Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.

Strength of TCR signal from self-peptide modulates autoreactive thymocyte deletion and Foxp3(+) Treg-cell formation.

Autoreactive CD4(+) CD8(-) (CD4SP) thymocytes can be subjected to deletion when they encounter self-peptide during their development, but they can also undergo selection to become CD4SPFoxp3(+) Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP-76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3(+) thymocytes. CD4SP thymocytes expressing TCR Vß-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3(+) thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg-cell formation in response to a defined self-peptide, SLP-76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3(+) Treg-cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg-cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg-cell formation.

Autoreactive Th1 cells activate monocytes to support regional Th17 responses in inflammatory arthritis.

We have examined mechanisms underlying the formation of pathologic Th17 cells using a transgenic mouse model in which autoreactive CD4(+) T cells recognize influenza virus hemagglutinin (HA) as a ubiquitously expressed self-Ag and induce inflammatory arthritis. The lymph nodes of arthritic mice contain elevated numbers of inflammatory monocytes (iMO) with an enhanced capacity to promote CD4(+) Th17 cell differentiation, and a regional inflammatory response develops in the paw-draining lymph nodes by an IL-17-dependent mechanism. The activation of these Th17-trophic iMO precedes arthritis development and occurs in the context of an autoreactive CD4(+) Th1 cell response. Adoptive transfer of HA-specific CD4(+) T cells into nonarthritic mice expressing HA as a self-Ag similarly led to the formation of Th1 cells and of iMO that could support Th17 cell formation, and, notably, the accumulation of these iMO in the lymph nodes was blocked by IFN-γ neutralization. These studies show that autoreactive CD4(+) Th1 cells directed to a systemically distributed self-Ag can promote the development of a regional Th17 cell inflammatory response by driving the recruitment of Th17-trophic iMO to the lymph nodes.

Requirement for diverse TCR specificities determines regulatory T cell activity in a mouse model of autoimmune arthritis.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are required to restrain the immune system from mounting an autoaggressive systemic inflammatory response, but why their activity can prevent (or allow) organ-specific autoimmunity remains poorly understood. We have examined how TCR specificity contributes to Treg activity using a mouse model of spontaneous autoimmune arthritis, in which CD4(+) T cells expressing a clonotypic TCR induce disease by an IL-17-dependent mechanism. Administration of polyclonal Tregs suppressed Th17 cell formation and prevented arthritis development; notably, Tregs expressing the clonotypic TCR did not. These clonotypic Tregs exerted Ag-specific suppression of effector CD4(+) T cells using the clonotypic TCR in vivo, but failed to mediate bystander suppression and did not prevent Th17 cells using nonclonotypic TCRs from accumulating in joint-draining lymph nodes of arthritic mice. These studies indicate that the availability of Tregs with diverse TCR specificities can be crucial to their activity in autoimmune arthritis.

CD4⁺CD25⁺Foxp3⁺ regulatory T cell formation requires more specific recognition of a self-peptide than thymocyte deletion.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are generated during thymocyte development and play a crucial role in preventing the immune system from attacking the body's cells and tissues. However, how the formation of these cells is directed by T-cell receptor (TCR) recognition of self-peptide:major histocompatibility complex (MHC) ligands remains poorly understood. We show that an agonist self-peptide with which a TCR is strongly reactive can induce a combination of thymocyte deletion and CD4(+)CD25(+)Foxp3(+) Treg cell formation in vivo. A weakly cross-reactive partial agonist self-peptide could similarly induce thymocyte deletion, but failed to induce Treg cell formation. These studies indicate that CD4(+)CD25(+)Foxp3(+) Treg cell formation can require highly stringent recognition of an agonist self-peptide by developing thymocytes. They also refine the "avidity" model of thymocyte selection by demonstrating that the quality of the signal mediated by agonist self-peptides, rather than the overall intensity of TCR signaling, can be a critical factor in directing autoreactive thymocytes to undergo CD4(+)CD25(+)Foxp3(+) Treg cell formation and/or deletion during their development.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

Comparative evaluation of itaconate and its derivatives reveals divergent inflammasome and type I interferon regulation in macrophages.

Following activation, macrophages undergo extensive metabolic rewiring1,2. Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process3. Itaconate inhibits succinate dehydrogenase4,5, has electrophilic properties6 and is associated with a change in cytokine production4. Here, we compare the metabolic, electrophilic and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild-type and Irg1-/- macrophages, we show that neither dimethyl itaconate, 4-octyl itaconate nor 4-monoethyl itaconate are converted to intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only dimethyl itaconate and 4-octyl itaconate induce a strong electrophilic stress response, in contrast to itaconate and 4-monoethyl itaconate. This correlates with their immunosuppressive phenotype: dimethyl itaconate and 4-octyl itaconate inhibited IκBζ and pro-interleukin (IL)-1ß induction, as well as IL-6, IL-10 and interferon-ß secretion, in an NRF2-independent manner. In contrast, itaconate treatment suppressed IL-1ß secretion but not pro-IL-1ß levels and, surprisingly, strongly enhanced lipopolysaccharide-induced interferon-ß secretion. Consistently, Irg1-/- macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive, metabolite and highlights the importance of using unmodified itaconate in future studies.

Sub-mitogenic phorbol myristate acetate co-stimulation rescues the PHA-induced activation of both naïve and memory T cells cultured in the rotating-wall vessel bioreactor.

T lymphocytes are unresponsive to T cell receptor (TCR) stimulation during culture in spaceflight or ground-based microgravity analogs such as the rotating-wall vessel (RWV) bioreactor. The TCR-induced activation of a subset of T cells can be rescued in the RWV by co-stimulation with sub-mitogenic doses of phorbol ester (PMA). We report that PMA co-stimulation of primary human T cells cultured in the RWV rescues the phytohemagglutinin (PHA)-induced activation of the CD8+ and CD4+ T cell subsets as well as naïve and memory CD4+ T cells. Importantly, T cells activated in the RWV by PHA+PMA contained these subsets in proportions strikingly similar to control cultures activated with PHA alone. The data indicate that rescuing T cell activation with PMA co-stimulation does not significantly perturb the heterogeneity of the responding cells, and represent an important proof of principle for the design of immune-boosting agents for use in spaceflight.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Flow cytometric profiling of mature and developing regulatory T cells in the thymus.

Natural Regulatory T (Treg) cells are a subset of CD4(+) T cells characterized by expression of the transcription factor Foxp3 and the ability to suppress immune responses. Treg cells develop in the thymus in response to highly specific interactions between the T cell receptor (TCR) and self-antigens. These processes can be recapitulated in antigen-specific systems using transgenic mice that coexpress a TCR with its cognate peptide as a neoself-antigen. Here, we describe a method for using such a system to establish a flow cytometric profile of phenotype markers expressed by developing and mature Treg cells in the thymus. Our approach is to compare antigen-specific thymocytes developing in the presence or absence of Treg cell-selecting ligands to identify phenotypic changes that characterize thymocytes undergoing selection into the Treg cell lineage.

How specificity for self-peptides shapes the development and function of regulatory T cells.

The cataclysmic disease that develops in mice and humans lacking CD4+ T cells expressing the transcription factor Foxp3 has provided abundant evidence that Foxp3+CD4+ Tregs are required to suppress a latent autoreactivity of the immune system. There is also evidence for the existence of tissue-specific Tregs that can act to suppress regional autoimmune responses, suggesting that Tregs exert their effects, in part, through responding to self-peptides. However, how the immune system generates a repertoire of Tregs that is designed to recognize and direct regulatory function to self-peptides is incompletely understood. This review describes studies aimed at determining how T cell recognition of self-peptide(s) directs Treg formation in the thymus, including discussion of a modified "avidity" model of thymocyte development. Studies aimed at determining how TCR specificity contributes to the ability of Tregs to suppress autoimmune diseases are also discussed.

Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application.

In the present study, mouse embryonic stem cells (ESCs) were differentiated into alveolar epithelial type II (AEII) cells for endotracheal injection. These enriched lung-like populations expressed lung epithelial markers SP-A, SP-B, SP-C, and CC10. First we show that rapid differentiation of ESCs requires a dissociated seeding method instead of an embryoid body culture method. We then investigated a two-step differentiation of ESCs into definitive endoderm by activin or A549-conditioned medium as a precursor to lung epithelial cells. When conditioned medium from A549 cells was used to derive endoderm, yield was increased above that of activin alone. Further studies showed that Wnt3a may be one of the secreted factors produced by A549 cells and promotes definitive endoderm differentiation, in part, through suppression of primitive endoderm. Activin and Wnt3a together at appropriate doses with dissociated cell seeding promoted greater endoderm yield than activin alone. Next, fibroblast growth factor 2 was shown to induce a dose-dependent expression of SPC, and these cells contained lamellar bodies characteristic of mature AEII cells from ESC-derived endoderm. Finally, ES-derived lung cells were endotracheally injected into preterm mice with evidence of AEII distribution within the lung parenchyma. This study concludes that a recapitulation of development may enhance derivation of an enriched population of lung-like cells for use in cell-based therapy.