Treg-mediated Immunosuppression Research Articles

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-β7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-β7, LAG3, TGFβ/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-β7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-β7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Abstract 100: Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer

Abstract Background: Cancer immune evasion remains a major barrier in developing enduring therapies for epithelial ovarian cancer (EOC). Expression of programmed death-ligand 1 (PD-L1) is known for its immunosuppressive role in cancer, thus interferences to PD-L1 have shown marked efficacy against many advanced cancers. However, the mechanisms by which PD-L1-negative cancers evade immune response remain unelucidated. We have found chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) to up-regulate in PD-L1-negative EOC. Therefore, the present study aims to investigate blockade of CCL5/CXCL10 signaling to overcome immune evasion of EOC. Methods: Isogenic BRCA2-mutated PEO1 and BRCA2-wild type PEO4 EOC cells were treated with IFNγ, olaparib, or both in combination for western blot and flow cytometric analyses of PD-L1 expression. The conditioned medium of PEO1 and PEO4 cells was analyzed for immune array analysis to determine and compare the levels of 36 cytokines. PEO1 and PEO4 cells were co-cultured with CD3/CD28 antibodies-activated human peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stained with anti-CD8 or CD4 and CD25 antibodies for flow cytometric analyses of cytotoxic or helper T cells, respectively. PEO1 and PEO4 cells were co-cultured with activated PBMCs in the presence and absence of TAK-779 or AMG-487. Thereafter, PBMCs were stained with anti-CD4, CD25, and FOXP3 antibodies for flow cytometric analyses of regulatory T cells (Tregs). PEO1 and PEO4 cells were transfected with CCL5- or CXCL10-siRNA and subsequently co-cultured with activated SUPT1 cells or PBMCs. The conditioned medium was collected and analyzed for the levels of IFNγ by ELISA. Results: PEO1 cells exhibited an up-regulation of basal and IFNγ-induced PD-L1 compared with PEO4 cells. PEO4 cells displayed a pronounced increase in the CCL5 and IFNγ-induced CXCL10 compared with PEO1 cells. Co-culture of PBMCs with PEO1 and PEO4 cells led to a significant decrease in activated CD8+ effector T and CD4+ helper T cells. However, co-culture of PBMCs with PEO4 cells caused a significant increase in activated CD4+ helper T cells. Co-culture of PBMCs with PEO4 cells caused a significant increase in FOXP3+CD4+CD25+ Tregs. This increase in Tregs was significantly reduced by the treatment with the CCL5/CCR5 antagonist TAK-779 or the CXCL10/CXCR3 antagonist AMG-487. Silencing of CCL5 or CXCL10 in PEO4 cells by siRNA significantly increased IFNγ production by SUPT1 cells and PBMCs. Conclusions: PEO4 cells secrete CCL5 and IFNγ-induced CXCL10 to activate Tregs, enabling tumor cells to evade host immune response by dysregulating their microenvironment. PEO4 cells-induced Tregs are reduced by CCL5/CXCL10 signaling blockade with TAG-779 or AMG-487. Furthermore, silencing of CCL5 or CXCL10 in PEO4 cells with siRNA abolishes Treg-mediated immunosuppression and restores the antitumor activity of effector T cells. Citation Format: Annabelle C. Lin, Jake Moscarelli, Z. Ping Lin, Elena S. Ratner. Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 100.

The type 1 diabetes susceptibility locus Idd5 favours robust neonatal development of highly autoreactive regulatory T cells in the NOD mouse.

Regulatory T lymphocytes expressing the transcription factor Foxp3 (Tregs) play an important role in the prevention of autoimmune diseases and other immunopathologies. Aberrations in Treg-mediated immunosuppression are therefore thought to be involved in the development of autoimmune pathologies, but few have been documented. Recent reports indicated a central role for Tregs developing during the neonatal period in the prevention of autoimmune pathology. We therefore investigated the development of Tregs in neonatal NOD mice, an important animal model for autoimmune type 1 diabetes. Surprisingly, we found that, as compared with seven other commonly studied inbred mouse strains, in neonatal NOD mice, exceptionally large proportions of developing Tregs express high levels of GITR and PD-1. The latter phenotype was previously associated with high Treg autoreactivity in C57BL/6 mice, which we here confirm for NOD animals. The proportions of newly developing GITRhighPD-1+ Tregs rapidly drop during the first week of age. A genome-wide genetic screen indicated the involvement of several diabetes susceptibility loci in this trait. Analysis of a congenic mouse strain confirmed that Idd5 contributes to the genetic control of GITRhighPD-1+ Treg development in neonates. Our data thus demonstrate an intriguing and paradoxical correlation between an idiosyncrasy in Treg development in NOD mice and their susceptibility to type 1 diabetes.

Manipulating regulatory T cells: is it the key to unlocking effective immunotherapy for pancreatic ductal adenocarcinoma?

The five-year survival rates for pancreatic ductal adenocarcinoma (PDAC) have scarcely improved over the last half-century. It is inherently resistant to FDA-approved immunotherapies, which have transformed the outlook for patients with other advanced solid tumours. Accumulating evidence relates this resistance to its hallmark immunosuppressive milieu, which instils progressive dysfunction among tumour-infiltrating effector T cells. This milieu is established at the inception of neoplasia by immunosuppressive cellular populations, including regulatory T cells (Tregs), which accumulate in parallel with the progression to malignant PDAC. Thus, the therapeutic manipulation of Tregs has captured significant scientific and commercial attention, bolstered by the discovery that an abundance of tumour-infiltrating Tregs correlates with a poor prognosis in PDAC patients. Herein, we propose a mechanism for the resistance of PDAC to anti-PD-1 and CTLA-4 immunotherapies and re-assess the rationale for pursuing Treg-targeted therapies in light of recent studies that profiled the immune landscape of patient-derived tumour samples. We evaluate strategies that are emerging to limit Treg-mediated immunosuppression for the treatment of PDAC, and signpost early-stage trials that provide preliminary evidence of clinical activity. In this context, we find a compelling argument for investment in the ongoing development of Treg-targeted immunotherapies for PDAC.

Multi-Omics profiling identifies aldehyde dehydrogenase 2 as a critical mediator in the crosstalk between Treg-mediated immunosuppression microenvironment and hepatocellular carcinoma.

Dysregulation of the aldehyde dehydrogenase (ALDH) family has been implicated in various pathological conditions, including cancer. However, a systematic evaluation of ALDH alterations and their therapeutic relevance in hepatocellular carcinoma (HCC) remains lacking. Herein, we found that 15 of 19 ALDHs were transcriptionally dysregulated in HCC tissues compared to normal liver tissues. A four gene signature, including ALDH2, ALDH5A1, ALDH6A1, and ALDH8A1, robustly predicted prognosis and defined a high-risk subgroup exhibiting immunosuppressive features like regulatory T cell (Tregs) infiltration. Single-cell profiling revealed selective overexpression of tumor necrosis factor receptor superfamily member 18 (TNFRSF18) on Tregs, upregulated in high-risk HCC patients. We identified ALDH2 as a tumor suppressor in HCC, with three novel phosphorylation sites mediated by protein kinase C zeta that enhanced enzymatic activity. Mechanistically, ALDH2 suppressed Tregs differentiation by inhibiting β-catenin/TGF-β1 signaling in HCC. Collectively, our integrated multi-omics analysis defines an ALDH-Tregs-TNFRSF18 axis that contributes to HCC pathogenesis and represents potential therapeutic targets for this aggressive malignancy.

Elevated Metabolite Secretion Facilitates a Treg-Mediated Immunosuppressive Microenvironment in the Solid Tumor

The success of T cell immunotherapies in the solid tumor is impeded by challenges in part created by the secretion of metabolic byproducts from the tumor, which generate a hostile and immunosuppressive tumor microenvironment (TME) (Tan et al., 2021). Dysregulated tumor metabolism results in the accumulation of metabolites that are exported into the interstitial space in the tumor, where they act as signaling molecules promoting the immunosuppressive landscape, ultimately reducing anti-tumor responses to T cell therapies (Jiang et al., 2020; Joyce & Fearon, 2015). Tumor cells exhibit several advantages through their high metabolic plasticity, but metabolic aggression deprives surrounding immune cells of nutrients, leading to exhausted, non-functional, and suppressive phenotypes (Lim et al., 2020). Regulatory T cells (Tregs) are a central immunosuppressive component of the TME that can rewire their metabolism and obstruct anti-tumor immunity by suppressing proliferation and activation of CD8+ T cells and CD4+ helper T cells in the TME (Betts et al., 2012; McNally et al., 2011). High Treg accumulation is associated with reduced anti-tumor responses, reduced efficacy of TCR therapies and poor clinical outcomes (Onda et al., 2019; Preston et al., 2013; Tang et al., 2014). Therefore, there is an imminent clinical need for the development of more synergistic therapies that specifically target Tregs in the TME to successfully overcome the barriers of infiltration and function of antigen-specific T cells. Accumulation of succinate, a key mitochondrial metabolite, occurs in conditions of low oxygen and increased energy demand, such the TME of solid tumors, and mutations in the gene that encoding succinate dehydrogenase ( Sdh) have been identified in a wide variety of solid tumors (Killian et al., 2013; Roh et al., 2019),resulting further an elevation of succinate levels. Accumulated succinate is secreted into the interstitial space (Garrigue et al., 2017) where it can stimulate the membrane bound succinate receptor (SUCNR1) on neighboring cells, further polarizing their immunosuppressive phenotype, and skewing macrophages to an M2 anti-inflammatory phenotype (Trauelsen et al., 2021; Wu et al., 2020). However, the effect of succinate on Tregs has not been explored. We hypothesized that succinate drives immunosuppression in the solid tumor by promoting Treg proliferation and function, leading to reduced anti-tumor responses to antigen-specific T cells. Consistent with this hypothesis, we confirmed that CD4+ T cells express the SUCNR1 and demonstrated that succinate promotes Treg numbers in CD4+ T cells isolated from healthy donor PBMCs ( Figure 1A). This effect was blocked upon administration of the SUCNR1 antagonist, NF-56-EJ40 ( Figure 1A). Deletion of the Sdhb gene, which encodes a subunit of the succinate processing enzyme, leads to intracellular accumulation of succinate. We deleted Sdhb in the non-small cell lung carcinoma cell line (H1299) and a melanoma cells line (A375) and confirmed that Sdhb -/- tumors secreted higher levels of succinate than WT. We translated these findings to a pre-clinical immunodeficient mouse model and determined that high succinate producing H1299 and A375 tumors had higher numbers of infiltrated Tregs in the TME compared with WT tumors ( Figure 1B), indicating a central role for succinate in the immunosuppressive landscape of the TME and identifying the suppression of succinate signaling as a potential therapeutic strategy to decrease immunosuppressive Tregs. Targeting Tregs in the TME is an attractive therapeutic strategy and regulating the induction and function of Tregs has the potential to increase the efficacy of effector T cells, thus improving anti-tumor responses by removing a critical suppressive barrier. Based on these findings, we propose a novel mechanism of Treg-mediated immunosuppression driven by succinate, whereby succinate promotes a Treg-rich environment in the TME and pushes Tregs towards a Th1-suppressing phenotype. These studies identify a previously unknown molecular regulation of Tregs by extracellular succinate and provide a novel therapeutic target that has the potential to enhance the effectiveness of TCR therapy in solid tumors thereby laying a framework for translational therapeutic development and innovation, that will limit suppression of and enhance tumor-specific T cell cytotoxicity.

Inhibition of glycolysis-driven immunosuppression with a nano-assembly enhances response to immune checkpoint blockade therapy in triple negative breast cancer

Immune-checkpoint inhibitors (ICI) are promising modalities for treating triple negative breast cancer (TNBC). However, hyperglycolysis, a hallmark of TNBC cells, may drive tumor-intrinsic PD-L1 glycosylation and boost regulatory T cell function to impair ICI efficacy. Herein, we report a tumor microenvironment-activatable nanoassembly based on self-assembled aptamer-polymer conjugates for the targeted delivery of glucose transporter 1 inhibitor BAY-876 (DNA-PAE@BAY-876), which remodels the immunosuppressive TME to enhance ICI response. Poly β-amino ester (PAE)-modified PD-L1 and CTLA-4-antagonizing aptamers (aptPD-L1 and aptCTLA-4) are synthesized and co-assembled into supramolecular nanoassemblies for carrying BAY-876. The acidic tumor microenvironment causes PAE protonation and triggers nanoassembly dissociation to initiate BAY-876 and aptamer release. BAY-876 selectively inhibits TNBC glycolysis to deprive uridine diphosphate N-acetylglucosamine and downregulate PD-L1 N-linked glycosylation, thus facilitating PD-L1 recognition of aptPD-L1 to boost anti-PD-L1 therapy. Meanwhile, BAY-876 treatment also elevates glucose supply to tumor-residing regulatory T cells (Tregs) for metabolically rewiring them into an immunostimulatory state, thus cooperating with aptCTLA-4-mediated immune-checkpoint inhibition to abolish Treg-mediated immunosuppression. DNA-PAE@BAY-876 effectively reprograms the immunosuppressive microenvironment in preclinical models of TNBC in female mice and provides a distinct approach for TNBC immunotherapy in the clinics.

Tumors recycle glucocorticoids to drive Treg-mediated immunosuppression.

Suppression of antitumor immunity is a prominent feature of the tumor microenvironment. In this issue of the JCI, Taves, Otsuka, and authors show that glucocorticoids (GCs), which are potent immunosuppressive hormones mainly produced by the adrenals, can be reconverted from their inactive form to active metabolites via the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme expressed by murine tumor cell lines. In the tumor microenvironment, GCs acted on CD4+ regulatory T cells to enhance their immunosuppressive function and promote tumor growth. The findings suggest that targeting GC recycling as a strategy for modulating tumor immunosuppression has the potential to improve therapeutic efficacy of immune checkpoint blockade.

Selective targeting of IL2Rβγ combined with radiotherapy triggers CD8- and NK-mediated immunity, abrogating metastasis in HNSCC

The implementation of cancer immunotherapies has seen limited clinical success in head and neck squamous cell carcinoma (HNSCC). Interleukin-2 (IL-2), which modulates the survival and functionality of lymphocytes, is an attractive target for new immunotherapies but one that is limited by presence of regulatory Tcells (Tregs) expressing the high-affinity IL-2Rα. The bispecific immunocytokine PD1-IL2v preferentially delivers IL-2 signaling through IL-2Rβγ on PD-1-expressing cells. Selectively targeting the intermediate-affinity IL-2Rβγ can be leveraged to induce anti-tumor immune responses in effector Tcells and natural killer (NK) cells while limiting the negative regulation of IL-2Rα activation on Tregs. Using radiation therapy (RT) in combination with PD1-IL2v improves local tumor control and survival, and controls metastatic spread in orthotopic HNSCC tumor models. PD1-IL2v drives systemic activation and expansion of circulating and tumor-infiltrating cytotoxic Tcells and NK cells while limiting Treg-mediated immunosuppression. These data show that PD1-L2v induces durable systemic tumor control in HNSCC.

Functional interrogation of lymphocyte subsets in alopecia areata using single-cell RNA sequencing

Alopecia areata (AA) is among the most prevalent autoimmune diseases, but the development of innovative therapeutic strategies has lagged due to an incomplete understanding of the immunological underpinnings of disease. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the graft-induced C3H/HeJ mouse model of AA, coupled with antibody-based depletion to interrogate the functional role of specific cell types in AA in vivo. Since AA is predominantly T cell-mediated, we focused on dissecting lymphocyte function in AA. Both our scRNAseq and functional studies established CD8+ T cells as the primary disease-driving cell type in AA. Only the depletion of CD8+ T cells, but not CD4+ T cells, NK, B, or γδ T cells, was sufficient to prevent and reverse AA. Selective depletion of regulatory T cells (Treg) showed that Treg are protective against AA in C3H/HeJ mice, suggesting that failure of Treg-mediated immunosuppression is not a major disease mechanism in AA. Focused analyses of CD8+ T cells revealed five subsets, whose heterogeneity is defined by an "effectorness gradient" of interrelated transcriptional states that culminate in increased effector function and tissue residency. scRNAseq of human AA skin showed that CD8+ T cells in human AA follow a similar trajectory, underscoring that shared mechanisms drive disease in both murine and human AA. Our study represents a comprehensive, systematic interrogation of lymphocyte heterogeneity in AA and uncovers a novel framework for AA-associated CD8+ T cells with implications for the design of future therapeutics.

Redox modulation with a perfluorocarbon nanoparticle to reverse Treg-mediated immunosuppression and enhance anti-tumor immunity.

Tumor hypoxia and high glutathione (GSH) expression promote regulatory T cell (Treg) infiltration and maintain its immunosuppressive function, which significantly reduces the response rate of cancer immunotherapy. Here, we developed an immunomodulatory nano-formulation (FEM@PFC) to reverse Treg-mediated immunosuppression by redox regulation in the tumor microenvironment (TME). Oxygen carried in perfluorocarbon (PFC) was delivered to the TME, thus relieving the hypoxic condition and inhibiting Treg infiltration. More importantly, GSH depletion by the prodrug efficiently restricted the Foxp3 expression and immunosuppressive function of Tregs, thus breaking the shackles of tumor immunosuppression. Additionally, the supplement of oxygen cooperated with the consumption of GSH to enhance the irradiation-induced immunogenic cell death and subsequent dendritic cell (DC) maturation, thereby efficiently promoting the activation of effector T cells and restricting the immunosuppression of Tregs. Collectively, the FEM@PFC nano-formulation reverses Treg-mediated immunosuppression and regulates the redox balance in the TME to boost anti-tumor immunity and prolong the survival of tumor-bearing mice, which provides a new immunoregulatory strategy from the perspective of redox modulation.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Abstract CT250: An open-label, phase 1a/b study of AB248, a CD8+ selective IL-2 mutein fusion protein, alone or in combination with pembrolizumab in patients with advanced solid tumors

Abstract Background: High-dose interleukin-2 (HD IL-2) induces durable clinical responses in a subset of patients with melanoma and RCC, but severe toxicity limits its therapeutic utility. Early clinical data from several not-α IL-2Rβγ agonists suggest better tolerability but lower objective response rates compared to historical HD IL-2 data.1-2 In patients, these not-α IL-2s demonstrate a profound NK cell bias, while retaining activity on Tregs, and exhibiting limited CD8+ T cell expansion.3-5AB248 is a CD8+ selective IL-2 that demonstrates more than 500-fold selectivity for CD8+ T cells over other immune cell types. AB248 has demonstrated a highly differentiated preclinical profile, with compelling anti-tumor activity and less toxicity when given alone and in combination with anti-PD1 in multiple murine tumor models. These data suggest that AB248 may have an improved therapeutic index compared to broad-acting IL-2Rβγ agonists by increasing CD8+ T cell expansion and activation, avoiding NK cell-driven toxicity and avoiding Treg-mediated immunosuppression. Here we introduce the first-in-human study which aims to investigate AB248 when administered alone and in combination with pembrolizumab in subjects with advanced solid tumors who failed prior standard of care therapies. Methods: This open-label phase 1a/b study consisting of a dose escalation and expansion phase aims to investigate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity of AB248 alone or in combination with pembrolizumab in subjects with locally advanced/metastatic tumors, including melanoma, renal cell carcinoma, NSCLC and SCCHN, who failed prior therapies, including prior anti-PD(L)1, as well as in first-line SCCHN during the expansion phase. Key eligibility criteria include age ≥18 years, ECOG ≤1, measurable disease per RECIST v1.1, adequate end-organ function, and no autoimmune disease. The dose escalation phase will follow the Bayesian Optimal Interval (BOIN) design and enroll subjects at multiple dose levels and schedules for the AB248 monotherapy and pembrolizumab combination portion. Upon identifying suitable dose and schedule based on the totality of cumulative data including safety, PK, PD and preliminary efficacy, additional subjects will be enrolled in indication specific cohorts in the expansion phase according to the Simon 2-stage design. The primary objectives for this study are to assess the safety and tolerability of AB248 alone or in combination with pembrolizumab. Secondary objectives include assessing the PK, PD changes, immunogenicity, and antitumor activity. Exploratory objectives include evaluating the potential response-predictive and/or associated changes in immune cells, blood, and tissue biomarkers. Tumor assessments will be performed every 6 weeks for the first 30 weeks and every 9 weeks thereafter. Adverse events will be assessed by CTCAE v5.0. The study is currently open for enrollment in the dose escalation phase at multiple sites in the US. Citation Format: Elizabeth I. Buchbinder, David R. Spigel, Costantine Albany, Michael Chisamore, Kelly D. Moynihan, Xiaohan Liu, Christopher DelNagro, Matt Axt, Andrea Pirzkall. An open-label, phase 1a/b study of AB248, a CD8+ selective IL-2 mutein fusion protein, alone or in combination with pembrolizumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT250.

GPR52 regulates cAMP in T cells but is dispensable for encephalitogenic responses.

G-protein coupled receptors (GPCR) regulate 3',5'-cyclic adenosine monophosphate (cAMP) levels in T cells. cAMP as ubiquitous second messenger is crucial for adequate physiology of T cells by mediating effector T cell (Teff) function as well as regulatory T cell (Treg)-mediated immunosuppression. Several GPCRs have been identified to be crucial for Teff and Treg function. However, the role of the orphan, constitutively active Gs-coupled GPCR GPR52 is unknown. Here we show that GPR52 regulates cAMP levels in T cells but does not affect T cell function. We found that stimulation of transfected HEK cells or primary T cells with a GPR52 agonist results in a rise of intracellular cAMP. However, neither Gpr52 deficiency nor pharmacological modulation of GPR52 by antagonists or agonists affected T cell activation, differentiation, and proliferation or Treg-mediated immunosuppression. Moreover, Gpr52 deletion did not modify the clinical disease course of experimental autoimmune encephalomyelitis (EAE). Our results demonstrate that a modulation of cAMP levels in T cells does not inevitably result in altered T cell function. While we could not identify an obvious role of GPR52 in in vitro T cell assays and in vivo CNS autoimmunity, it might regulate T cell function in a different context or affect the function of other GPR52-expressing cells.

BATF reprograms the tumor immune status

The AP-1 family member BATF maintains immune cell function. Two recent papers by Zhang etal.1 and Itahashi etal.2 investigated the role of this protein in cancer immunity, finding that its depletion in CAR-T cells augments anti-tumor activity and it contributes to Treg-mediated immunosuppression in the tumor microenvironment.

Effective and Safe Immune Checkpoint Blockade Induces Complete Responses in Murine PTCL Model of ALCL

Anaplastic large cell lymphoma (ALCL) is a rare T-cell non-Hodgkin lymphoma. In the majority of cases the disease is driven by a chromosomal translocation t(2;5)(p23;35) leading to the fusion of the anaplastic lymphoma kinase (ALK) gene with the nucleophosmin gene (NPM), constitutively activating ALK kinase. In the last decade immunotherapy turned out to be a real game-changer in cancer treatment, especially in the field of solid tumors. Regarding T-cell lymphomas (TCL), the significance of immune checkpoint inhibition is still highly debated. The outcomes in preclinical models as well as clinical trials are ranging from response to hyperprogression. Consequently, to provide a better understanding of the role and underlying mechanisms of targeting immune checkpoint axes, preclinical models of TCL are urgently needed to identify patients that benefit from immunotherapy as well as potential biomarkers. To examine disease biology and therapy effects of ALCL in vivo, we use a syngeneic retroviral bone marrow transplantation mouse model of ALK+ ALCL that closely resembles the disease in humans. In order to study the effect of blocking the PD-1/PD-L1-axis in ALCL in vivo, serial transplantations of lymphoma cells from a murine thymus were performed. We further investigated both spontaneous and immunotherapy-induced immune responses and characterized the interaction of innate and adaptive immune master players with flow cytometry, cytokine analysis, histology and transcriptome analysis. In our mouse model of ALK+ ALCL we confirmed the upregulation of PD-L1 on ALK+ lymphoma cells in vivo. Immune checkpoint inhibition of PD-L1 in vivo leads to high complete response rates, prolonged survival and significantly reduced lymphoma burden of treated animals. Flow cytometry analysis revealed that PD-L1 blockade reverses Treg-mediated immunosuppression and causes augmented circulation of CD8+ effector T-lymphocytes. Treatment with depletion antibodies of lymphocyte subpopulations unveils CD4+ T-cells and NK-cells but not CD8+ T-cells as master players of checkpoint-mediated immune response to ALK+ ALCL. Accordingly, we identified exhausted CD4+ T-cells in non-responders to checkpoint inhibition underlining the central role of CD4+ T-cells in ALCL immune surveillance and response. These results are contributing to a broader understanding of the immune surveillance and response in ALCL, with the outmost goal of better identifying patients that benefit from immunotherapy.

Correlations between Circulating and Tumor-Infiltrating CD4+ Treg Subsets with Immune Checkpoints in Colorectal Cancer Patients with Early and Advanced Stages.

The existence of various T regulatory cell (Treg) subsets in colorectal cancer (CRC) could play a variety of functions in the regulation of anti-cancer immunity. We studied correlations between CD4+ Treg subsets with the expression of immunological checkpoints on CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 in CRC patients with early and advanced TNM staging. Strong positive correlations were found between frequencies of FoxP3+ Tregs and FoxP3+Helios+ Tregs with frequencies of various immune checkpoint-expressing CD4+ T cells in the tumor microenvironment (TME). However, there were strong negative correlations between frequencies of FoxP3−Helios− T cells and these immune checkpoint-expressing CD4+ T cells. Specifically, in the TME, we found that the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, FoxP3+Helios− Tregs, and FoxP3−Helios− T cells with CD4+LAG-3+ T cells and CD4+CTLA-4+ T cells were higher in patients with early stages, suggesting the potential of these highly immunosuppressive cells in inhibiting inflammatory responses in the TME. However, the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, and FoxP3−Helios− T cells with CD4+TIM-3+ T cells were higher in patients with advanced stages. This is the first study to explore correlations of Treg subpopulations with immune checkpoint-expressing CD4+ T cells in CRC based on clinicopathological features of CRC patients. The findings of our study provide a justification for focusing on these cells that possess highly immunosuppressive features. Understanding the correlations between different immune checkpoints and Treg subsets in CRC patients has the potential to enhance our understanding of core mechanisms of Treg-mediated immunosuppression in cancer.

Elastic Nanovaccine Enhances Dendritic Cell‐Mediated Tumor Immunotherapy (Small 32/2022)

Elastic Nanovaccines In article number 2201108, Zhaogang Teng, Heng Dong, Yongbin Mou, and co-workers report a soft mesoporous organosilica-based nanovaccine (SMONV) and demonstrate that elastic SMONV evokes robust antitumor immune cascades, including lymphatic drainage, antigen internalization and cross-presentation, and tumor-specific T-cell immune responses. Treg-mediated immunosuppression is inhibited after immunization with SMONV. Furthermore, combined administration of anti-PD-L1 antibodies induces tumor regression. The paper provides insight into nanoparticle elasticity and dendritic cell-mediated immunotherapy.

T-cell activation and bacterial infection in skin wounds of recessive dystrophic epidermolysis bullosa patients.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell‐mediated immunity in RDEB wounds. Using a non‐invasive approach for sampling of wound‐associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen‐specific activation of RDEB wound‐derived T cells were investigated by fluorescence‐activated cell sorting‐based immune‐phenotyping and T‐cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB‐derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound‐associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus‐infected RDEB keratinocytes and respond by producing interleukin‐2 (IL‐2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB‐derived T cells to microbial antigens in vitro does not trigger PD‐1‐mediated T‐cell exhaustion but induces differentiation of the CD4high population into CD4highCD25+FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell‐mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD‐1/Treg‐mediated immuno‐suppression in RDEB.

Excessive immunosuppression by regulatory T cells antagonizes T cell response to schistosome infection in PD-1-deficient mice.

Schistosomiasis is caused by parasitic flatworms known as schistosomes and affects over 200 million people worldwide. Prevention of T cell exhaustion by blockade of PD-1 results in clinical benefits to cancer patients and clearance of viral infections, however it remains largely unknown whether loss of PD-1 could prevent or cure schistosomiasis in susceptible mice. In this study, we found that S. japonicum infection dramatically induced PD-1 expression in T cells of the liver where the parasites chronically inhabit and elicit deadly inflammation. Even in mice infected by non-egg-producing unisex parasites, we still observed potent induction of PD-1 in liver T cells of C57BL/6 mice following S. japonicum infection. To determine the function of PD-1 in schistosomiasis, we generated PD-1-deficient mice by CRISPR/Cas9 and found that loss of PD-1 markedly increased T cell count in the liver and spleen of infected mice. IL-4 secreting Th2 cells were significantly decreased in the infected PD-1-deficient mice whereas IFN-γ secreting CD4+ and CD8+ T cells were markedly increased. Surprisingly, such beneficial changes of T cell response did not result in eradication of parasites or in lowering the pathogen burden. In further experiments, we found that loss of PD-1 resulted in both beneficial T cell responses and amplification of regulatory T cells that prevented PD-1-deficient T cells from unleashing anti-parasite activity. Moreover, such PD-1-deficient Tregs exert excessive immunosuppression and express larger amounts of adenosine receptors CD39 and CD73 that are crucial for Treg-mediated immunosuppression. Our experimental results have elucidated the function of PD-1 in schistosomiasis and provide novel insights into prevention and treatment of schistosomiasis on the basis of modulating host adaptive immunity.