Islet allografts expressing a PD-L1 and IDO fusion protein evade immune rejection and reverse preexisting diabetes in immunocompetent mice without systemic immunosuppression.

Allogeneic islet transplantation is a promising experimental therapy for poorly controlled diabetes. Despite pharmacological immunosuppression, long-term islet engraftment remains elusive. Here, we designed a synthetic fusion transgene coupling PD-L1 and indoleamine dioxygenase [hereafter PIDO] whose constitutive expression prevents immune destruction of genetically engineered islet allograft transplanted in immunocompetent mice. PIDO expressing murine islets maintain robust dynamic insulin secretion in vitro and when transplanted in allogeneic hyperglycemic murine recipients reverse pre-existing streptozotocin-induced and autoimmune diabetes in the absence of pharmacological immunosuppression for more than 50 and 8 weeks, respectively, and is dependent on host CD4 competence. Additionally, PIDO expression in allografts preserves endocrine functional viability of islets and promotes a localized tolerogenic milieu characterized by the suppression of host CD8 T cell and phagocyte recruitment and accumulation of FOXP3+ Tregs. Furthermore, in the canine model of xenogeneic islet transplantation, muscle implanted PIDO-expressing porcine islets displayed physiological glucose-responsive insulin secretion competency in euglycemic recipient for up to 20 weeks. In conclusion, the PIDO transgenic technology enables host CD4+ T cell-modulated immune evasiveness and long-term functional viability of islet allo- and xenografts in immune-competent recipients without the need for pharmacological immune suppression and would allow for improved outcomes for tissue transplantation.

Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination.

The unmet medical need of patients with multiple sclerosis (MS) is the inexorable loss of CNS myelin and latterly neurons leading to permanent neurologic disability. Solicitation of endogenous oligodendrocytes progenitor cells, the precursor of oligodendrocytes, to remyelinate axons may abort the onset of disability. In female mice with experimental autoimmune encephalomyelitis (EAE), a murine model of MS, adoptive transfer of IL-10+ regulatory B cells (Bregs) has been shown to reverse EAE by promoting the expansion of peripheral and CNS-infiltrating IL-10+ T cells. Here, we examined whether Bregs treatment and its bystander effect on regulatory T cells are associated with CNS repair as reflected by oligodendrogenesis and remyelination. We have found that transfusion of Bregs reverses established clinical EAE and that clinical improvement is associated with a significant increase in spinal cord remyelination as reflected by g-ratio analysis within the thoracic and lumbar spine. We further observed in the spinal cords of EAE Bregs-treated mice that CNS resident CD11b/CD45intLy6C- microglia, and infiltrating CD11b+/CD45high monocytes/macrophages content reverts to normal and polarize to a M2-like CD206+ phenotype. Concurrently, there was a substantial increase in neo-oligodendrogenesis as manifest by an increase in CD45-/low CNS cells expressing A2B5, an early marker in oligodendrocytes progenitor cell differentiation as well as GalC+/O1+ premyelinating and myelin basic protein+/myelin oligodendrocyte glycoprotein+ mature oligodendrocytes with reciprocal downregulation of paired related homeobox protein 1. These results demonstrate that the clinical benefit of Bregs is associated with normalization of CNS immune milieu and concurrent activation of oligodendrocyte progenitor cells with subsequent remyelination.SIGNIFICANCE STATEMENT In multiple sclerosis patients, demyelination progresses with aging and disease course, leading to irreversible disability. In this study, we have discovered, using a mouse model of multiple sclerosis, that the transfusion of autologous regulatory B cells (Bregs) is able to ameliorate, cure, and sustain the durable remission of the disease. We show that the adoptive transfer of Bregs dramatically decreased the frequency of myeloid-derived cells, both infiltrating monocytes/macrophages and resident microglia, and converted their phenotype to an immunosuppressive-like phenotype. Moreover, we showed that CNS oligodendrocyte progenitor cells are activated following Bregs treatment and differentiate into myelinating oligodendrocytes, which results in neo-oligodendrogenesis and remyelination of spinal cords.

T-cells expressing a chimeric-PD1-Dap10-CD3zeta receptor reduce tumour burden in multiple murine syngeneic models of solid cancer.

Adoptive transfer of T-cells is a promising therapy for many cancers. To enhance tumour recognition by T-cells, chimeric antigen receptors (CARs) consisting of signalling domains fused to receptors that recognize tumour-associated antigens can be expressed in T-cells. While CAR T-cells have shown clinical success for treating haematopoietic malignancies, using CAR T-cells to treat solid tumours remains a challenge. We developed a chimeric PD1 (chPD1) receptor that recognizes the ligands for the PD1 receptor that are expressed on many types of solid cancer. To determine if this novel CAR could target a wide variety of tumour types, the anti-tumour efficacy of chPD1 T-cells against syngeneic murine models of melanoma, renal, pancreatic, liver, colon, breast, prostate and bladder cancer was measured. Of the 14 cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T-cells. ChPD1 T-cells lysed the tumour cells and secreted pro-inflammatory cytokines [interferon (IFN)γ, tumour necrosis factor (TNF)α, interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-17 and IL-21], but did not secrete the anti-inflammatory cytokine IL-10. Furthermore, T-cells expressing chPD1 receptors reduced an established tumour burden and led to long-term tumour-free survival in all types of solid tumours tested. ChPD1 T-cells did not survive longer than 14 days in vivo; however, treatment with chPD1 T-cells induced protective host anti-tumour memory responses in tumour-bearing mice. Therefore, adoptive transfer of chPD1 T-cells could be a novel therapeutic strategy to treat multiple types of solid cancer.

Inclusion of Dap10 or 4-1BB costimulation domains in the chPD1 receptor enhances anti-tumor efficacy of T cells in murine models of lymphoma and melanoma.

Chimeric antigen receptors (CAR) utilize costimulatory domains to enhance anti-tumor efficacy. However, it is unclear which costimulatory domain is preferable. Therefore, the intracellular domains of CD28, Dap10, 41BB, GITR, ICOS, or OX40 were compared in a murine chimeric PD1 (chPD1) receptor that targets tumor-associated PD1 ligands. Upon antigen restimulation, T cells expressing chPD1-CD28 receptors had reduced lytic capacity. While most of the chPD1 T cell receptors secreted pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1-Dap10 did not secrete IL-10. Furthermore, chPD1-Dap10 and -41BB receptors induced a memory precursor phenotype, had enhanced persistence in vivo, and superior therapeutic efficacy in murine models of T cell lymphoma and melanoma compared to chPD1-CD28 or chPD1-GITR expressing T cells. Therefore, each costimulatory domain induces differential effects in CAR-expressing T cells and inclusion of Dap10 or 4-1BB costimulatory domains may induce a preferential cytokine profile and differentiation for cancer therapy.

Adoptive transfer of murine T cells expressing a chimeric-PD1-Dap10 receptor as an immunotherapy for lymphoma.

Adoptive transfer of T cells is a promising cancer therapy and expression of chimeric antigen receptors can enhance tumour recognition and T-cell effector functions. The programmed death protein 1 (PD1) receptor is a prospective target for a chimeric antigen receptor because PD1 ligands are expressed on many cancer types, including lymphoma. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 extracellular domain fused to the cytoplasmic domain of CD3ζ. Additionally, chimeric antigen receptor therapies use various co-stimulatory domains to enhance efficacy. Hence, the inclusion of a Dap10 or CD28 co-stimulatory domain in the chPD1 receptor was compared to determine which domain induced optimal anti-tumour immunity in a mouse model of lymphoma. The chPD1 T cells secreted pro-inflammatory cytokines and lysed RMA lymphoma cells. Adoptive transfer of chPD1 T cells significantly reduced established tumours and led to tumour-free survival in lymphoma-bearing mice. When comparing chPD1 receptors containing a Dap10 or CD28 domain, both receptors induced secretion of pro-inflammatory cytokines; however, chPD1-CD28 T cells also secreted anti-inflammatory cytokines whereas chPD1-Dap10 T cells did not. Additionally, chPD1-Dap10 induced a central memory T-cell phenotype compared with chPD1-CD28, which induced an effector memory phenotype. The chPD1-Dap10 T cells also had enhanced in vivo persistence and anti-tumour efficacy compared with chPD1-CD28 T cells. Therefore, adoptive transfer of chPD1 T cells could be a novel therapy for lymphoma and inclusion of the Dap10 co-stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T-cell differentiation phenotype for anti-tumour therapies.

CCL2 and CXCL12 Derived from Mesenchymal Stromal Cells Cooperatively Polarize IL-10+ Tissue Macrophages to Mitigate Gut Injury.

Mesenchymal stromal cell (MSC)-based therapy for inflammatory diseases involves paracrine and efferocytotic activation of immunosuppressive interleukin-10+ (IL-10+) macrophages. The paracrine pathway for MSC-mediated IL-10+ macrophage functionality and response to tissue injury is not fully understood. In our present study, clodronate pre-treatment of colitic mice confirms the essential role of endogenous macrophages in bone-marrow-derived MSC (BM-MSC)-mediated clinical rescue of dextran sulfate sodium (DSS)-induced colitis. We identify that BM-MSC-secreted chemokine ligand 2 (CCL2) and C-X-C motif chemokine 12 (CXCL12) cooperate as a heterodimer to upregulate IL-10 expression in CCR2+ macrophages in vitro and that CCL2 expression by MSC is required for IL-10+ polarization of intestinal and peritoneal resident macrophages in vivo. We observe that tissue macrophage IL-10 polarization in vivo is widespread involving extra-intestinal tissues and secondarily leads to bystander IL-10 expression in intestine-resident B and T cells. In conclusion, the BM-MSC-derived chemokine interactome dictates an IL-10+-macrophage-amplified anti-inflammatory response in toxic colitis.