Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Regulatory CD8 T cells that recognize Qa-1 expressed by CD4 T-helper cells inhibit rejection of heart allografts.

Induction of longstanding immunologic tolerance is essential for survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.

mTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.

Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases-due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

Increased Dopamine Type 2 Gene Expression in the Dorsal Striatum in Individuals With Autism Spectrum Disorder Suggests Alterations in Indirect Pathway Signaling and Circuitry.

Autism spectrum disorder (ASD) is behaviorally defined and diagnosed by delayed and/or impeded language, stereotyped repetitive behaviors, and difficulties with social interactions. Additionally, there are disruptions in motor processing, which includes the intent to execute movements, interrupted/inhibited action chain sequences, impaired execution of speech, and repetitive motor behaviors. Cortical loops through basal ganglia (BG) structures are known to play critical roles in the typical functioning of these actions. Specifically, corticostriate projections to the dorsal striatum (caudate and putamen) convey abundant input from motor, cognitive and limbic cortices and subsequently project to other BG structures. Excitatory dopamine (DA) type 1 receptors are predominantly expressed on GABAergic medium spiny neurons (MSNs) in the dorsal striatum as part of the "direct pathway" to GPi and SNpr whereas inhibitory DA type 2 receptors are predominantly expressed on MSNs that primarily project to GPe. This study aimed to better understand how this circuitry may be altered in ASD, especially concerning the neurochemical modulation of GABAergic MSNs within the two major BG pathways. We utilized two classical methods to analyze the postmortem BG in ASD in comparison to neurotypical cases: ligand binding autoradiography to quantify densities of GABA-A, GABA-B, 5-HT2, and DA type 1 and 2 receptors and in situ hybridization histochemistry (ISHH) to quantify mRNA for D1, D2 receptors and three key GABAergic subunits (α1, ß2, and γ2), as well as the GABA synthesizing enzymes (GAD65/67). Results demonstrated significant increases in D2 mRNA within MSNs in both the caudate and putamen, which was further verified by proenkephalin mRNA that is co-expressed with the D2 receptor in the indirect pathway MSNs. In contrast, all other GABAergic, serotonergic and dopaminergic markers in the dorsal striatum had comparable labeling densities. These results indicate alterations in the indirect pathway of the BG, with possible implications for the execution of competing motor programs and E/I imbalance in the direct/indirect motor feedback pathways through thalamic and motor cortical areas. Results also provide insights regarding the efficacy of FDA-approved drugs used to treat individuals with ASD acting on specific DA and 5-HT receptor subtypes.

Regulatory T cells engineered with TCR signaling-responsive IL-2 nanogels suppress alloimmunity in sites of antigen encounter.

Adoptive cell transfer of ex vivo expanded regulatory T cells (Tregs) has shown immense potential in animal models of auto- and alloimmunity. However, the effective translation of such Treg therapies to the clinic has been slow. Because Treg homeostasis is known to require continuous T cell receptor (TCR) ligation and exogenous interleukin-2 (IL-2), some investigators have explored the use of low-dose IL-2 injections to increase endogenous Treg responses. Systemic IL-2 immunotherapy, however, can also lead to the activation of cytotoxic T lymphocytes and natural killer cells, causing adverse therapeutic outcomes. Here, we describe a drug delivery platform, which can be engineered to autostimulate Tregs with IL-2 in response to TCR-dependent activation, and thus activate these cells in sites of antigen encounter. To this end, protein nanogels (NGs) were synthesized with cleavable bis(N-hydroxysuccinimide) cross-linkers and IL-2/Fc fusion (IL-2) proteins to form particles that release IL-2 under reducing conditions, as found at the surface of T cells receiving stimulation through the TCR. Tregs surface-conjugated with IL-2 NGs were found to have preferential, allograft-protective effects relative to unmodified Tregs or Tregs stimulated with systemic IL-2. We demonstrate that murine and human NG-modified Tregs carrying an IL-2 cargo perform better than conventional Tregs in suppressing alloimmunity in murine and humanized mouse allotransplantation models. In all, the technology presented in this study has the potential to improve Treg transfer therapy by enabling the regulated spatiotemporal provision of IL-2 to antigen-primed Tregs.