Mucosal tissues contain large numbers of memory CD4+ T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4+ T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα+DR3+CD4+ T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα+DR3+CD4+ T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα+DR3+ CD4+ T cells may contribute to antigen-independent innate responses at barrier surfaces.

Although CD8+ T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)–derived antigen involves the generation and suppressive function of FoxP3+CD8+ T cells. FoxP3+CD8+ Treg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFβ, and retinoic acid contributed to the generation of gut-tropic CCR9+CD103+FoxP3+CD8+ Tregs. Last, CD103-deficient CD8+ T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3+CD8+ Treg function. Our results describe a role for FoxP3+CD8+ Tregs in cross-tolerance in the intestine for which development requires intestinal cDC1.

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated ‘cold’ tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VVGM-αCTLA-4).ResultsThe identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VVGM-αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VVGM-αCTLA-4 synergized with αPD-1 to reject cold tumors.ConclusionOur findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VVGM-αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced.

The pleiotropic TNF-α:TNFR axis plays a central role in the immune system. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells and especially high on T regs. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated but the mechanism-of-action (MoA) of these reagents remains unclear, with conflicting data reported by different investigators. To gain further insight, we identified and characterized a wide panel of human and mouse TNFR2-specific antibodies, generated from the n-CoDeR F.I.R.S.T™ phage display platform. Based on their ability to block TNF-α:TNFR2 binding and to agonize or antagonize TNFR2 signaling, we identified parallel human and mouse TNFR2-specific antibodies with activities ranging from agonist to antagonist, and from complete ligand blocking to non-blocking. Two antibody variants with distinctly opposing in vitro activities (complete ligand-blocking antagonist versus non-blocking agonist), were expressed in various IgG formats preferentially engaging activating FcγR (mIgG2a), inhibitory FcγR (mIgG1), or no FcγR (N297A Fc-mutated) and screened for in vivo antitumor activity. Both anti-TNFR2 antibody clones displayed anti-tumor efficacy but showed strikingly different FcγR-dependence for optimal antitumor activity. Further characterization demonstrated potent anti-tumor efficacy across several syngeneic in vivo cancer models (CT26, MC38 and B16), both as single agents, and when combined with anti-PD-1. In vivo mode-of-action studies indicated different initial events are evoked by the two antibodies but that they ultimately converge to elicit a similar immune modulation within the tumor that is associated with anti-tumor efficacy. The antagonist antibody caused early intra-tumoral T reg depletion, while the agonist caused dramatic CD8+ T cell increases. Over time, both antibodies induce an increase in effector T cells at the tumor site, improved CD8/T reg ratios, and tumor regression. In addition, the two antibodies similarly modulated the tumor myeloid content. Based on careful MoA-characterization, two human lead candidate anti-TNFR2 antibodies are being developed for treatment of solid cancer; BI-1808, a ligand-blocking T reg depleting antibody and BI-1910, a TNFR2 agonist. Citation Format: Linda Mårtensson, Kirstie Cleary, Monika Semmrich, Mathilda Kovacek, Petra Holmkvist, Caroline Svensson, Mimoza Demiri, Therese Blidberg, Ulla-Carin Thornberg, Vincentiu Pitic, Osman Dadas, Sean H. Lim, Stephen A. Beers, Mark S. Cragg, Björn Frendéus, Ingrid Teige. Targeting TNFR2 for cancer immunotherapy: Ligand blocking depletors versus receptor agonists [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 936.