CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Acute myeloid leukemia (AML), the most common adult acute leukemia in the United States, has the poorest survival rate, with 26% of patients surviving 5 years. Adoptive immunotherapy with T cells genetically modified to recognize tumors is a promising and evolving treatment option. However, antitumor activity, particularly in the context of progressive leukemia, can be dampened both by limited costimulation and triggering of immunoregulatory checkpoints that attenuate T-cell responses. Expression of CD200 (OX2), a negative regulator of T-cell function that binds CD200 receptor (CD200R), is commonly increased in leukemia and other malignancies and is associated with poor prognosis in leukemia patients. To appropriate and redirect the inhibitory effects of CD200R signaling on transferred CD8 T cells, we engineered CD200R immunomodulatory fusion proteins (IFPs) with the cytoplasmic tail replaced by the signaling domain of the costimulatory receptor, CD28. An analysis of a panel of CD200R-CD28 IFP constructs revealed that the most effective costimulation was achieved in IFPs containing a dimerizing motif and a predicted tumor-T-cell distance that facilitates localization to the immunological synapse. T cells transduced with the optimized CD200R-CD28 IFPs exhibited enhanced proliferation and effector function in response to CD200 leukemic cells in vitro. In adoptive therapy of disseminated leukemia, CD200R-CD28-transduced leukemia-specific CD8 T cells eradicated otherwise lethal disease more efficiently than wild-type cells and bypassed the requirement for interleukin-2 administration to sustain in vivo activity. The transduction of human primary T cells with the equivalent human IFPs increased proliferation and cytokine production in response to CD200 leukemia cells, supporting clinical translation. This trial was registered at www.clinicaltrials.gov as #NCT01640301.

Adoptive T cell therapy (ACT) with genetically modified T cells has shown impressive results against some hematologic cancers, but efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs). For example, Fas ligand is commonly overexpressed in TMEs and induces apoptosis in tumor-infiltrating, Fas receptor–positive lymphocytes. We engineered immunomodulatory fusion proteins (IFPs) to enhance ACT efficacy, combining an inhibitory receptor ectodomain with a costimulatory endodomain to convert negative into positive signals. We developed a Fas-4-1BB IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function, and altered metabolism in vitro. In vivo, Fas-4-1BB ACT eradicated leukemia and significantly improved survival in the aggressive KPC pancreatic cancer model. Fas-4-1BB IFP expression also enhanced primary human T cell function in vitro. Thus, Fas-4-1BB IFP expression is a novel strategy to improve multiple T cell functions and enhance ACT against solid tumors and hematologic malignancies.

Adoptive T-cell therapy using high-affinity T-cell receptors (TCRs) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and immunohistochemical analysis of human HGSOC tumors and disseminated mouse ID8 VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a highaffinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8 VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2 + HGSOC lines. Immunohistochemistry and gene expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell

The ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), is a novel inducer of adaptive Tregs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent AHR ligand, induces adaptive CD4+CD25+ Tregs during an acute graft-versus-host (GvH) response and prevents the generation of allospecific cytotoxic T lymphocytes. TCDD also suppresses the induction of experimental autoimmune encephalitis in association with an expanded population of Foxp3+ Tregs. In this study, we show that chronic treatment of NOD mice with TCDD potently suppresses the development of autoimmune Type 1 diabetes in parallel with greatly reduced pancreatic islet insulitis and an expanded population of CD4+CD25+Foxp3+ cells in the pancreatic lymph nodes. When treatment with TCDD was terminated after 15 weeks (23 weeks of age), mice developed diabetes over the next 8 weeks in association with lower numbers of Tregs and decreased activation of AHR. Analysis of the expression levels of several genes associated with inflammation, T-cell activation and/or Treg function in pancreatic lymph node cells failed to reveal any differences associated with TCDD treatment. Taken together, the data suggest that AHR activation by TCDD-like ligands may represent a novel avenue for treatment of immune-mediated diseases.

No abstract

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.