Costimulatory ICOS Research Articles

ICOS costimulation in combination with CTLA-4 blockade remodels tumor-associated macrophages toward an antitumor phenotype.

We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.

ICOS costimulation is indispensable for the differentiation of T follicular regulatory cells.

ICOS is a T-cell costimulatory receptor critical for Tfh cell generation and function. However, the role of ICOS in Tfr cell differentiation remains unclear. Using Foxp3-Cre-mediated ICOS knockout (ICOS FC) mice, we show that ICOS deficiency in Treg-lineage cells drastically reduces the number of Tfr cells during GC reactions but has a minimal impact on conventional Treg cells. Single-cell transcriptome analysis of Foxp3+ cells at an early stage of the GC reaction suggests that ICOS normally inhibits Klf2 expression to promote follicular features including Bcl6 up-regulation. Furthermore, ICOS costimulation promotes nuclear localization of NFAT2, a known driver of CXCR5 expression. Notably, ICOS FC mice had an unaltered overall GC B-cell output but showed signs of expanded autoreactive B cells along with elevated autoantibody titers. Thus, our study demonstrates that ICOS costimulation is critical for Tfr cell differentiation and highlights the importance of Tfr cells in maintaining humoral immune tolerance during GC reactions.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

Abstract 1849: ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control

Abstract In recent years, antibody-based immunotherapies targeting immune checkpoint proteins cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have revolutionized the treatment of cancer. Despite durable clinical responses in many indications, a significant number of patients fail to yield meaningful benefit, emphasizing the need to identify novel and/or complementary therapeutic interventions (e.g., alternative immune checkpoints, cytotoxic agents, etc.). Inducible T cell Co-Stimulator (ICOS/CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells shortly after T cell receptor (TCR) activation. A growing body of non-clinical and clinical evidence supports the concept of antibody-mediated induction of ICOS signaling as a means to promote durable antitumor responses, particularly in collaboration with other checkpoint immunotherapies. To support ongoing and future clinical studies for feladilimab (GSK3359609), a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials, we conducted a series of in vivo studies using a rodent surrogate (7E.17G9 mouse [m]IgG1; analogous non-depleting Fc) in ICOS-sensitive (EMT6 breast carcinoma) and ICOS-insensitive (CT26 colon carcinoma) tumor models. Female BALB/c mice with pre-established EMT6 tumors, exhibited partial responses to GSK'609 surrogate/tool antibody (7E.17G9 mIgG1), with ~20% (range 0-40%) of mice demonstrating tumor clearance. Concurrent administration of anti-PD-1 (RMP1-14 rat [r]IgG2a) and CTLA-4 (9D9 mIgG2b) dramatically improved tumor growth inhibition and survival. Notably, a triplet of TIM-3 blockade (RMT3-23 rIgG2a) in concert with ICOS and PD-1 mAbs further enhanced the survival of EMT6 tumor-bearing mice, suggesting complementary therapeutic mechanisms. In addition to combination with immune checkpoint blockade, we also explored the potential of ICOS co-stimulation with different focal irradiation (FRT) doses and frequencies in both EMT6 and CT26 tumor models. Intriguingly, we observed dramatic differences in response to concurrent FRT and ICOS mAb depending on the tumor model and associated FRT dose and frequency. Further, we identified a FRT dose threshold required for ICOS agonist appreciable combination responses, enabling exploration of additional immunotherapeutic combinations on top of ICOS/FRT. Collectively, these data support the combination of ICOS co-stimulation with different checkpoint immunotherapies (doublets and triplets), several of which are currently being evaluated in clinical studies. Moreover, these data demonstrate potential of ICOS co-stimulation with cytotoxic strategies, such as FRT - providing various dosing considerations and a framework future therapeutic intervention strategies. Citation Format: Jeremy D. Waight, Meixia Bi, Chris Hopson, Sapna Yadavilli, Kenneth W. Hance, Marc Ballas, Axel Hoos. ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1849.

Fully Human Tandem CD22-CD19 CAR-T Cells with Superior Sensitivity to Low Antigen Density Derived by Optimization of Co-Stimulation and CAR Architecture

In the treatment of B cell leukemia, relapse due to antigen to loss or downregulation remains a major challenge. Tumor antigen escape may be mitigated by multi-targeting CAR T cells redirected to CD19 and CD22, and possessing a superior sensitivity to low-density antigens. Using lentiviral transduction of primary T cells, flow cytometry, cell-based assays, and xenograft mouse models, we systematically optimized 22-19 CAR architecture and co-stimulatory domains for best functionality. Fully-human tandem 22-19 CARs with co-stimulatory domains derived from 4-1BB, CD28, ICOS, OX40 or CD27, and hinge and transmembrane domains derived from CD8, CD28, or OX40 were evaluated. The tandem targeting ScFv domain orientation 22-19 was selected based on greater expression and cytotoxicity vs 19-22. All CARs achieved high T cell expression (mean 50-90%), and efficient dose-dependent killing of RajiCD19+CD22+, 293TCD19+, 293TCD22+, but not 293TCD19-CD22- target cells, and elaborated IL-2, IFN-γ, and TNF-a in antigen-dependent manner. CARs' potency in vitro varied by co-stimulatory domain: 4-1BB< OX40, ICOS, CD27<CD28, 4-1BB-CD28. The 22-19 tandem CAR with hinge and transmembrane domains (H/TM) derived from CD8 (CD8/CD8) performed as well as the CAR with H/TM CD28/CD28 configuration, whereas configuration OX40/OX40 was superior to CD8/OX40. In RajiCD19+CD22+ mouse xenografts, the potency of tumor rejection by the 2nd generation tandem 22-19 CARs was also dependent on co-stimulatory domain, ranking 4-1 BB<CD27, ICOS<OX40<CD28. Tandem 3rd generation CAR 22-19, combining CD28 and 4-1BB co-stimulation, achieved the greatest anti-tumor effect. In antigen-heterogeneous Raji xenografts of a mixture of CD19-CD22+, CD19+CD22-, and CD19+CD22+ clones, the 22-19 tandem CARs with CD28, OX40 or ICOS co-stimulation mediated rapid and complete tumor rejection, whereas the conventional single-targeting CAR19 or CAR22 enabled tumor progression due to antigen escape. Low antigen density Raji clones were generated by CRISPR-Cas9-mediated disruption of both CD19 and CD22 expression, followed by lentiviral transduction to express a limited number of antigen molecules on the cell surface. When challenged with Raji CD22 low clone in vitro, 2nd generation tandem CARs with CD28 and ICOS co-stimulation, and the 3rd generation tandem CAR combining CD28 and 4-1BB co-stimulatory domains were more effective than 4-1BB-, CD27-, or OX40-containing tandem CARs. Against RajiCD19 low clone, CARs with CD27 and OX40 domains were more effective than CARs with 4-1BB, and CARs with ICOS or CD28 co-stimulation were the most potent. In summary, the fully-human tandem 22-19 CARs incorporating ICOS and CD28 co-stimulatory domains mitigate tumor antigen escape, exhibit robust anti-tumor function in pre-clinical models, enable superior lysis of CD22low and CD19low tumor clones, and may help improve clinical outcomes. Disclosures Hu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Xiong:Lentigen, a Miltenyi Biotec Company: Current Employment. Wu:Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blockade therapy is able to induce long-lasting antitumor responses in a fraction of cancer patients. Nonetheless, there is still room for improvement in the quest for new therapeutic combinations. ICOS costimulation has been underscored as a possible target to include with CTLA-4 blocking treatment. Herein, we describe an ICOS agonistic aptamer that potentiates T cell activation and induces stronger antitumor responses when locally injected at the tumor site in combination with anti-CTLA-4 antibody in different tumor models. Furthermore, ICOS agonistic aptamer was engineered as a bi-specific tumor-targeting aptamer to reach any disseminated tumor lesions after systemic injection. Treatment with the bi-specific aptamer in combination with CTLA-4 blockade showed strong antitumor immunity, even in a melanoma tumor model where CTLA-4 treatment alone did not display any significant therapeutic benefit. Thus, this work provides strong support for the development of combinatorial therapies involving anti-CTLA-4 blockade and ICOS agonist tumor-targeting agents.

PD-1 Primarily Targets TCR Signal in the Inhibition of Functional T Cell Activation.

Cancer-immunotherapy targeting programmed cell death 1 (PD-1) activates tumor-specific T cells and provides clinical benefits in various cancers. However, the molecular basis of PD-1 function is still enigmatic. Especially, it is unclear which signaling pathway PD-1 primarily targets. Besides, the capacity of PD-1 to inhibit the T cell receptor (TCR)-dependent activation of T cells in the presence of co-stimulation is also controversial. Here we used co-culture systems of T cells and antigen-presenting cells with targeted deletion and overexpression of co-receptors and ligands and examined the inhibitory potency of PD-1 against T cell activation upon TCR stimulation with CD28 and ICOS co-stimulation. As an unambiguous criterion of T cell activation, we used the acquisition of cytokine production capacity, which represents one of the most important functions of T cells. PD-1 inhibited functional T cell activation upon TCR stimulation in the absence as well as in the presence of CD28 co-stimulation, indicating that PD-1 can directly inhibit TCR signal. Notably, CD28 co-stimulation rather attenuated the efficiency of PD-1 in inhibiting TCR-dependent functional T cell activation. In addition, PD-1 inhibited TCR-dependent functional T cell activation with ICOS co-stimulation as efficiently as that with CD28 co-stimulation. Furthermore, we found that the maintenance of antigen-induced follicular helper T (TFH) cells that required ICOS co-stimulation was persistently restrained by PD-1 in vivo. These findings indicate that PD-1 primarily targets TCR signal in the inhibition of functional T cell activation. Thus, PD-1 functions as the rheostat of T cell activation rather than an inhibitor of a specific stimulatory co-receptor.

Acute Myeloid Leukemia (AML) Blasts Influence the Gene Expression Signature and Co-Signaling Receptor Expression of CD8+ T Cells

Abstract Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments. Methods: Serial peripheral blood (PB) samples (at diagnosis and at the recovery after induction chemotherapy) were collected. To study transcriptional signatures, RNA isolated from FACS-purified PB CD8+ T cells from 6 patients [3 responders (R) and 3 non-responders (NR)] and 4 healthy controls (HC) was analyzed with the Human Prime View Gene Expression Array (Affymetrix). The data were normalized and log transformed. Expression fold change (FC), p values and false discovery rate were determined. Enrichment of canonical pathways was determined using Ingenuity Pathway Analysis (IPA, QIAGEN). To study AML blast-T cell interactions, we FACS-purified T cells and primary AML blasts at diagnosis (n=13) and T cells from HC (n=12). T cells were cultured in vitro for 3 days in the presence or absence of blasts (T cell:blast ratio 1:10) and analyzed by flow cytometry. Results: The transcriptional profile of CD8+ T cells at AML diagnosis significantly differed from that of HC. Genes were selected based on >2 FC between patient and HC, and p< 0.01. We identified a total of 453 dysregulated genes, of which 237 were up- and 216 down-regulated. Upregulated genes included immune inhibitory receptors LILRB1, 2B4, KLRG1, CD160, the transcription factors EOMES, TBET, TIGIT and cytokines (granzyme-A/B/K). In contrast, co-stimulatory receptor genes were downregulated, including CD40LG, CD28, CD30LG and CD28H. Canonical pathways analysis with IPA revealed that the NFAT pathway (involved in T cell differentiation and self-tolerance) was highly upregulated, while co-stimulatory CD28, ICOS and OX40 signaling pathways were downregulated in CD8+ T cells at AML diagnosis. Next, we compared R to NR after induction chemotherapy. There were a total of 351 dysregulated genes; 108/243 genes were up-/down-regulated, respectively. R patients upregulated immune stimulatory receptor genes like ICOS, whereas the top expressed genes for NR patients included the co-inhibitory receptor TIM3; several members of the inhibitory LIR receptor family; LST1 (involved in inhibition of lymphocyte proliferation); TWEAK-APRIL (associated with T cell apoptosis); and CD39 (terminally exhausted CD8+ T cells). In line with these findings, IPA showed that the co-stimulatory ICOS and OX40 signaling pathways were enriched in R patients. In contrast, the NFAT pathway, which had been highly upregulated at diagnosis, remained enriched in NR, but not in R patients. Results were confirmed by qPCR. The culture assay showed that the presence of primary AML blasts significantly reduced the viability of both AML and HC T cells (p <0.005 in both cases). The presence of AML blasts also significantly decreased the frequency of primary AML T cells expressing co-stimulatory receptors 41BB, ICOS and OX40, while it increased the frequency of HC T cells expressing co-inhibitory receptor 2B4 and the senescence/exhaustion marker CD57 compared to their counterparts cultured without blasts. Conclusions: Our study provides insight into the genomic CD8+ T cell signatures of AML patients at diagnosis and following chemotherapy. At diagnosis, T cells overexpressed genes that negatively regulate T cell immune responses, while genes that positively regulate immune responses were downregulated. Interestingly, after induction chemotherapy these changes persisted in NR only. Additionally, a pattern of decreased viability and co-stimulatory receptor expression was seen after in vitro co-culture of T cells with AML blasts, whereas immune inhibitory receptor expression was increased. Our data suggests that the blasts themselves influence the T cell phenotype and genotype in AML patients and that remission is associated with reversion to HC pattern. Disclosures Levis: Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Millennium: Consultancy, Research Funding.

Phenotypic Tfh development promoted by CXCR5-controlled re-localization and IL-6 from radiation-resistant cells.

How follicular T-helper (Tfh) cells develop is incompletely understood. We find that, upon antigen exposure in vivo, both naïve and antigen-experienced T cells sequentially upregulate CXCR5 and Bcl6 within the first 24 h, relocate to the T-B border, and give rise to phenotypic Bcl6+CXCR5+ Tfh cells before the first cell division. CXCR5 upregulation is more dependent on ICOS costimulation than that of Bcl6, and early Bcl6 induction requires T-cell expression of CXCR5 and, presumably, relocation toward the follicle. This early and rapid upregulation of CXCR5 and Bcl6 depends on IL-6 produced by radiation-resistant cells. These results suggest that a Bcl6hiCXCR5hi phenotype does not automatically define a Tfh lineage but might reflect a state of antigen exposure and non-commitment to terminal effector fates and that niches in the T-B border and/or the follicle are important for optimal Bcl6 induction and maintenance.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0210-0) contains supplementary material, which is available to authorized users.

A Novel Th17-Prone CD146+CCR5+ T-Cell Population As an Early Marker of Intestinal Graft-Versus-Host Disease

GVHD of the gastrointestinal tract (GI) is associated with high mortality. We have identified systemic, skin- and GI-specific plasma biomarkers present at clinical GVHD onset, and more recently biomarkers for treatment responsiveness. However, in order to identify early GI-specific biomarkers prior to GVHD onset, we performed state-of-the-art proteomics on plasma samples taken 14 days prior to clinical manifestation of GI GVHD. We selected candidates that were increased at least 1.5 fold in plasma from GI GVHD patients compared to HSCT patients without GVHD at matched time points. We identified two lead proteins: CD146, a cell adhesion and trafficking molecule expressed on a subset of CD4+ T cells and endothelial cells, and the chemokine (C-C motif) ligand 14 that binds to the chemokine receptor CCR5 on T cells. As these proteins have not been previously identified in proteomics experiments and antibodies for their corresponding receptors on T cells were available, we analyzed their expression profiles on PB cells from 214 HSCT patients (71 GI GVHD, 48 no GVHD, 33 non-GVHD enteritis, 22 skin first GVHD, 40 isolated skin GVHD) at the onset of symptoms. The frequency of CD146+CCR5+ T cells was significantly increased in GI GVHD patients compared to patients without GVHD, non-GVHD enteritis, or with isolated skin GVHD, as well as increased in patients who first experienced skin and then GI GVHD (Fig. 1). When using the median % of CD146+CCR5+ T cells detected in GI GVHD patients to classify patients into low and high-risk groups, patients in the high-risk group had higher 6-month non-relapse mortality (42 vs. 20%, p = 0.02). Importantly, CD146+CCR5+ T cells at onset of clinical symptoms were not correlated with GI histologic severity, suggesting that these cells are not mucosa damage products but rather systemic effectors. Thus, we measured their frequencies in samples taken at a median of 19 days post-transplant and with a median interval of 14 days prior to clinical symptoms and found that CD146+CCR5+ T cells circulate in patients before GI GVHD clinical onset.In GI GVHD patients, these cells expressed a Th1 and Th17 phenotype and expressed high levels of the activation marker ICOS known to be critical for the development of human Th17 cells. To test the hypothesis that CD146+ T cells are Th17-prone, we next investigated whether in vitro polarization of CD4 T cells with defined stimulation conditions will increase the CD146+CCR5+ expression as well as the production of Th1 and Th17 cytokines. Fig. 2A demonstrates that CD4 T cells differentiated with both Th17-inducing cytokines and ICOS co-stimulation had a significantly higher percentage of CD146+CCR5+ T cells and co-expressed more IL-17A+IFNγ+ than T-cells stimulated with Th1-inducing cytokines or by CD28.We then analyzed colonic mucosa biopsies from patients with GI GVHD (N = 18) and non-GVHD enteritis (N = 10) for the expression of CD146 by immunohistochemistry. CD146 expression was detected on CD3 lymphoid cells and was strongly present on the endothelium. The CD146+ vessel count in GI GVHD tissues was significantly higher than in non-GVHD enteritis tissues (p < 0.001).Th17 cells migrated more efficiently through endothelial cell monolayers than their Th1 counterparts (Fig. 2B). Lentivirus-mediated shRNA knock-down of CD146 in either cell type demonstrated that reduced CD146 expression on CD4+ T cells, but not on endothelial cells significantly reduced the T-cell transendothelial migration (Fig. 2C), suggesting that CD146 on T cells is paramount for promoting infiltration of pathogenic T cells into GVHD target organs. As proof of principle for this hypothesis, we tested donor CD146-/- T cells in an allogeneic murine GVHD model and did not find any difference in GVHD severity when compared to wild type CD146+/+ T cells. Finally, we used a xenogeneic GVHD mouse model with injection of human CD4+ T cells lentivirally transduced with CD146 or control shRNA. In comparison to the vector control group, mice transplanted with CD146 shRNA transduced T cells did not lose weight (Fig. 3A), had similar human T cells engraftment (B), had less splenic CD146+CCR5+ T cells (C), and expressed less TBET 53 days after transplant (D).In conclusion, early quantification of a novel CD146+CCR5+ Th17-prone and ICOS-induced population may allow identification of patients at risk for GI GVHD development and subsequent mortality. Targeting CD146 may represent a new avenue to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PL02-03: Immune modulation for cancer therapy: Assessing antagonists and agonists

Abstract PL02-03: Immune modulation for cancer therapy: Assessing antagonists and agonists

The number of responding CD4 T cells and the dose of antigen conjointly determine the TH1/TH2 phenotype by modulating B7/CD28 interactions.

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

PI3K-Independent Signaling Pathways Contribute to ICOS-Mediated T-Cell Costimulation in Acute Graft-Versus-Host Disease in Mice.

AbstractAbstract 3003ICOS provides an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for the generation of follicular helper T cells. We also observed that, in both CD4+ and CD8+ T cells, ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner in vitro. Although ICOS can potentiate TCR-mediated calcium flux independent of PI3K, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. Severity of acute graft-versus-host disease (GVHD) was evaluated based on recipient survival, body weight change, and pathologic scores. Consistent with the data previously published by us and others, ICOS KO T cells had significantly reduced ability to cause acute GVHD as compared to WT T cells. We further observed that YF T cells were significantly more capable in causing GVHD than KO T cells, but less capable than WT T cells. Mechanically, the levels of serum TNFa and IFNg were similar in the recipients of YF or KO T cells, but significantly lower than those of WT T cells. However, on the per-cell basis, YF CD8+ T cells expressed similar levels of intracellular IFNg as WT T cells, but significantly higher than KO T cells. We further compared the ability of CD4+ or CD8+ T cells alone in the induction of acute GVHD, and found that CD4+ T cells from YF and KO mice were similarly impaired in their capacity to induce acute GVHD. In contrast, the pathogenic capacity of CD8+ T cells from YF mice was comparable to that of WT cells, whereas KO CD8+ T cells were significantly less pathogenic. These results suggest that although both CD4+ and CD8+ T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4+ T cells depend on ICOS-PI3K signaling whereas CD8+ T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in T cell activation and GVHD induction. Disclosures:No relevant conflicts of interest to declare.

ICOS promotes T cell activation and function beyond phosphoinositide 3-kinase signaling (126.11)

Abstract ICOS delivers an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for generation of follicular helper T cells. Although ICOS can potentiate TCR-mediated calcium flux in a PI3K-independent manner, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. We found that CD4 T cells from ICOS-YF and KO mice were similarly impaired in their capacity to induce acute graft-versus-host disease (GVHD). In contrast, the pathogenic capacity of CD8 T cells from ICOS-YF mice was comparable to that of WT cells, whereas ICOS KO CD8 T cells were less pathogenic. In vitro, we observed that in both CD4 and CD8 T cells ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner. These results suggest that although both CD4 and CD8 T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4 T cells depend on ICOS-PI3K signaling whereas CD8 T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in GVHD.

Defects of mitogen-activated protein kinase in ICOS signaling pathway lead to CD4 + and CD8 + T-cell dysfunction in patients with active SLE

In this study, hypoproliferation and defects of effectors and cytokines in CD4 + and CD8 + T-cells via ICOS costimulation were found in active SLE patients, relative to normal individuals and RA patient controls. Exogenous IL-2 can partially reverse those defects. In addition, low level of ERK phosphorylation in ICOS-mediated signaling pathway was discovered in lupus CD4 + and CD8 + T-cells. When blocked with ERK-specific chemical inhibitor PD98059, cell proliferation and IL-2 production via ICOS costimulation from both CD4 + and CD8 + T-cells will be severely inhibited. These findings confirmed the dysfunction of both CD4 + and CD8 + T-cells after ICOS costimulation in lupus patients and most importantly pointed out that impairment of ERK activation might be one of the critical factors involved in ICOS-mediated IL-2 and T-cell hypoproliferation in active SLE.

A Costimulation-Initiated Signaling Pathway Regulates NFATc1 Transcription in T Lymphocytes

T cell activation and differentiation is accompanied and mediated by transcriptional reprogramming. The NFATc1 transcription factor is strongly induced upon T cell activation and controls numerous genes involved in the T cell effector function. However, its regulation by physiological stimuli in primary T cells has not been well understood. We previously found that ICOS synergizes with TCR and CD28 to greatly enhance NFATc1 expression in primary T cells. In this study, we have examined the signaling mechanisms whereby costimulation regulates NFATc1 expression. We found that CD28 and ICOS regulate sustained PI3K activity in primary T cells, which is required for NFATc1 up-regulation. CD28 and ICOS costimulation, possibly through Itk, a Tec kinase downstream of the PI3K, enhanced phosphorylation of phospholipase C gamma1 and increased and sustained Ca(2+) flux in T cells. Costimulation of T cells potentiated transcription of the Nfatc1 gene P1 promoter in a PI3K-dependent manner. This work demonstrates an important role for costimulatory receptors in sustaining T cell activation programs leading to Nfatc1 gene transcription and has implications in our understanding of the immune response and tolerance.

Inducible costimulator protein controls the protective T cell response against Listeria monocytogenes.

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.

Modulation of TCR-induced transcriptional profiles by ligation of CD28, ICOS, and CTLA-4 receptors.

Signals generated by T cell receptor (TCR) and CD28 engagement are required for optimal T cell activation, but how these signals integrate within the cell is still largely unknown. We have used near genome-scale expression profiling to monitor T cell signal transduction pathways triggered via TCR and/or costimulatory receptors. Ligation of CD28 alone induced a set of short-lived early response transcripts in both Jurkat T cells and primary CD4 T cells, thus providing evidence that CD28 engagement can affect gene regulation independently of TCR engagement. Simultaneous signaling through both the TCR and CD28 resulted in altered expression of several thousand genes following several distinct temporal patterns. Most of these gene regulations were induced by TCR signaling alone and were augmented to varying degrees by CD28 costimulation. CD28 and ICOS costimulation had nearly identical effects on gene regulation, but a few transcripts (e.g., IL2, IL9) were significantly more affected by CD28. Therefore, the distinctive functional outcomes of costimulation via CD28 and ICOS are accompanied by relatively few distinct differences in gene expression. Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement selectively blocked augmentation of gene regulations by CD28-mediated costimulation, but did not ablate gene regulation induced by TCR triggering alone.