Maria-Luisa Alegre

The role of conventional type 1 DCs (cDC1s) in tolerance induction to solid organ allografts is unknown and important for strategies that seek to prolong allograft viability. Using a murine model deficient in cDC1s, we report cDC1s are required for donor antigen and costimulation blockade (DST + CoB) tolerance induction and survival of cardiac allografts. cDC1 deficiency led to decreases in CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells within allograft and spleen tissue of transplant recipients, and this was found to be antigen specific. Donor antigen stimulation induced TGF-β1 expression in both in vivo cDC1s and in vitro Flt3L-derived cDC1s. Genetic deletion of TGF- β 1 in cDC1s prevented induction of antigen-specific CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells and was associated with cardiac allograft rejection. In parallel, single-cell RNA sequencing and metabolic analysis revealed upregulation of cDC1 mitochondrial metabolic signatures after in vivo exposure to DST + CoB. Genetic inactivation of cDC1 mitochondrial metabolism reduced expression of cDC1 TGF-β1, decreased antigen-specific Treg populations, and impaired allograft tolerance. Taken together, our findings implicate cDC1s in strategies to preserve solid organ allografts and also implicate mitochondrial metabolism of cDC1s as a molecular mechanism to enhance the generation of antigen-specific CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells through TGF-β1.

Current asthma treatments manage disease symptoms but fail to address the underlying cause of allergic disease. Allergen immunotherapy holds the promise for durable disease control by establishing allergen-specific tolerance through repeated introduction of native allergens; however, its efficacy can be limited by long interventions, reactions upon administration, and poor patient compliance. Here, we developed a rapid, safe, and effective liver-targeted allergen immunotherapy (LIT) to provide long-term disease control. We developed LIT tolerogens from native respiratory allergens, which induced antigen-specific regulatory T (T _reg ) cells in vivo with only two interventions. Synthetic mannosylation of native allergens prevented antibody-mediated recognition and subsequent life-threatening anaphylaxis upon administration. Protein engineering prevented sensitization events that occurred because of the proteolytic activity of native respiratory allergens, which limit the effectiveness of allergen immunotherapy. In preclinical models of allergic asthma, LIT ameliorated clinical, pathological, and serological features, and protection was dependent on antigen-specific T _reg cells. In sensitized mice, LIT provided a year-long control of allergic asthma symptoms in the absence of additional intervention. Last, LIT induced antigen-specific T _reg cells against Der p 1, a major protein in the clinically relevant house dust mite (HDM) respiratory allergen. In mice with established HDM allergy, LIT was well tolerated and provided allergic symptom relief. Together, our data provide a proof of concept that LIT with synthetically mannosylated tolerogens provides a rapid, safe, and effective approach to allergen immunotherapy and holds promise for durable control of allergic asthma.

Successful allograft-specific tolerance induction would eliminate the need for daily immunosuppression and improve posttransplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific chimeric antigen receptors (CAR Tregs) is a promising strategy but, as monotherapy, cannot prolong survival with allografts with multiple MHC mismatches. Using an HLA-A2–transgenic haplo-mismatched heart transplantation model in immunocompetent C57BL/6 recipients, we showed that HLA-A2–specific CAR (A2.CAR) Tregs were able to synergize with a low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited donor-specific T cell, B cell, and antibody responses and promoted a substantial increase in endogenous FOXP3 ⁺ Tregs with indirect donor specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FOXP3 ^– A2.CAR T cells preferentially accumulated in rejecting allografts, and endogenous donor-specific responses were not controlled. This study therefore provides evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity toward allograft tolerance.

Alloreactive memory, unlike naive, CD8 ⁺ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semiallogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8 ⁺ T cells (T _FGS ) toward hypofunction. Female C57BL/6 mice harboring memory CD8 ⁺ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable with naive controls. Postpartum CD8 ⁺ T _FGS from skin-sensitized dams upregulated expression of T cell exhaustion (T _EX ) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical T _EX genes in postpartum memory T _FGS and revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory T _FGS , whereas minimal epigenetic modifications were observed in naive T _FGS . Finally, postpartum memory T _FGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared with memory T _FGS . These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8 ⁺ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.