this study was to define the role of MPO in murine lupus nephritis (LN). LN was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO(-/-) ) mice by intraperitoneal injection of pristane. Autoimmunity and glomerulonephritis were assessed 20 and 40 weeks after pristane administration. Cell apoptosis, leukocyte accumulation, and cytokine levels in the peritoneal cavity of WT and MPO(-/-) mice were assessed 3 or 6 days after pristane injection. MPO(-/-) mice developed more severe nephritis than did WT mice 20 and 40 weeks after pristane injection, despite having reduced glomerular deposition of antibody and complement and diminished levels of markers of oxidative stress (oxidized DNA and glutathione sulfonamide). Enhancement of renal disease in MPO-deficient mice correlated with increased accumulation of CD4+ T cells and macrophages in glomeruli, which, in turn, was associated with augmented generation of CD4+ T cell responses and increased activation and migration of dendritic cells in secondary lymphoid organs. In addition, the enhanced renal injury in MPO(-/-) mice was associated with increased glomerular accumulation of neutrophils and deposition of neutrophil extracellular traps. MPO deficiency also increased early cell apoptosis, leukocyte accumulation, and proinflammatory cytokine expression in the peritoneum. MPO attenuates pristane-induced LN by inhibiting early inflammatory responses in the peritoneum and limiting the generation of CD4+ T cell autoimmunity in secondary lymphoid organs.

Homeostatic regulatory T cell trafficking in peripheral lymph nodes (LYM2P.731)

Abstract Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. As widely studied as CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed. Our laboratory has previously described the dynamics of naïve T cell trafficking through SLOs and scanning of dendritic cells, revealing that CD4+ T cells and CD8+ T cells have distinct dynamic behaviors in the lymph node (LN). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. In this study, we use flow cytometry and 2-photon laser scanning microscopy to quantitate the trafficking kinetics and surveillance strategy of naïve Tregs in C57BL/6 mice LNs and to compare their behavior to conventional T cells. Our data reveal differences in naïve Treg trafficking in mesenteric versus other peripheral LNs. We also show that self-pMHC recognition regulates the scanning behavior and localization of Tregs in peripheral LNs. In addition, we describe a novel imaging platform for high-resolution intravital microscopy of T cell behavior within mesenteric LNs. These results add to mounting evidence that self-pMHC interactions dynamically control physiologic Treg retainment and contact-dependent suppressive function in peripheral LNs.

Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model

Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.

Complement receptor type 1 (CR1/CD35) expressed on activated human CD4+ T cells contributes to generation of regulatory T cells

The role of complement in the regulation of T cell immunity has been highlighted recently by several groups. We were prompted to reinvestigate the role of complement receptor type 1 (CR1, CD35) in human T cells based on our earlier data showing that activated human T cells produce C3 (Torok et al. (2012) [48]) and also by results demonstrating that engagement of Membrane Cofactor Protein (MCP, CD46) induces a switch of anti-CD3-activated helper T cells into regulatory T cells (Kemper et al. (2003) [17]). We demonstrate here that co-ligation of CD46 and CD35, the two C3b-binding structures present on activated CD4+ human T cells significantly enhances CD25 expression, elevates granzyme B production and synergistically augments cell proliferation. The role of CR1 in the development of the Treg phenotype was further confirmed by demonstrating that its engagement enhances IL-10 production and reduces IFNγ release by the activated CD4+ T cells in the presence of excess IL-2. The functional in vivo relevance of our findings was highlighted by the immunohistochemical staining of tonsils, revealing the presence of CD4/CD35 double positive lymphocytes mainly in the inter-follicular regions where direct contact between CD4+ T cells and B lymphocytes occurs.Regarding the in vivo relevance of the complement-dependent generation of regulatory T cells in secondary lymphoid organs we propose a scenario shown in the figure. The depicted process involves the sequential binding of locally produced C3 fragments to CD46 and CD35 expressed on activated T cells, which – in the presence of excess IL-2 – leads to the development of Treg cells.

CCR7 expressing mesenchymal stem cells potently inhibit graft-versus-host disease by spoiling the fourth supplemental Billingham's tenet.

The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham’s tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.

Delta-like Ligands Expressed By Stromal Cells in Secondary Lymphoid Organs Deliver an Early Pulse of Notch Signaling and Drive T Cell Pathogenicity in Acute Graft-Versus-Host Disease

Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands results in near-complete protection from acute GVHD in mouse models of allogeneic bone marrow transplantation. Notch-deprived alloreactive T cells proliferate and accumulate in vivo, but produce dramatically reduced levels of the proinflammatory cytokines IFNγ, TNFα and interleukin-2 (IL-2) (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., J Clin Invest 2013). In this study, we sought to: 1) determine the kinetic requirements for Notch signaling in the pathogenesis of acute GVHD; 2) identify the essential cellular compartment that delivers Dll1 and/or Dll4 ligands to incoming alloreactive T cells. In the B6 anti-BALB/c major histocompatibility complex-mismatched model, a single dose of Dll1 and Dll4 blocking antibodies at the time of transplantation abolished alloreactive T cell production of IFNγ, TNFα, and IL-2, increased regulatory T cell numbers (as assessed at day 10), and conferred long-term protection from GVHD. Conversely, delaying antibody administration by only two days after transplantation resulted in persistent T cell cytokine production, no changes in regulatory T cell numbers, and loss of long-term protection from GVHD. These findings identify a critical early window of Notch activity that promotes the pathogenesis of acute GVHD. To identify the dominant cellular source of Dll1 and Dll4, we assessed the impact of Cre-mediated Dll1 and Dll4 inactivation within host hematopoietic, donor hematopoietic, or host non-hematopoietic tissues. Bone marrow chimeras that lacked Dll1 and Dll4 solely within the host hematopoietic system were generated from poly(I:C)-induced Mx1-Cre;Dll1fl/fl;Dll4fl/fl donor mice. Both donor chimerism and Cre-mediated excision efficiency were >97%. Unlike systemic Dll1/4 blockade, Dll1 and Dll4 inactivation within the host hematopoietic system failed to decrease GVHD mortality or severity. Likewise, Mx1-Cre-mediated deletion of Dll1 and Dll4 within the donor hematopoietic system had minimal effects on T cell proinflammatory cytokines. In contrast, Ccl19-Cre-mediated Dll1 and Dll4 inactivation within host stromal cells profoundly impaired donor T cell production of IFNγ, TNFα, and IL-2, and resulted in long-term protection from GVHD. Lineage tracing in Ccl19-Cre x ROSA26-YFP mice revealed Cre activity within a small subset of CD45-negative lymph node and spleen stromal cells, but not in professional hematopoietic antigen-presenting cells. These data suggest that a specialized subset of non-hematopoietic stromal cells delivers an early pulse of Notch signaling to alloreactive T cells during acute GVHD. To our knowledge, these results provide the first in vivo evidence for non-motile secondary lymphoid-resident stromal cells as critical drivers of T cell-mediated immune pathology, with a central role for Notch signaling in this process. Transient interference with Notch ligand function or with their expression by the stromal cell niche in the peri-transplant period could serve as a novel therapeutic strategy for GVHD. DisclosuresYan:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.