Effector Cytokines In Response Research Articles

Upregulation, Functional Association, and Correlated Expressions of TRPV1 and TRPA1 During Telmisartan-Driven Immunosuppression of T Cells

ABSTRACT TRPV1 and TRPA1, are known to be functionally expressed in T cells, where these two channels differentially regulate effector immune responses. Telmisartan (TM), an anti-hypertension drug, has been recently repurposed to suppress various inflammatory responses. However, the possible involvement of TRP channels during TM-driven suppression of T cells responses has not been explored yet. In this study, we investigated the potential role of TRPV1 and TRPA1 during TM-driven immunosuppression of T cells in vitro. We observed a significant elevation of both TRPV1 and TRPA1 during TM-induced immunosuppression of T cells.We found that TRPA1 activation-driven suppression of T cell activation and effector cytokine responses during TM treatment is partially, yet significantly overridden by TRPV1 activation. Moreover, the expressions of TRPV1 and TRPA1 were highly correlated in various conditions of T cell. Mechanistically, it might be suggested that TRPV1 and TRPA1 are differentially involved in regulating T cell activation despite the co-elevation of both these TRP channels’ expressions in the presence of TM. T cell activation was delineated by CD69 and CD25 expressions along with the effector cytokine levels (IFN-γ and TNF) in TM-driven suppression of T cell. These findings could have broad implications for designing possible future immunotherapeutic strategies, especially in the repurposing of TM for T cell-TRP-directed immune disorders.

Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation.

Antibiotic treatment can lead to elimination of both pathogenic bacteria and beneficial commensals, as well as to altered host immune responses. Here, we investigated the influence of prolonged antibiotic treatment (Abx) on effector, memory and recall Th2 immune responses during the primary infection, memory phase and secondary infection with the small intestinal nematode Heligmosomoides polygyrus. Abx treatment significantly reduced gut bacterial loads, but neither worm burdens, nor worm fecundity in primary infection were affected, only worm burdens in secondary infection were elevated in Abx treated mice. Abx mice displayed trends for elevated effector and memory Th2 responses during primary infection, but overall frequencies of Th2 cells in the siLP, PEC, mLN and in the spleen were similar between Abx treated and untreated groups. Gata3+ effector and memory Th2 cytokine responses also remained unimpaired by prolonged Abx treatment. Similarly, the energy production and defence mechanisms of the host tissue and the parasite depicted by NAD(P)H fluorescence lifetime imaging (FLIM) did not change by the prolonged use of antibiotics. We show evidence that the host Th2 response to intestinal nematodes, as well as host and parasite metabolic pathways are robust and remain unimpaired by host microbiota abrogation.

CD69+ Bone Marrow-Resident CD8+ T Cells Represent a Functionally Impaired Tumor-Reactive Population and Can be Restored By Blocking PD-1 and Tigit in Multiple Myeloma

Recently, novel anti-myeloma therapies utilizing T cell-mediated immune responses, including chimeric antigen receptor (CAR) T cells or bispecific antibodies engaging T cells to target tumor cells, have been actively studied in clinical trials and shown significant clinical efficacy. To enhance the clinical activity of T cell-exploiting immunotherapies in MM further, a better understanding of immune evasion mechanisms by characterizing myeloma-specific T cells and the strategic reinvigoration of the impaired T cell functions are essential prerequisites. In the present study, we demonstrated that bone marrow (BM)-resident CD69-expressing CD8+ T cells showed more prominent pathologic features including tumor-antigen associated exhaustion and impaired function in MM patients. In addition, using direct ex vivo experimental techniques, we examined the strategy how to restore anti-myeloma responses of the tumor-specific T cells, based on the characteristics of BM-resident CD69+ CD8+ T cell population. We first tried to evaluate various immunophenotypes of CD8+ T cells in BM and peripheral blood (PB) compartments of MM patients and found that the frequency of CD69+ cells among CD8+ T cells was significantly higher in BM than in peripheral circulation. These CD69+CD8+ T cells in BM had an effector memory phenotype negative for CCR7. Also, CD8+ T cells obtained from BM of normal controls contained similar frequency of CD69+ cells to the CD8+ T cells from BM of MM patients, suggesting that the CD69+ population could represent a BM-resident memory population even in a physiologic condition. To this end, we isolated CD69+ and CD69- memory CD8+ T cells from BM of MM patients and analyzed their transcriptomic difference using RNA-sequencing analysis. CD69, EPAS1, EOMES, PDCD1, TIGIT, and TOX were significantly upregulated in CD69+ cells when compared to CD69- cells among memory CD8+ T cells. On the contrary, S1PR1, KLF2, KLF4, SELL, GZMB, and GNLY were downregulated in CD69+ cells when compared to CD69- cells. These results indicate that transcriptomic signatures of CD69+ cells were similar to previously known features of tissue-resident memory T cells and functionally exhausted CD8+ tumor-infiltrating lymphocytes. We then compared the surface expression level of immune checkpoint receptors between CD69+ and CD69- cells among BM CD8+ T cells. CD69+ cells showed significantly higher positive frequencies of PD-1, Lag-3 and TIGIT than CD69- cells, but expression of Tim-3 did not differ between two populations. Notably, TOX, the key transcription factor regulating T cell exhaustion, was higher in CD69+ cells than in CD69- cells. When we examined the expression of other transcription factors as indicators of T cell exhaustion, the EomeshiT-betlo cells were significantly enriched in CD69+ population, indicating that CD69+ cells have features of terminally exhausted CD8+ T cells. More importantly, the myeloma antigen (NY-ESO-1157-165 and HM1.2422-30)-specific CD8+ T cells in BM of MM patients, identified with the MHC-multimer technique, also enriched in CD69+ population. When we compared the expression of immune checkpoint receptors among myeloma antigen-specific CD8+ T cells, the frequencies of PD-1+, TIGIT+ and PD-1+TIGIT+ cells were higher in CD69+ cells than CD69- cells. In fact, CD69+CD8+ T cells from BM of MM patients showed reduced production of effector cytokines in response to direct ex vivo TCR stimulation, compared to CD69- counterpart. Next, we investigated whether blocking PD-1 and/or TIGIT could restore the function of BM-resident CD8+ T cells of MM patients. Although a single blockade using either anti-PD-1 or anti-TIGIT antibody was not effective, their combination resulted in significant increase of IFN-γ+ cells in total CD8+ T cells and CD69+CD8+ T cells but not in CD69-CD8+ T cells Taken together, we demonstrated that BM-resident CD69+CD8+ T cells represent a population of exhausted T cells which are functionally impaired but potentially restorable. Indeed, we provide an ex vivo evidence of dual blockade of immune checkpoint receptors to enhance anti-tumor T cell responses in MM. Detailed understanding of BM-resident CD8+ T cells could provide more fundamental information for improving the current T cell-based immunotherapeutic approaches against MM.

TGFβ2 Induces the Soluble Isoform of CTLA-4 - Implications for CTLA-4 Based Checkpoint Inhibitor Antibodies in Malignant Melanoma.

Malignant melanoma is an aggressive form of cancer, which can be treated with anti-CTLA-4 and anti-PD-1 checkpoint inhibitor antibodies but while anti-CTLA-4 antibodies have clear benefits for some patients with melanoma, productive responses are difficult to predict and often associated with serious immune related adverse events. Antibodies specific to CTLA-4 bind two major isoforms of CTLA-4 in humans, the receptor isoform and a second naturally secretable, soluble isoform - sCTLA-4. The primary aim here was to examine the effect of selectively blocking the function of sCTLA-4 on in vitro immune responses from volunteer healthy or melanoma patient PBMC samples. Addition of recombinant sCTLA-4 to healthy PBMC samples demonstrated sCTLA-4 to have immunosuppressive capacity comparable to recombinant CTLA4-Ig, partially reversible upon antibody blockade. Further, we identified a mechanistic relationship where melanoma patient TGFβ2 serum levels correlated with sCTLA-4 levels and provided the basis for a novel protocol to enhance sCTLA-4 production and secretion by T cells with TGFβ2. Finally, a comparison of selective antibody blockade of sCTLA-4 demonstrated that both healthy and melanoma patient effector cytokine responses can be significantly increased. Overall, the data support the notion that sCTLA-4 is a contributory factor in cancer immune evasion.

Abstract LB-391: Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC is urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs in African-American and Latina patients. Since hormone receptors, like insulin-like growth factor-1/insulin (IGF1R/IR, are known to be enriched and to cross-communicate in subtypes of TNBC, we assessed effects of IGF1R/IR antagonists on proliferation of human and murine TNBC cells in vitro. Combination treatment of BMS-754807 (IGF1R/IR) with NVP-AEW541 (IGF1R inhibitor) reduced proliferation of human MDA-MB-231, BT549, HCC1937 and murine 4T1 TNBC cells in vitro (P<0.001). Moreover, combination treatment using BMS-754807 and NVP-AEW541 significantly reduced MDA-MB-231 tumor xenograft progression in vivo (P<0.001). IHC study of tumor tissues from in vivo experiments confirmed reduced Ki-67 expression with combination treatments. Among breast cancer subtypes, TNBC is characterized by significant numbers of tumor infiltrating lymphocytes (TILs), a predictive marker for response to immunotherapy. Further, recent reports from clinical trials using immune checkpoint inhibitors demonstrate that a subset of TNBC patients respond to treatment with immune checkpoint inhibitors (ICIs). It is also notable that increased expression of IGF1Rs are identified on immune cells responsible for mounting adaptive and humoral immune responses. Using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/c mice, we find that combination treatment with BMS-754807 and anti-PD-L1 antibody significantly blocks tumor progression as compared to both controls and with either agent used alone (P<0.0001). In addition, single cell analysis of the tumor microenvironment after treatment with BMS-754807 alone or combined with nti-PD-L1 antibody combination therapy shows a significant reduction in myeloid-derived suppressor cells (MDSCs). Moreover, BMS-754807 combined with anti-PD-L1 antibody increased infiltrating effector T cells and significantly enhanced the T central memory population (Tcm) (P<0.01). Functionally, it is reported that Tcm subsets of immune cells produce effector cytokines in response to antigens and can readily proliferate and differentiate into effector cells. Use of immunotherapy is a promising management option for a subset of TNBC patients, and combination treatments using IGF1R antagonists with immune checkpoint inhibitor may constitute a new treatment strategy to combat this deadly disease. [Funding by NCI U54 CA1433930; California Breast Cancer Research Program; UCLA Jonsson Citation Format: Nalo M. Hamilton, Diana C. Marquez-Garban, Lorena P. Burton, Begonya Comin-Anduix, Alejandro J. Garcia, Jaydutt V. Vadgama. Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-391.

A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings

The imbalance in immune tolerance may cause the variety of reproductive failures. An intravenous immunoglobulin infusion (IVIg) therapy is used to improve the live birth rate in women suffering from recurrent pregnancy loss, recurrent spontaneous abortions and recurrent implantation failures. However, the results of IVIg studies are still inconclusive as IVIg infusion in women suffering from pregnancy loss is sometimes ineffective. One of the mechanisms of action of this treatment is inhibition of B cells differentiation and expansion of Tregs and secretion of interleukin 10. It was proposed that immunomodulatory effects of IVIg may be attributed to tregitopes - self-IgG-derived epitopes present in the structure of immunoglobulins. Similarly to IVIg, tregitopes cause the expansion of Tregs and secretion of antigen-specific effector cytokine response. Here, we studied whether the administration of mouse tregitope 167 and/or 289 can prevent abortions in mouse abortion-prone mouse matings. We revealed that tregitopes reduce the foetal death rate. This may be driven by observed higher pool of peripheral Tregs, increased production of IL-10 by Tregs and Bregs and/or maintaining the tolerogenic phenotype of antigen-presenting cells. We believe that our findings may indicate a potential alternative to IVIg for therapeutic intervention in case of pregnancy failures.

T Cell Immunity Toward Viral- and Tumor-Antigens Is Preserved in MDS Patients

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

Next Generation CRISPR Gene-Edited and Off-the-Shelf Virus-Specific T-Cells for the Immunocompromised Patient

Introduction: A number of Clinical trials have demonstrated the feasibility, safety and efficacy of cell and gene therapy for cancer, autoimmune disorders and infectious disease. Strategies that enhance the function and survival of immune cells are critical for the success of immunotherapy. We have developed a strategy for the ex vivo expansion of off-the-shelf viral-specific T cells (VSRs) from healthy donor buffy coat which have been extremely effective in eradicating refractory cytomegalovirus (CMV), polyomavirus and adenovirus infections in immunocompromised patients. Glucocorticoids commonly used to treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are a common cause of iatrogenically-induced immunosuppression and contribute the risk of life-threatening viral-infections. To render VSTs resistant to the lymphocytotoxic effect of glucocorticoids, we have developed a novel strategy to silence the expression of the glucocorticoid receptor using RNA-guided endonucleases CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) 9 gene editing.. Methods: The technique involves the expansion from donor blood of CMV, BKV or adenoviral-specific T cells using peptide libraries from the immunodominant viral proteins followed by CRISPR knockout of exon 2 of the GR gene on chromosome 5 of the human NR3C1 gene. Cells are electroporated with the RNP (Cas9 plus guide RNA) complex (IDT pre-designed alt-R crispr Cas9 platform) using Neon electroporation and the Amaxa 4-D nucleofector system. Results: GR knockout efficiency in ex vivo expanded virus-specific T cells was consistently > 90%. In vitro experiments confirmed the resistance of VSTs to corticosteroid treatment as assessed by annexin V assay. GR KO VSTs maintained potent antiviral activity as assessed by their ability to proliferate and release effector cytokines in response to viral antigens. Conclusions: CRISPR gene-editing to knock-out the glucocorticoid receptor gene in viral-specific T cells can preserve the activity of VSTs in the presence of corticosteroid-induced immunosuppression. Engineering runs using GMP-compliant Cas9 protein and gRNA are underway in anticipation of a clinical trial. Disclosures Champlin: Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Characterization of Donor-Specific Alloreactive CD4+ and CD8+ Cellular Immune T Cell Responses in the Lung Allograft and Blood in Lung Transplant Recipients

Purpose Lung transplantation remains the only therapeutic option for select patients with end-stage lung diseases, however chronic lung allograft dysfunction (CLAD) significantly limits long-term survival in lung transplant recipients (LTRs). Episodes of acute cellular rejection (ACR) are common and the major risk factor for developing CLAD, however little is known about donor-specific cellular T cell responses, as these have not been previously characterized in LTRs. Methods We used a novel ex vivo flow cytometric assay to assess donor-specific alloimmune responses from LTRs cells in lung allograft resident effector T cells (BAL-derived) and PBMC. Using a 6h in vitro re-stimulation protocol with either irradiated donor cells or donor lysate, we measured the frequencies of effector responses (IFN-γ, TNF-α, the cytotoxic marker CD107a, IL-17a, IL-13, IL-2 and the costimulation surface molecule, CD154) from CD4+ and CD8+ lung resident or blood compartment T cells. Results Overall the predominant alloreactive effector responses were donor-specific CD154 surface expression following in vitro re-stimulation with donor lysate in lung resident CD4+ T cells compared to the PBMC compartment, with minimal to absent expression on CD8+ T cells. Expression of surface CD154 was highly co-expressed with allospecific CD4+ T cells producing the Type-1 cytokines IFN-γ, TNF-α and CD107a suggesting CD154 as a marker for Type-1 effector function, but not Type-2 or Type-17 responses. In fact, donor-specific IL-13 and IL-17 responses were detectable in some patients but at significantly lower frequencies compared to Type-1 effector responses, suggesting a hierarchy of alloeffector immune responses. Comparison between the lung resident T cells and blood T cells revealed consistently increased donor-specific alloreactive frequencies in the lung allograft versus the periphery. Ongoing experiments are assessing the proliferative capacities of donor-specific alloreactive T cell populations in the blood compartment. Conclusion Together, these data indicate donor-specific alloreactive effector CD4+CD154+ lung resident T cells activated via the indirect allorecognition pathway are present in high frequencies in LTRs with histologic evidence or history of ACR and segregate to Type-1 effector cytokine responses.

CD47 Expression in Natural Killer Cells Regulates Homeostasis and Modulates Immune Response to Lymphocytic Choriomeningitis Virus

CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-α. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47−/− mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47−/− NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47−/− mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47−/− NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection.

Myeloid-Derived Suppressor Cells Contribute to Susceptibility to Trypanosoma congolense Infection by Suppressing CD4+ T Cell Proliferation and IFN-γ Production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ-producing CD4+ T cells from the spleen of T. congolense-infected mice. Furthermore, MDSCs from T. congolense-infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1-dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1-dependent manner.

Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients

BackgroundCellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet–deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. MethodsOrthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. ResultsWe show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet–deficient mice. ConclusionsOur results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

A Protective Role for NKG2D–H60a Interaction via Homotypic T Cell Contact in Nonobese Diabetic Autoimmune Diabetes Pathogenesis

The NK group 2 member D (NKG2D) immune receptor is implicated in both human and mouse autoimmune diabetes. However, the significance of NKG2D in diabetes pathogenesis has been unclear due to conflicting reports as to the importance of this receptor in the NOD mouse model. In this study we demonstrate that NKG2D expression affects NOD diabetes development by at least two previously undescribed, and opposing, mechanisms. First, we demonstrate that the NKG2D ligand H60a is induced on activated NOD T cells, and that NKG2D–H60a interaction during CD8+ T cell differentiation into CTLs generally decreases the subsequent CTL effector cytokine response. This corresponds to an increase in diabetes development in NKG2D-deficient compared with wild-type NOD mice under microbiota-depleted conditions. Second, we demonstrate that NKG2D promotes NOD diabetes development through interaction with the microbiota. Together these findings reveal a previously undescribed role for NKG2D ligand expression by activated T cells in CTL development. Further, they demonstrate that NKG2D has both diabetogenic and antidiabetogenic roles in NOD diabetes development.

Radiotherapy in Combination with αOX40 and Actla-4 Immunotherapy Reverses Anergy and Prevents Development of Functional Exhaustion within Tumor-Specific CD8 T Cells

The immunosuppressive tumor microenvironment and chronic T cell stimulation that occurs in the presence of cancer results in CD8 T cell dysfunction that has been difficult to reverse with immunotherapy (IT) alone. This dysfunction can be separated into two categories of anergy and exhaustion. Anergy is a lack of proliferative response to stimulation with cytokine or antigen resulting from inadequate co-stimulation or suboptimal T cell receptor (TCR) stimulation during T cell priming. Functional exhaustion is the inability of T cells to kill or produce effector cytokines in response to stimulation. We hypothesized the addition of ionizing radiotherapy (RT) to IT with agonist αOX40 & blocking αCTLA-4 antibodies would reverse tumor associated CD8 T cell anergy and exhaustion. We utilized an established model of CD8 T cell anergy in which transgenic, OVA-specific, OT-I CD8 T cells are adoptively transferred into centrally tolerant POET-1 mice bearing OVA expressing tumors. The development of anergy/exhaustion was monitored within this T cell population and animals were treated with a single 20 Gy fraction of RT using a SARRP, αOX40/αCTLA-4, or combined IT/RT 21 days after transfer. To assess anergy/exhaustion in endogenous CD8 T cell responses CT26 colon carcinoma bearing BALB/c mice were treated with IT, RT, or combined IT/RT either 10 or 17 days after tumor implantation. The frequency/function of tumor-specific (AH1 or OT-I) CD8 T cells was monitored in blood, lymph node (LN), and tumor infiltrating lymphocytes (TIL) in these models. Reporter Nur77-GFP-OT-I CD8 T cells expressing green fluorescent protein in response to TCR ligation were used in to measure the presence/strength of TCR signaling following IT or RT. We demonstrate OT-I cells become anergic/exhausted and produce few effector cytokines 21 days after adoptive transfer. Combination therapy with IT/RT resulted in increased TCR stimulation (measured by GFP expression) and reversed anergy (measured by proliferation) within OT-I T cells in LN and TIL compared to IT or RT alone. Combination therapy was able to reverse functional exhaustion (measured by cytokine production) within LN but not TIL in this model. In CT26 tumor bearing mice combination IT/RT significantly increased (up to 4-fold) the frequency of tumor-specific endogenous AH1 CD8 TIL and prevented development of functional exhaustion within TIL when compared to IT or RT alone. Combined ablative RT and IT with αOX40/αCTLA-4 results in increased CD8 TCR signaling, reversal of T cell anergy, and can prevent or reverse functional exhaustion. These novel results suggest the addition of RT to IT can effectively reverse tumor-associated T cell dysfunction where IT alone is insufficient and provide rationale for early phase clinical trial design.

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Abstract Here we report a peptide-MHC (pMHC) dodecamer as a “next generation” technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8+ αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4+CD8+ double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.

Off-the-Shelf Natural Killer Cell Immunotherapy for Enhanced Antibody Directed Cellular Cytotoxicity

Natural Killer (NK) cells play a crucial role in immunosurveillance and form a first line of defense against cancer. In comparison to other lymphocytes, NK cells are unique in their capability to elicit tumoricidal responses without the need for antigen presentation or prior sensitization. Clinical data from bone marrow transplant and allogeneic NK immunotherapy suggest that MHC mismatch is advantageous in promoting graft-versus-leukemia without eliciting graft-versus-host, providing evidence that NK cells hold promisa as an allogeneic, universal immunotherapeutic. Further, the anti-tumor effect of many monoclonal antibodies is mediated through binding of the low-affinity Fc receptor CD16 on NK cells, which induces tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC). Thus, NK cells represent a unique source of effector cells that can be combined with monoclonal antibodies, bispecific engagers or chimeric antigen receptors to direct tumor specificity and enhance cytotoxicity. Despite the significant potential of NK cell therapy, current clinical practices are limited by the need for large numbers of healthy NK cells, lack of in vivo persistence, and a burdensome manufacturing strategy that requires donor cell extraction, modulation, expansion and re-introduction per each patient.The ability to generate universally histocompatible and genetically-enhanced NK cells from continuously renewable human induced pluripotent stem cell (hiPSC) lines offers the potential to develop a true "off-the-shelf" cellular immunotherapy. While NK differentiation from hiPSC has been demonstrated, the clonal derivation of engineered hiPSCs to improve effector function has been challenging and the scalability and robustness of the differentiation method has been limited by skewed development towards primitive hematopoiesis and the cumbersome use of embryoid bodies.Here we highlight our "off-the-shelf" NK cell therapy preclinical program by demonstrating robust and highly scalable generation of functionally mature, genetically targeted and universally histocompatible NK cells. This program utilizes our previously described naïve hiPSC platform where we uniquely create clonal lines of precisely engineered, renewable hiPSCs and drive definitive hematopoiesis in a highly scalable manner. Because hiPSC differentiation is lineage directed, minimal cellular contamination is seen, including the lack of T and B cells, in the final product. Through precise genetic engineering of naïve hiPSC lines, we have engineered HLA-class I deficient NK cells uniformly expressing a high affinity, non-cleavable version of the Fc receptor CD16 (NcHaCD16-NK). The hiPSC-derived NcHaCD16-NKs display markers of maturity, including CD16, KIR, NCRs, and CD94. When compared to conventional cord blood and peripheral blood sourced NK cells, NcHaCD16-NKs exhibit superior cytotoxicity and production of effector cytokines in response to both solid and liquid tumor cell challenge in vitro. NcHaCD16-NKs exhibit augmented cytokine response following Fc-mediated stimulation, demonstrating function competence of the engineered CD16 construct. Because surface expression of CD16 is resistant to activation-induced shedding, NcHaCD16-NKs continuously maintain enhanced ADCC while retaining the capacity for general cytotoxicity. Importantly, the hiPSC-derived hematopoietic cells can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Preliminary data also shows NcHaCD16-NKs elicit preferred specificity for cancer stem cells as defined by expression of ALDH1 and surface markers such as CD24. In conclusion, the outlined preclinical data demonstrate the potential therapeutic utility of NK cells developed via precision genetic engineering of a renewable, scalable hiPSC platform, and highlights the therapeutic value of NcHaCD16-NKs as an ideal ADCC-mediated "off-the-shelf" NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity and preclinical efficacy. DisclosuresBjordahl:Fate Therapeutics, Inc: Employment. Clarke:Fate Therapeutics: Employment. Gaidarova:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics, Inc.: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics, Inc.: Employment. Kim:Fate Therapeutics, Inc.: Employment. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Abstract B124: Roles for Tnfsf11 expression by group 3 innate lymphoid cells

Abstract Innate lymphoid cells (ILC) are important sources of effector cytokines during homeostasis and diverse inflammatory conditions. A specialized group of ILC, the Group 3 ILC (ILC3), produces IL-22 and IL-17A, and contributes to maintenance of the gut epithelial barrier and the host response to extracellular microbes. ILC3s express Tnfsf11, a type II receptor required for osteoclastogenesis. The functions of Tnfsf11 in ILC3s are not fully understood. Here we show that Tnfsf11-deficient ILC3s are hyperproliferative and express altered cell surface markers at steady state. Additionally, Tnfsf11-deficient ILC3s produce increased amounts of effector cytokines in response to the activating cytokine IL-23. Although mature osteoclast-, lymph node-, and M cell-development are dysfunctional in Tnfsf11−/− mice, Tnfsf11 expression is not required in ILC3s for these developmental pathways. Thus, Tnfsf11 regulates ILC3 homeostasis in the gut. Citation Format: Jennifer K. Bando, Christina Song, Susan Gilfillan, Marco Colonna. Roles for Tnfsf11 expression by group 3 innate lymphoid 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 B124.

NKG2D-H60a interaction via homotypic T cell contact delays NOD diabetes development.

Abstract Type I diabetes is an autoimmune disorder mediated by T cells that destroy the insulin-secreting β-islet cells in the pancreas. Both human and murine studies implicate the immune receptor natural killer group 2 member D (NKG2D) in enhancing type 1 diabetes development. However, a mechanism for NKG2D influence in type 1 diabetes development is not clear. Utilizing NKG2D-deficient non-obese diabetic (NOD) mice, we demonstrate that NKG2D expression affects diabetes development via multiple mechanisms in multiple anatomical locations, with both pro- and anti-diabetic effects. First, we found NKG2D influences NOD diabetes development via alteration in the microbiota composition. When we eliminated this microbiome effect, NKG2D-deficient mice had enhanced diabetes development, demonstrating a protective role for NKG2D in this disease. Our in vitro studies suggest this protection is mediated via a previously undescribed interaction between NKG2D and the NKG2D ligand H60a, both expressed by activated NOD CD8+ T cells, during diabetogenic cytotoxic T cell (CTL) generation. We show that during CD8+ T cell differentiation into CTLs, NKG2D-H60a interaction generally decreases the subsequent CTL effector cytokine response. Taken together, these findings demonstrate there is a protective role for NKG2D during diabetes development that is likely mediated by NKG2D-ligand interaction during diabetogenic CTL differentiation.

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4(+)and CD8(+)αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4(+)CD8(+)double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.