Oligoclonal T Cells Transiently Expand and Express Tim-3 and PD-1 Following Anti-CD19 CAR T Cell Therapy: A Case Report.

Clinical trials of chimeric antigen receptor (CAR) T cells in hematologic malignancy associate remissions with two profiles of CAR T cell proliferation kinetics, which differ based upon costimulatory domain. Additional T cell intrinsic factors that influence or predict clinical response remain unclear. To address this gap, we report the case of a 68-year-old woman with refractory/relapsed diffuse large B cell lymphoma (DLBCL), treated with tisagenlecleucel (anti-CD19), with a CD137 costimulatory domain (4-1BB) on an investigational new drug application (#16944). For two months post-infusion, the patient experienced dramatic regression of subcutaneous nodules of DLBCL. Unfortunately, her CAR T exhibited kinetics unassociated with remission, and she died of DLBCL-related sequelae. Serial phenotypic analysis of peripheral blood alongside sequencing of the ß-peptide variable region of the T cell receptor (TCRß) revealed distinct waves of oligoclonal T cell expansion with dynamic expression of immune checkpoint molecules. One week prior to CAR T cell contraction, T cell immunoglobulin mucin domain 3 (Tim-3) and programmed cell death protein 1 (PD-1) exhibited peak expressions on both the CD8 T cell (Tim-3 ≈ 50%; PD-1 ≈ 17%) and CAR T cell subsets (Tim-3 ≈ 78%; PD-1 ≈ 40%). These correlative observations draw attention to Tim-3 and PD-1 signaling pathways in context of CAR T cell exhaustion.

Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell's multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells' ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the "stemness" of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.

CHANGE-seq reveals genetic and epigenetic effects on CRISPR-Cas9 genome-wide activity.

Current methods can illuminate the genome-wide activity of CRISPR-Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe 'circularization for high-throughput analysis of nuclease genome-wide effects by sequencing' (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 single guide RNA targets across 13 therapeutically relevant loci in human primary T cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers and transcribed regions. Finally, CHANGE-seq analysis of six targets across eight individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive and scalable approach to understanding the specificity of genome editors.

Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma.

High grade gliomas (HGG) comprise a heterogeneous group of brain malignancies with dismal prognosis. Current standard-of-care includes radiation, chemotherapy, and surgical resection when possible. Despite advances in each of these treatment modalities, survival rates for pediatric and adult HGG patients has remained largely unchanged over the course of several years. This is in stark contrast to the significant survival increases seen recently for a variety of hematological and other solid malignancies. The introduction and widespread use of immunotherapies have contributed significantly to these survival increases, and as such these therapies have been explored for use in the treatment of HGG. In particular, chimeric antigen receptor (CAR) T cell therapy has shown promise in clinical trials in HGG patients. However, unlike the tremendous success CAR T cell therapy has seen in B cell leukemia and lymphoma treatment, the success in HGG patients has been modest at best. This is largely due to the unique tumor microenvironment in the central nervous system, difficulty in accessing the tumor site, and heterogeneity in target antigen expression. The results of these features are poor CAR T cell proliferation, poor persistence, suboptimal cytokine secretion, and the emergence of antigen-loss tumor variants. These issues have called for the development of "next generation" CAR T cells designed to circumvent the barriers that have limited the success of current CAR T cell technologies in HGG treatment. Rapid advancements in gene editing technologies have provided several avenues for CAR T cell modification to enhance their efficacy. Among these are cytokine overexpression, gene knock-out and knock-in, targeting of multiple antigens simultaneously, and precise control of CAR expression and signaling. These "next generation" CAR T cells have shown promising results in pre-clinical models and may be the key to harnessing the full potential of CAR T cells in the treatment of HGG.

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists.

Adoptive therapy with ex vivo-expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo-expanded cancer-specific T cells.

Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity.

Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease._Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/-vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs._Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs.

Administration of a vasoactive intestinal peptide antagonist enhances the autologous anti-leukemia T cell response in murine models of acute leukemia.

Vasoactive intestinal peptide (VIP) is a neuroendocrine peptide hormone that has potent anti-inflammatory activities. VIP signaling through its receptor VPAC1 on T cells leads to reduced proliferation and a reduction in pro-inflammatory cytokine secretion. We report here that inhibition of the VIP pathway with a peptide antagonist significantly enhances a T-cell-dependent, autologous anti-leukemia response in murine models of acute myeloid leukemia and T lymphoblastic leukemia. Subcutaneous administration of the VIP antagonist, VIPhyb, resulted in reduced tumor burden and significantly enhanced survival (30-50% survival) over vehicle-treated controls (0-20% survival). The T cells in mice treated with VIPhyb expressed lower levels of the co-inhibitory PD-1 and secreted higher levels of IFNγ. Furthermore, T cells from VIPhyb-treated survivors were protective against C1498 following adoptive transfer. These data highlight the potential for the VIP pathway as a novel target for immunomodulation in settings of hematological malignancies.

Modulation of Immune Checkpoints and Graft-versus-Leukemia in Allogeneic Transplants by Antagonizing Vasoactive Intestinal Peptide Signaling.

The goal of allogeneic bone marrow transplantation (allo-BMT) is elimination of leukemia cells through the graft-versus-leukemia (GvL) activity of donor cells, while limiting graft-versus-host disease (GvHD). Immune checkpoint pathways regulate GvL and GvHD activities, but blocking antibodies or genetic inactivation of these pathways can cause lethal GVHD. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide that regulates coinhibitory pathways; its role in allo-BMT has not been studied. We found VIP transiently expressed in donor NK, NK-T, dendritic cells, and T cells after allo transplant, as well as host leukocytes. A peptide antagonist of VIP signaling (VIPhyb) increased T-cell proliferation in vitro and reduced IL10 expression in donor T cells. Treatment of allo-BMT recipients with VIPhyb, or transplanting donor grafts lacking VIP (VIP-KO), activated donor T-cells in lymphoid organs, reduced T-cell homing to GvHD target organs, and enhanced GvL without increasing GvHD in multiple allo-BMT models. Genetic or ex vivo depletion of donor NK cells or CD8+ T cells from allografts abrogated the VIPhyb-enhanced GvL activity. VIPhyb treatment led to downregulation of PD-1 and PD-L1 expression on donor immune cells, increased effector molecule expression, and expanded oligoclonal CD8+ T cells that protected secondary allo transplant recipients from leukemia. Blocking VIP signaling thus represents a novel pharmacologic approach to separate GvL from GvHD and enhance adaptive T-cell responses to leukemia-associated antigens in allo-BMT. Cancer Res; 76(23); 6802-15. ©2016 AACR.