Expansion Of Effector Research Articles

IL-10 and ICOS Differentially Regulate T Cell Responses in the Brain during Chronic Toxoplasma gondii Infection.

Control of chronic CNS infection with the parasite Toxoplasma gondii requires ongoing T cell responses in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokines exclusively during the chronic phase of infection, we blocked IL-10R in chronically infected mice. Consistent with previous reports, IL-10R blockade led to severe, fatal tissue destruction associated with widespread changes in the inflammatory response, including increased APC activation, expansion of CD4+ T cells, and neutrophil recruitment to the brain. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS, a molecule implicated in IL-10 production. Unexpectedly, ICOS ligand (ICOSL) blockade led to a local expansion of effector T cells in the brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. We observed increased expression of IL-2-associated signaling molecules CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain, along with decreased apoptosis. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also, unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cells or neutrophil recruitment to the brain and no severe tissue destruction. Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection.

Immunome Single Cell Profiling Reveals T Cell Exhaustion with Upregulation of Checkpoint Inhibitors LAG3 and Tigit on Marrow Infiltrating T Lymphocytes in Daratumumab and IMiDs Resistant Patients

Background: The addition of immunomodulatory (IMiDs) drugs to monoclonal antibodies targeting the transmembrane glycoprotein CD38 has demonstrated very encouraging and durable responses in myeloma patients. It is believed that the synergistic effects observed with anti-CD38 antibodies and IMiDs are derived from their co-modulation of the host adaptive and innate immunity and therefore it is plausible to speculate that acquired resistance to daratumumab and IMiDs may be largely immune-mediated. The aim of the present study was to 1) interrogate at the single cell level the bone marrow immune repertoire of daratumumab sensitive and resistant patients, 2) identify cellular mediators of resistance to anti-CD38 antibodies and 3) define potential means to reinstate sensitivity to daratumumab and IMiDs.Methods and Results: Serial BM aspirates (n=44) were collected from patients treated with single agent daratumumab (MMY3012 trial) or daratumumab + pomalidomide (MM014 trial) prior to initiation of therapy, C3D1 and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD138pos and CD138neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Paired-end sequencing was performed on Illumina NEXTseq and NOVAseq platforms. Cell Ranger Single and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting and data analysis. Sequencing data were analyzed by principal component analysis (PCA), clustering with multi-sample batch correction and then visualized by t-distributed stochastic neighbor embedding (t-SNE) projection. Comparison of the single cell transcriptomes of CD138neg cells from responding patients pre- and post- treatment revealed that Daratumumab and Pomalidomide dramatically modify the immune cells composition (immunome) of the bone marrow niches leading to: 1) expansion of effector T cells (KLRG1high, GZMAhigh, CCL5high), 2) significant depletion of CD38high / FCGR3Ahigh NK cells with retained population of cytotoxic NK cells (CD27high, KLRB1high, NCR3high, GZMApos, PRF1pos), 3) depletion of FCGR3Ahigh / CD14low monocytes, 4) expansion of M1 inflammatory macrophages and depletion of plasmacytoid dendritic cells. Similar changes were seen in patients treated with single agent daratumumab (without IMiDs) however with a lesser expansion of effector T cells and in particular reduced marrow infiltrating inflammatory macrophages. In contrast, the immunome of daratumumab and pomalidomide resistant patients was characterized by a reduced central memory T cells (TCM), and a largely exhausted effector T cells populations that are CD28neg and expressing checkpoint inhibitors (LAG3and TIGIT significantly more than PDCD1) as well as high expression of TIM3 (HAVCR2) on marrow macrophages. Upregulation of LAG3 and TIGIT expression on T cells was also confirmed at the antigenic level by flow cytometry. Consistent with the non-bystander and suppressive effect of LAG3 and TIGIT on the function of effector T cells, activation (CD107a expression) and proliferation of LAGpos and/or TIGITpos sorted bone marrow T cells from resistant patients were significantly reduced in response to autologous myeloma cells stimulation or CD3/CD28 crosslinking. Lastly, a higher proportion and number of clonal T cell (through single cell TCR sequencing) was also observed in responding (≥ PR) vs non-responding (< PR) patients. Interrogation of the myeloma cells transcriptome, showed little to no loss of CD38 transcript at the time of acquired resistance with rather upregulation of complement inhibitory molecule CD59 and NFκB signature genes.Conclusion: A systematic unsupervised interrogation of the bone marrow immunome of daratumumab and IMiDs treated MM patients demonstrated a significant activation of adaptive and innate immunity in responding patients and revealed an expansion of exhausted T cells with upregulation of the checkpoint inhibitors LAG3 and TIGIT in resistant patients. Our findings warrant the exploration of LAG3- and/or TIGIT-blocking strategies as potential means to reinstate sensitivity to daratumumab and IMiDs in myeloma patients. DisclosuresNeri:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. McCulloch:Celgene: Honoraria; Takeda: Other: Travel expenses. Thakurta:Celgene Corporation: Employment, Equity Ownership. Bahlis:Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

Abstract 3566: Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214

Abstract NKTR-214 is a CD122-biased cytokine agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand CD8 T and natural killer cells (NK) over Tregs in the tumor. We evaluated the tumor immunology, biodistribution and anti-tumor activity of NKTR-214 combined with ACT in the pmel-1 ACT/B16F10 melanoma tumor model. NKTR-214+ACT provides a robust and durable anti-tumor response compared to IL-2+ACT with less frequent dosing in the aggressive B16F10 model. NKTR-214+ACT led to significant tumor growth inhibition at day 14 compared with IL-2+ACT, 174mm3 vs 484mm3 tumor volume, respectively (p&amp;lt;0.05, n=12). Tumors of animals receiving IL-2+ACT grew to the endpoint of 1,500 mm3 18 days after treatment, while NKTR-214+ACT significantly improved the survival to 35 days (p&amp;lt;0.0001). Bioluminescence imaging (BLI) showed that NKTR-214+ACT treatment significantly increased T cells expansion in the spleen from day 5 to day 9 compared to IL-2+ACT (p&amp;lt;0.0001, n=5). At day 5, quantification of BLI of serial images with region of interest analysis at the site of spleen revealed an average radiance 14 folds higher in NKTR+ACT than in IL-2+ACT treated mice (1.3*10^7 vs 9.5*10^5 p/s/cm²/sr). BLI showed a stronger peak of activity of tumor-infiltrating effector T cells in the ACT+NKTR-214 group versus the ACT+IL-2 from day 5 to day 7 (p&amp;lt;0.0001, n =5). The second dose of NKTR-214 at day 9 triggered a second expansion of effector T cells in the spleen and tumor from day 12 to day 17, while no effect in the group treated with 3 doses IL-2 was observed (p&amp;lt;0.0001, n =5). The peak of signal was reached at day 14 with an average radiance 10 folds higher in the NKTR-214+ACT group compared to the IL2+act group (4.7*10^6 vs 4.5*10^5 p/s/cm²/sr). These data are supported by immuno-PET imaging using minibody (Mb) targeting CD8 in vivo. Ex vivo biodistribution analysis showed a signal in the spleen 5-folds higher in the ACT+NKTR-214 group compared with the ACT+IL-2 group on day 5 after treatment, 87% and 17% injected dose per gram, respectively (p&amp;lt;0.05, n=3). Flow cytometry analysis performed at the same time point showed that NKTR-214 treatment significantly increased pmel-1 CD8 T cells and amplified the CD8/Treg ratio compared to IL-2 both in spleen and tumor (3 and 6 folds, respectively, p&amp;lt;0.05, n=3). In conclusion, NKTR-214 + ACT is well tolerated and robustly mobilizes T cells into tumors where they durably persist, supporting NKTR-214 potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Felix Bergara, Paige Krystofinski, Ruixue Zhang, Cristina Puig Saus, Siwen Hu-Lieskovan, Begonya Comin-Anduix, Anna Wu, Deborah H. Charych, Antoni Ribas. Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214 [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 3566.

Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.

The mucosal surfaces of both eyes are immunologically linked by a neurogenic inflammatory reflex involving TRPV1 and substance P.

Immunological interdependence between the two eyes has been reported for the cornea and the retina but not for the ocular mucosal surface. Intriguingly, patients frequently report ocular surface-related symptoms in the other eye after unilateral ocular surgery. Here we show how unilateral eye injuries in mice affect the mucosal immune response of the opposite ocular surface. We report that, despite the lack of lymphatic cross-drainage, a neurogenic inflammatory reflex in the contralateral conjunctiva is sufficient to increase, first, epithelial nuclear factor kappa B signaling, then, dendritic cell maturation, and finally, expansion of effector, instead of regulatory, T cells in the draining lymph node, leading to disrupted ocular mucosal tolerance. We also show that damage to ocular surface nerves is required. Using pharmacological inhibitors and agonists, we identified transient receptor potential vanilloid 1 (TRPV1) channel as the receptor sensing tissue damage in the injured eye and substance P released in the opposite ocular surface as the effector of the sympathetic response. Finally, blocking either step prevented subsequent ocular allergic reactions in the opposite eye in a unilateral corneal alkali burn model. This study demonstrates that both ocular surfaces are immunologically linked and suggests potential therapeutic targets for intervention.

Formyl peptide receptor activation inhibits the expansion of effector T cells and synovial fibroblasts and attenuates joint injury in models of rheumatoid arthritis

AbstractThe effects of formyl peptide receptors (FPRs) on effector T cells and inflammation-causing tissue-resident cells are not well known. Here, we explored the effect of FPR activation on efferent T cell responses in models of rheumatoid arthritis (RA) and on the expansion of fibroblast-like synoviocytes (FLS). Compound 43 (Cpd43; FPR1/2 agonist) was administered to mice with collagen-induced arthritis (CIA) or antigen-induced arthritis (AIA) after disease onset. Joint inflammation/damage and immunity were assessed. FLS were cultured with Cpd43 to test its effects on cell apoptosis and proliferation. To explore the effects of endogenous FPR2 ligands on FLS proliferation, FLS FPR2 was blocked or Annexin A1 (AnxA1) expression silenced. Cpd43 reduced arthritis severity in both models. In CIA, Cpd43 decreased CD4 T cell proliferation and survival and increased the production of the protective cytokine, IFNγ, in lymph nodes. In AIA, Cpd43 increased CD4 apoptosis and production of the anti-inflammatory IL-4, while augmenting the proportion of splenic regulatory T cells and their expression of IL-2Rα. In both models, Cpd43 increased CD4 IL-17A production, without affecting humoral immunity. FPR2 inhibitors reversed Cpd43-mediated effects on AIA and T cell immunity. Cpd43 decreased TNF-induced FLS proliferation and augmented FLS apoptosis in association with intracellular FPR2 accumulation, while endogenous AnxA1 and FPR2 reduced FLS proliferation via the ERK and NFκB pathways. Overall, FPR activation inhibits the expansion of arthritogenic effector CD4 T cells and FLS, and reduces joint injury in experimental arthritis. This suggests the therapeutic potential of FPR ligation for the treatment of RA.

Redirection to the bone marrow improves T cell persistence and antitumor functions.

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15–dependent homeostatic expansion and promoted the differentiation of memory precursor–like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

Regulatory T cells control toxicity in a humanized model of IL-2 therapy

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ Tcells within the tumor and decreased the frequency of regulatory Tcells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Human Zika infection induces a reduction of IFN-\u03b3 producing CD4 T-cells and a parallel expansion of effector V\u03b42 T-cells

The definition of the immunological response to Zika (ZIKV) infection in humans represents a key issue to identify protective profile useful for vaccine development and for pathogenesis studies. No data are available on the cellular immune response in the acute phase of human ZIKV infection, and its role in the protection and/or pathogenesis needs to be clarified. We studied and compared the phenotype and functionality of T-cells in patients with acute ZIKV and Dengue viral (DENV) infections. A significant activation of T-cells was observed during both ZIKV and DENV infections. ZIKV infection was characterized by a CD4 T cell differentiation toward effector cells and by a lower frequency of IFN-γ producing CD4 T cells. Moreover, a substantial expansion of CD3+CD4−CD8− T-cell subset expressing Vδ2 TCR was specifically observed in ZIKV patients. Vδ2 T cells presented a terminally differentiated profile, expressed granzyme B and maintained their ability to produce IFN-γ. These findings provide new knowledge on the immune response profile during self-limited infection that may help in vaccine efficacy definition, and in identifying possible immuno-pathogenetic mechanisms of severe infection.

TLR-Induced Murine Dendritic Cell (DC) Activation Requires DC-Intrinsic Complement.

Induction of proinflammatory T cell immunity is augmented by innate dendritic cell (DC) maturation commonly initiated by TLR signaling. We demonstrate that ligation of TLR3, TLR4, and TLR9 induces murine DC production of complement components and local production of the anaphylatoxin C5a. In vitro, ex vivo, and in vivo analyses show that TLR-induced DC maturation, as assessed by surface phenotype, expression profiling by gene array, and functional ability to stimulate T cell responses, requires autocrine C3a receptor and C5a receptor (C3ar1/C5ar1) signaling. Studies using bone marrow chimeric animals and Foxp3-GFP/ERT2-Cre/dTomato fate-mapping mice show that TLR-initiated DC autocrine C3ar1/C5ar1 signaling causes expansion of effector T cells and instability of regulatory T cells and contributes to T cell-dependent transplant rejection. Together, our data position immune cell-derived complement production and autocrine/paracrine C3ar1/C5ar1 signaling as crucial intermediary processes that link TLR stimulation to DC maturation and the subsequent development of effector T cell responses.

Abstract 962: Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies

Abstract Background: A recognized and potent subtype of regulatory T cells (Tregs) expresses tumor necrosis factor receptor 2 (TNFR2), a member of the tumor necrosis factor (TNF) superfamily. TNFR2 expressing Tregs are overabundant in human and murine tumors and are the most potent suppressors of host immune responses. In addition, recent data in diverse human tumors show that TNFR2-expressing host T cells are the dominant Tregs in the tumor microenvironment, expressed far in excess of OX40 and GITR. Unlike many tumor markers, TNFR2 has limited expression in the immune system and almost no expression on parenchymal cells. This makes TNFR2 Tregs an attractive target for anticancer therapy. Methods: We produced monoclonal antibodies to human TNFR2 with the goal of inactivating the potent host Tregs that express TNFR2, competing with the agonism of TNF and inactivating infiltating Tregs of human tumors. Tregs from fresh ovarian ascites fluid were compared to Tregs from normal human blood donors for antibody potency. Results: Using a 48-hr cell-based Treg assay, we showed that TNFR2 antagonistic antibodies can inhibit Treg proliferation, suppress soluble TNFR2 secretion on normal cells and allow T effector expansion. The antagonism is dominant, succeeding even in the presence of TNF, which is a TNFR2 agonist. Furthermore, the structural biology of dominant TNFR2 receptor antagonism uncovers a unique conformation that equally inhibits downstream NFkB signaling. We also showed that dominant TNFR2 antibodies in a dose dependent fashion kill Tregs in ovarian cancer infiltrates more strongly than Tregs from healthy donors. Treg killing in the cancer microenvironment was dependent on cell proliferation and turnover the Tregs. Conclusion: In sum, blocking TNFR2 signaling on the most potent Tregs of the tumor microenvironment with a dominant antagonist antibody selectively heightens death of the immunosuppressive Tregs in the tumor microenvironment, at least in ovarian cancer. With the expanding knowledge that TNFR2 Tregs are the most potent host cells of the immune response, their dominance in diverse human tumor infiltrates and the natural low expression of this marker on normal lymphoid cells makes targeting of TNFR2 in the tumor microenvironment attractive. Citation Format: Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies [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 962. doi:10.1158/1538-7445.AM2017-962

GITR ligand fusion protein agonist enhances the tumor antigen\u2013specific CD8 T-cell response and leads to long-lasting memory

BackgroundThe expansion of antigen-specific CD8 T cells is important in generating an effective and long-lasting immune response to tumors and viruses. Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) is a co-stimulatory receptor that binds the GITR ligand (GITRL). Agonism of GITR can produce important signals that drive expansion of effector T cell populations.MethodsWe explored two separate murine tumor models, CT26 and TC-1, for responsiveness to GITR Ligand Fusion Protein(GITRL-FP) monotherapy. In TC-1, GITRL-FP was also combined with concurrent administration of an E7-SLP vaccine. We evaluated tumor growth inhibition by tumor volume measurements as well as changes in CD8 T cell populations and function including cytokine production using flow cytometry. Additionally, we interrogated how these therapies resulted in tumor antigen-specific responses using MHC-I dextramer staining and antigen-specific restimulations.ResultsIn this study, we demonstrate that a GITR ligand fusion protein (GITRL-FP) is an effective modulator of antigen-specific CD8 T cells. In a CT26 mouse tumor model, GITRL-FP promoted expansion of antigen-specific T cells, depletion of regulatory T cells (Tregs), and generation of long-lasting CD8 T cell memory. This memory expansion was dependent on the dose of GITRL-FP and resulted in complete tumor clearance and protection from tumor rechallenge. In contrast, in TC-1 tumor–bearing mice, GITRL-FP monotherapy could not prime an antigen-specific CD8 T cell response and was unable to deplete Tregs. However, when combined with a vaccine targeting E7, treatment with GITRL-FP resulted in an augmentation of the vaccine-induced antigen-specific CD8 T cells, the depletion of Tregs, and a potent antitumor immune response. In both model systems, GITR levels on antigen-specific CD8 T cells were higher than on all other CD8 T cells, and GITRL-FP interacted directly with primed antigen-specific CD8 T cells.ConclusionsWhen taken together, our results demonstrate that the delivery of GITRL-FP as a therapeutic can promote anti-tumor responses in the presence of tumor-specific CD8 T cells. These findings support further study into combination partners for GITRL-FP that may augment CD8 T-cell priming as well as provide hypotheses that can be tested in human clinical trials exploring GITR agonists including GITRL-FP.

Induction Therapy With Basiliximab in Heart-Transplanted Children Impairs Regulatory T-Cell Population (Treg)

Introduction: Following transplantation, many transplant programs employ induction immunosuppression, a relatively intense prophylactic therapy used at the time of transplant to prevent early acute rejection. Of the induction drugs, the interleukin-2 receptor (CD25) antagonist basiliximab (BXM) is frequently used. The targets of BXM are activated effector T cells, that upregulate CD25 upon activation and undergo IL-2 mediated T-cell proliferation, compromising allograft survival [1]. However, regulatory T cells (Treg) also express high levels of CD25 constitutively, and they rely on CD25 not only for their own proliferation and survival but also for detection of excessive proliferation of effector cells [2][3]. Therefore, BXM may be able to prevent acute rejection by hindering IL-2 mediated T effector expansion, but it could also compromise the likelihood of tolerance by impairing Treg proliferation and function. Methods: We enrolled 6 children who received a heart transplant and were treated with an established immunosuppression regimen. Two of the children were treated with BXM (20 mg on Day 0 and +4 after transplant), and 4 children did not receive BXM as induction therapy. We performed an exhaustive immunologic analysis at different time points including Tregs to analyze the effect of BXM on immune cells. Results: We show that the use of BXM during the first 4 days post-transplant completely blocks CD25 on T cells including Tregs for at least 45 days. Moreover, Treg numbers and Foxp3 expression were decreased in BXM-treated patients compared to patients not treated with BXM. We confirm the direct effect of BXM on Tregs by treating in vitro PBMC from healthy donors with BXM (n = 6). Basiliximab produced a marked Treg deregulation inducing a decrease in Foxp3 expression, a reduction in IL-10 production and a diminution in Treg proliferation under α-CD3/α-CD28 activation. Treg cells were more sensitive to the inhibitory effect of BXM than total CD4+ or CD8+ T cells. Conclusion: The use of basiliximab induction in transplanted children could compromise Treg-mediated tolerance during the first months post-transplant, which is the period with highest incidence of acute rejection, and could contribute to deregulation of immune homeostasis. This impact of BXM must be considered when making clinical decisions regarding induction immunosuppression.FIGURE 1: Frequency of Foxp3+CD25+CD4+ Treg cells in heart transplated children receiving induction therapy with Basiliximab (+BXM, n=2), or patients not treated with basilimab (-BXM, n=4). Mean values ±SEM are showed.This work was supported by grants from Instituto de Salud Carlos III (ISCIII) co-financed by FEDER funds (PI15/00011; ICI14/00282).FIGURE 2: Changes (%) from pre-transplant values (BL=0) in absolute counts (cells per uL of blood) of Foxp3+CD4+ cells in heart transplanted children receiving induction therapy with basilimab (+BXM, n=2), or patients not treated with basiliximab (-BXM, n=4). Mean values ±SEM are showed.FIGURE 3: CFrequencies of CDA+Foxp3+ (Treg) cells, CD4+ and CD8+ T cells within untreated (clear box) or BXM treated (solid box) PBMC stimulated with anti-CD3/CD28 dynabeads for 72h. Average and error bars indicating the 90th and 10th percentiles of 6 experiments are represented. *=<0.05 when comparing BXM-treated and non treated conditions.

Transient FOXP3 upregulation protects human conventional T cells from restimulation-induced cell death during activation-induced proliferation

Abstract An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, followed by timely disposal of those cells in order to avoid damage to host tissues. Effector T cell proliferation is constrained by the action of forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) and restimulation-induced cell death (RICD), an apoptotic pathway triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Constitutive FOXP3 expression renders Tregs resistant to RICD, in part through repression of the signaling adaptor molecule SLAM-associated protein (SAP) and FAS ligand (FASL). Interestingly, FOXP3 is also expressed transiently in human conventional T cells (Tcons) during initial rounds of activation-induced proliferation, but its function in this context remains unclear. Here we uncover a new role for FOXP3 induction in protecting both CD4+ and CD8+ human Tcons from premature RICD during the expansion phase. SiRNA-mediated silencing of FOXP3 sensitized early effector Tcons to RICD. This increase in RICD was completely dependent on de novo transcription, but FOXP3 knockdown did not alter SAP or FASL expression in these cells. Instead, FOXP3 silencing increased glycolysis and reduced autophagy in Tcons, which we and others have linked to changes in apoptosis sensitivity. Surprisingly, FOXP3-dependent autophagy specifically protected CD4+ but not CD8+ Tcons from RICD. Our results suggest a fundamentally different mechanism connecting transient FOXP3 expression to metabolic activity and RICD resistance in CD4+ versus CD8+ T cells.

Adipose Tissue-Resident Regulatory T Cells.

Tissue-resident immune cells play critical roles in regulating tissue function and homeostasis. Obesity-associated visceral adipose tissue inflammation is attributed to the accumulation of M1 macrophages which produce inflammatory cytokines like TNF-α, IL-6, and expansion of effector T cells like Th1 cells, CD8+ cytotoxic T cells which produce interferon-γ to further add to the severity of inflammation in the visceral adipose tissue. Regulatory T cells have been reported to exert key roles in suppressing inflammation, thus maintaining the homeostasis of immune responses, and visceral adipose Tregs exert critical roles in defending against obesity-associated metabolic disorders. They inhibit the infiltration of effector T cells and facilitate the reconstitution of adipose tissue macrophages from M1 to M2 phenotype. What is more, they can take up lipids from the adipocytes through CD36 which is driven by PPARγ. Here we review the recent progress in adipose tissue-resident regulatory T cells (Tregs), a subpopulation of CD4+ T cells which suppress adipose tissue inflammation.

Alloresponses of Human T-Cells from Adult Peripheral Blood and Umbilical Cord Blood Are Differentially Impacted By Lenalidomide

Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB).We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling.We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p<0.001). Although pre-treatment of allogeneic stimulators and responders had a modest potentiating effect, lenalidomide was required during allogeneic co-culture for maximal potentiation of CD8+ alloresponses of APB T-cells. CD8+ T-cells had enhanced effector memory differentiation, were enriched for polyfunctional effectors (capable of producing two or more pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and/or expressing the lysosomal membrane-associated protein CD107a), and had a distinct gene expression profile with altered expression of key immunoregulatory genes and depletion of cellular ikaros levels.In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p<0.01) whereas alloproliferation of APB CD4+ T-cells was unaffected. The reduction in UCB CD4+ alloproliferation was accompanied by selectively expansion of CD4+CD25+FOXP3+ regulatory T-cells (median 7.6% (lenalidomide-treated) vs 5.2% (untreated), n=12, p 0.02), resulting in an overall reduction in net UCB T-cell alloproliferation after lenalidomide treatment.Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT. DisclosuresNo relevant conflicts of interest to declare.

Death Receptor 3 Promotes Chemokine-Directed Leukocyte Recruitment in Acute Resolving Inflammation and Is Essential for Pathological Development of Mesothelial Fibrosis in Chronic Disease

Death receptor 3 (DR3; TNFRSF25) and its tumor necrosis factor-like ligand TL1A (TNFSF15) control several processes in inflammatory diseases through the expansion of effector T cells and the induction of proinflammatory cytokines from myeloid and innate lymphoid cells. Using wild-type (DR3+/+) and DR3-knockout (DR3-/-) mice, we show that the DR3/TL1A pathway triggers the release of multiple chemokines after acute peritoneal inflammation initiated by a single application of Staphylococcus epidermidis supernatant, correlating with the infiltration of multiple leukocyte subsets. In contrast, leukocyte infiltration was not DR3 dependent after viral challenge with murine cytomegalovirus. DR3 expression was recorded on connective tissue stroma, which provided DR3-dependent release of chemokine (C-C motif) ligand (CCL) 2, CCL7, CXCL1, and CXCL13. CCL3, CCL4, and CXCL10 production was also DR3 dependent, but quantitative RT-PCR showed that their derivation was not stromal. Invitro cultures identified resident macrophages as a DR3-dependent source of CCL3. Whether DR3 signaling could contribute to a related peritoneal pathology was then tested using multiple applications of S. epidermidis supernatant, the repetitive inflammatory episodes of which lead to peritoneal membrane thickening and collagen deposition. Unlike their DR3+/+ counterparts, DR3-/- mice did not develop fibrosis of the mesothelial layer. Thus, this work describes both a novel function and essential requirement for the DR3/TL1A pathway in acute, resolving, and chronic inflammation in the peritoneal cavity.

CD4(+) T-cell help amplifies innate signals for primary CD8(+) T-cell immunity.

CD8(+) T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8(+) T-cell immunity have been shown to depend on CD4(+) T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4(+) T cells, various dendritic cell subsets, and CD8(+) T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8(+) T-cell expansion and differentiation. This modelfacilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity.

Regulatory T cells evade restimulation-induced cell death by FOXP3-mediated repression of the SAP promoter.

Abstract An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, paired with timely disposal of those cells in order to avoid damage to host tissues and maintain immune homeostasis. Restimulation-induced cell death (RICD) is one programmed apoptotic pathway that ensures effector T cell expansion remains in check, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). CD4+ regulatory T cells (Tregs) require IL-2 and experience frequent TCR stimulation, but are highly resistant to RICD. Although the mechanism remains unclear, previous work has shown that RICD resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcriptional repressor, the master regulator of Treg function. We have previously shown that T cells from patients with X-linked lymphoproliferative disease (XLP-1), caused by null mutations in the signaling adaptor molecule SLAM-associated protein (SAP), are resistant to RICD. In fact, SAP is required for optimal RICD in effector T cells. We now demonstrate that Tregs express very low levels of SAP compared to conventional T cells (Tconv). Using chromatin immunoprecipitation and reporter assays, we show that FOXP3 reduces SAP expression in Tregs by binding to and repressing the SH2D1A (SAP) promoter. Indeed, knockdown of FOXP3 restores SAP-driven RICD sensitivity in Tregs through a FAS ligand-dependent mechanism. Intriguingly, our preliminary data suggest that FOXP3 also confers RICD resistance in Tconv during initial rounds of activation-induced proliferation. Hence our results provide a mechanism by which FOXP3 confers resistance to RICD to help preserve Tregs and protect early effector Tconv cells.