Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

The recently described generation of a highly defined population of dendritic cells which express perforin and granzyme A (termed "perf-DCs") and their ability to selectively delete cognate CD8+ T cell has raised the possibility that these cells play a role in the maintenance of peripheral tolerance. Using bone marrow transplantation, we generated mice selectively lacking perforin expressing dendritic cells. These mice progressively gain weight and exhibit features resembling metabolic syndrome as well as an enhanced susceptibility to autoimmunity induction. Interestingly, these pathological phenotypes were reversed upon treatment with CD4/CD8 neutralizing antibodies. Thus, it appears that this rare subpopulation of dendritic cells (perf-DCs) displays a major regulatory role in adipose tissue inflammatory processes and in autoimmunity.

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The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

R.S.K and B.N.L contributed equally to this study. The induction of tolerance towards pancreas autoantigens is a promising approach in the treatment of type 1 diabetes mellitus (T1DM). This goal can be partially attained by immuno-ablation followed by autologous HSCT which mitigates the immune reaction leading to diabetes, at least temporarily, if performed soon after diagnosis. However, most T1DM patients transplanted with autologous HSCT eventually relapse (E Snarski et al., Bone marrow Transplantation; 2016; 51, 398-402). Thus, developing methodologies for allogeneic HSCT, to provide a durable non-autoimmune TCR repertoire, could be a promising treatment approach provided that such protocols can safely achieve donor type chimerism. Accordingly, transplantation of T cell-depleted allogeneic HSCT (TD-HSCT) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option. However, overcoming rejection after reduced conditioning in T1DM represents a major challenge. We previously demonstrated in wild type mice that rejection of TD-HSCT can be prevented using donor-derived veto cells. Here, we show proof of concept of the safety and efficacy of veto cell mediated non-myeloablative mismatched allogeneic TD-HSCT, in the established NOD mouse T1DM model. Veto activity, first defined by Miller (Miller, R. G; Nature; 1980; 287; 544-54), is based on the ability of specific cell populations to attack host CTL-precursors (CTLp) that are directed against the antigens presented by the veto cells themselves. This response spares cells that are not targeted against the veto cells, including those recognizing pathogens. Among different veto cell populations described in the literature, central memory CD8 T cells exhibit the most robust veto activity upon transplantation, but are also endowed with marked GvH activity. We overcame GvH reactivity by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, under culture conditions favoring expression of the central memory phenotype. Such anti-3rd party central memory CD8 T cells (Tcm) are endowed with marked veto activity, while effectively depleted of GVH reactivity in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin. Hematol. 2019; 56(3): 173-182). In this study, Tcm veto cells were generated from splenocytes obtained from Balb/c donors (H2d) cultured against irradiated third-party splenocytes (FVB; H2q), under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators under these conditions leads to selective 'death by neglect' of bystander anti-host T cell clones that could mediate GvHD; such cells are further diluted out by subsequent expansion of anti-3rd party T clones during further culture in the presence of IL15. In addition to causing selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be crucial for robust veto activity in vivo. To evaluate the safety and efficacy of such a transplant for diabetes therapy, 8 week old NOD mice (before diabetes onset) were treated with 4.5 Gy TBI conditioning at day -1, anti-3rd party veto Tcm and megadose nude bone marrow (NuBM) on day 0, and Rapamycin treatment from day -1 to day +4 (Scheme 1). Controls were untreated or received conditioning with no transplant. In four independent experiments (n= 35 NOD mice in the transplanted group), high chimerism from 83.5% to 99.6 % was found in all transplanted mice at 6 months post-transplant. Notably, with a follow-up of 200 days, 72.4% mice in the untreated group, and 96% in the conditioned group died of diabetes, while only 8.5% diabetes-associated mortality (Fig.1A) and no GvHD or other transplant-related mortality was observed in the transplanted mice (Fig.1B). Our results demonstrate a proof of concept for the safety and efficacy of non-myeloablative allogeneic TD-HSCT in type 1 diabetes. A clinical protocol testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is commencing at MD Anderson Cancer Center. If successful, our results support further extension of this platform to autoimmune diabetes and other autoimmune diseases. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.