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Background: A number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the 'Cancer Immunity Cycle'. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance., Methods: The antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs., Results: The addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs., Conclusion: These data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates., Competing Interests: Competing interests: ES and AN are founders of Nouscom. JZ and DHC are current or past employees and shareholders of Nektar Therapeutics. The remaining authors are employees of Nouscom., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

FOXP3(+) regulatory T (Treg) cells enforce immune self-tolerance and homeostasis, and variation in some aspects of Treg function may contribute to human autoimmune diseases. Here, we analyzed population-level Treg variability by performing genome-wide expression profiling of CD4(+) Treg and conventional CD4(+) T (Tconv) cells from 168 donors, healthy or with established type-1 diabetes (T1D) or type-2 diabetes (T2D), in relation to genetic and immunologic screening. There was a range of variability in Treg signature transcripts, some almost invariant, others more variable, with more extensive variability for genes that control effector function (ENTPD1, FCRL1) than for lineage-specification factors like FOXP3 or IKZF2. Network analysis of Treg signature genes identified coregulated clusters that respond similarly to genetic and environmental variation in Treg and Tconv cells, denoting qualitative differences in otherwise shared regulatory circuits whereas other clusters are coregulated in Treg, but not Tconv, cells, suggesting Treg-specific regulation of genes like CTLA4 or DUSP4. Dense genotyping identified 110 local genetic variants (cis-expression quantitative trait loci), some of which are specifically active in Treg, but not Tconv, cells. The Treg signature became sharper with age and with increasing body-mass index, suggesting a tuning of Treg function with repertoire selection and/or chronic inflammation. Some Treg signature transcripts correlated with FOXP3 mRNA and/or protein, suggesting transcriptional or posttranslational regulatory relationships. Although no single transcript showed significant association to diabetes, overall expression of the Treg signature was subtly perturbed in T1D, but not T2D, patients.

Immature thymocytes expressing autoreactive T-cell receptors (TCR) can adopt differing cell fates: clonal deletion by apoptosis or deviation into alternative lineages such as FoxP3(+) regulatory T cells (Treg). We revisited the role of the transcription factor Nr4a1 (Nur77), an immediate-early response gene induced by TCR engagement. Nr4a1KO mice show clear quantitative defects in antigen-induced clonal deletion. The impact of the Nr4a1 deletion is not enhanced by deletion of the proapoptotic factor Bim. In addition, Nr4a1 curtails initial differentiation into the Treg lineage in TCR transgenic mice and in nontransgenic mice. Transcriptional profiling of Nr4a1KO thymocytes under selection conditions reveals that Nr4a1 activates the transcription of several targets, consistent with these diverse actions: (i) Nr4a1 partakes in the induction of Bim after TCR triggering; (ii) perhaps paradoxically, Nr4a1 positively controls several transcripts of the Treg signature, in particular Ikzf2 and Tnfrsf9; (iii) consistent with its prosurvival and metabolic role in the liver, Nr4a1 is also required for the induction by TCR of a coordinated set of enzymes of the glycolytic and Krebs cycle pathways, which we propose may antagonize Treg selection as does activation of mTOR/Akt. Thus, Nr4a1 appears to act as a balancing molecule in fate determination at a critical juncture of T-cell differentiation.

Foxp3(+) regulatory T cells (Tregs) originate in the thymus, but the Treg phenotype can also be induced in peripheral lymphoid organs or in vitro by stimulation of conventional CD4(+) T cells with IL-2 and TGF-β. There have been divergent reports on the suppressive capacity of these TGF-Treg cells. We find that TGF-Tregs derived from diabetes-prone NOD mice, although expressing normal Foxp3 levels, are uniquely defective in suppressive activity, whereas TGF-Tregs from control strains (B6g7) or ex vivo Tregs from NOD mice all function normally. Most Treg-typical transcripts were shared by NOD or B6g7 TGF-Tregs, except for a small group of differentially expressed genes, including genes relevant for suppressive activity (Lrrc32, Ctla4, and Cd73). Many of these transcripts form a coregulated cluster in a broader analysis of T-cell differentiation. The defect does not map to idd3 or idd5 regions. Whereas Treg cells from NOD mice are normal in spleen and lymph nodes, the NOD defect is observed in locations that have been tied to pathogenesis of diabetes (small intestine lamina propria and pancreatic lymph node). Thus, a genetic defect uniquely affects a specific Treg subpopulation in NOD mice, in a manner consistent with a role in determining diabetes susceptibility.

FoxP3(+) regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes. NOD Tregs did show a defect in standard in vitro T cell suppression assays, particularly at low suppressor/effector ratios. Gene expression profiling revealed the vast majority of transcripts constituting the "Treg signature" to be normally distributed in NOD Tregs versus CD4(+) T conventional (Tconv) cells, although there were a few differences affecting one or the other population. According to results from criss-cross experiments, the functional inefficacy was not rooted in NOD Tregs, which suppressed as well as their C57BL/6 (B6) counterparts, but rather in NOD Tconv, which were less prone to suppression than were B6 Tconv cells. They also responded more effectively to anti-CD3/28 monoclonal antibody (mAb) stimulation in vitro or to a natural pancreatic antigen in vivo. This difference was independent of autoimmune inflammation, did not map to the idd3 region, and was not due to the overproduction of interleukin-21 in NOD mice. That the immune dysregulation in this T1D model is rooted in the ability of effector T cells to be regulated, rather than in Tregs themselves, has implications for proposed therapeutic interventions.