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Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8 + T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDO-overexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors. The tryptophan metabolite kynurenine is an endogenous ligand of the aryl hydrocarbon receptor (AHR). Here, the authors show that AHR targeting in IDO/TDO-expressing tumours counteracts a regulatory T cell/macrophage suppressive axis and synergizes with immune checkpoint blockade to hinder tumour growth.

Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.

BackgroundIn contrast to PD-1+CD8+ T cells, PD-1+CD4+ T cells and their impact in tumor progression and immunotherapy response remain relatively unexplored. We previously reported that PD-1hiFoxp3-CD4+ T cells (4PD1hi) from melanoma-bearing mice and patients with melanoma or non-small cell lung cancer (NSCLC) suppress T-cell function and correlate with unfavorable outcomes upon immune checkpoint blockade (ICB) therapy.1 CD4+PD-1+ T cells were also found to correlate with poor prognosis in other NSCLC patient cohorts.2–4 We showed that 4PD1hiup-regulate T-follicular-helper-cell(Tfh)-related genes.1 4PD1hi cells suppressing immunotherapy responses were recently described in mouse sarcoma models; however, in this setting, 4PD1hi did not over-express Tfh genes.5 Here, we sought to deconvolve the lineage commitment of 4PD1hi tumor-infiltrating lymphocytes (TILs) in relationship with their immune function and impact on ICB outcome.MethodsSingle-cell RNA-sequencing (scRNAseq) was performed in 4PD1hi, PD-1-Foxp3-CD4+ (4Dneg), and Foxp3+CD4+ T cells (Tregs) FACS-sorted from ICB-treated B16F10-melanoma bearing Foxp3-GFP mice. scRNAseq datasets of TILs from ICB-treated cancer patients6–9 were used to extract 4PD1hi and analyze their profiles. Tfh-deficient SAP knock-out (KO) and CD4KO:CXCR5KO mixed bone marrow (BM) transplanted RAGKO and control mice were implanted with B16F10, treated with ICB, and 4PD1hi, 4Dneg, and Tregs were quantified by flow cytometry and FACS-sorted for functional analyses.ResultsUsing prior-knowledge-based signatures and mutual-information-based cell-type classification,10 we found that spleen-derived 4PD1hi cells from tumor-bearing mice polarize toward Tfh, Tregs toward a canonical Treg phenotype, and 4Dneg toward Th1. Conversely, tumor-derived 4PD1hi were not significantly skewed toward these phenotypes but gained in Th1 polarization after an effective anti-CTLA-4 treatment. In human primary melanoma,7 NSCLC,6 and squamous/basal cell carcinoma,8 4PD1hi cells over-expressed Tfh-related genes. This was less clear in 4PD1hi from mixed NSCLC samples, encompassing primary tumors and different metastatic sites.9 However, 4PD1hi from ICB-non-responder patients in this study9 displayed the greatest Tfh-signature scores. Consistently, 4PD1hi TILs from ICB-non-responders in the other NSCLC and melanoma datasets up-regulated Tfh-related genes. To test 4PD1hi TIL Tfh polarization in ICB response, we used SAPKO and CD4KO:CXCR5KO BM chimera mice. Both Tfh-deficient models showed better tumor responses to a suboptimal anti-CTLA-4 treatment1; however, 4PD1hi TILs did not substantially decrease. In this setting, 4PD1hi TILs lost suppressive function, down-regulated Pdcd1 and Il10, and up-regulated Ifng, suggesting a Th1 phenotypic switch.ConclusionsThese results indicate that 4PD1hi TILs are heterogeneous and their Tfh/Th1 polarization influences immunotherapy responses possibly in a tumor-tissue dependent way. In melanoma, 4PD1hi TIL Tfh polarization drives immunosuppression and ICB resistance.AcknowledgementsThis study was supported in part by the Parker Institute for Cancer Immunotherapy. S. M. and A.O. contributed equally to this work.ReferencesZappasodi R, Budhu S, Hellmann MD, Postow MA, Senbabaoglu Y, Manne S, et al. Non-conventional Inhibitory CD4(+)Foxp3(-)PD-1(hi) T Cells as a Biomarker of Immune Checkpoint Blockade Activity. Cancer Cell. 2018;33(6):1017–32 e7.Zheng H, Liu X, Zhang J, Rice SJ, Wagman M, Kong Y, et al. Expression of PD-1 on CD4 + T cells in peripheral blood associates with poor clinical outcome in non-small cell lung cancer. Oncotarget. 2016;7(35).Arrieta O, Montes-Servín E, Hernandez-Martinez J-M, Cardona AF, Casas-Ruiz E, Crispín JC, et al. Expression of PD-1/PD-L1 and PD-L2 in peripheral T-cells from non-small cell lung cancer patients. Oncotarget. 2017;8(60).Duchemann B, Naigeon M, Auclin E, Ferrara R, Cassard L, Jouniaux JM, et al. CD8(+)PD-1(+) to CD4(+)PD-1(+) ratio (PERLS) is associated with prognosis of patients with advanced NSCLC treated with PD-(L)1 blockers. J Immunother Cancer. 2022;10(2).Hussein S, Kelly M, Yuang S, Samuel A, Ton S, Yik Andy Y, et al. 578 CD8-targeted IL-2 drives potent anti-tumor efficacy and promotes action of tumor specific vaccines. Journal for ImmunoTherapy of Cancer. 2021;9(Suppl 2):A607.Caushi JX, Zhang J, Ji Z, Vaghasia A, Zhang B, Hsiue EH, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature. 2021;596(7870):126–32.Schad SE, Chow A, Mangarin L, Pan H, Zhang J, Ceglia N, et al. Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions. J Exp Med. 2022;219(6).Yost KE, Satpathy AT, Wells DK, Qi Y, Wang C, Kageyama R, et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med. 2019;25(8):1251–9.Liu B, Hu X, Feng K, Gao R, Xue Z, Zhang S, et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nature Cancer. 2022;3(1):108–21.Ceglia N, Sethna Z, Freeman SS, Uhlitz F, Bojilova V, Rusk N, et al. GeneVector: Identification of transcriptional programs using dense vector representations defined by mutual information. bioRxiv. 2023:2022.04.22.487554.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53 dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

BackgroundTumor reliance on glycolytic metabolism represents a hallmark of cancer and a mechanism of resistance to immune based therapies.1 2 Competition for glucose between T cells and tumor cells as well as lactate-mediated immune suppression contribute to this resistance. Thus, immune checkpoint blockade (ICB) more effectively controls glycolysis-low tumors lacking lactate dehydrogenase (LDH), primarily due to enhanced glucose availability within the tumor microenvironment.3 The development of LDH inhibitors (LDHi) have been reported to reduce glucose uptake and growth of preclinical models of cancer,4 but their impact on infiltrating T cells within the tumor microenvironment remains unexplored.MethodsB16F10 and MC38 tumor-bearing C57BL/6 mice were treated with LDHi GNE-140 and/or anti-CTLA-4 and tumor volume was measured twice per week. Tumors were also processed for flow cytometric analysis of tumor-infiltrating lymphocytes to assess infiltration, activation, and function. Glucose uptake, LDH, and Glut1 expression was assessed in tumor and T cells by flow cytometry. Seahorse analysis was performed on mouse and human tumor and T cells.ResultsTumor cells exhibit higher basal levels of LDH and glucose consumption compared to tumor-infiltrating T cells, exposing a therapeutic window for tumor-specific targeting of glycolysis. We show that effective LDH inhibition relies on tumoral overexpression of LDH and the adaptive immune system to impede murine tumor progression. Mechanistically, we demonstrate that LDHi treatment 1) decreases tumor cell glucose uptake and expression of the glucose transporter GLUT1 while 2) increasing glucose uptake and GLUT1 expression in tumor-infiltrating CD8+ T cells. Compared to activated T cells, tumor cells display significantly higher metabolic sensitivity to LDH inhibition via reduction of glucose uptake and extracellular acidification rates while also downregulating LDH and GLUT1 expression. Strikingly, LDH inhibition combined with anti-CTLA-4 effectively controls murine melanoma and colon cancer progression. This dual therapy also promotes effector T cell infiltration and activation while functionally destabilizing regulatory T cells (Tregs). Accordingly, increasing glucose availability leads to improved T cell killing of tumor cells, and impaired Treg-mediated suppression of T cells.ConclusionsIncreasing intratumoral glucose levels contributes to anti-tumor efficacy of CTLA-4 blockade by supporting effector T cell function while impairing Tregs. Our results establish LDH inhibition as an effective tumor-specific strategy to reduce tumor glucose uptake and increase glucose availability within the tumor microenvironment, thereby boosting T cell glucose uptake. This study provides a comprehensive rationale for combining immune checkpoint blockade with inhibitors of glycolysis for patients with highly glycolytic cancers.ReferencesHanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674.Zappasodi R, Merghoub T, Wolchok JD. Emerging Concepts for Immune Checkpoint Blockade-Based Combination Therapies. Cancer Cell. 2018;34:690.Zappasodi R, et al. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours. Nature. 2021;591:652–658.Boudreau A, et al. Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition. Nat Chem Biol. 2016;12:779–786.

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8 + T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8 + T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Metastatic uveal melanoma (UM) remains challenging to treat, with objective response rates to immune checkpoint blockade (ICB) being much lower than in primary cutaneous melanoma (CM). Besides a lower mutational burden, the overall immune-excluded tumor microenvironment of UM might contribute to the poor response rate. We therefore aimed at targeting deficiency in argininosuccinate synthase 1, which is a key metabolic feature of UM. This study aims at investigating the safety and tolerability of a triple combination consisting of ipilimumab and nivolumab immunotherapy and the metabolic therapy, ADI-PEG 20. Nine patients were enrolled in this pilot study. The combination therapy was safe and tolerable with an absence of immune-related adverse events (irAE) of special interest, but with four of nine patients experiencing a CTCAE grade 3 AE. No objective responses were observed. All except one patient developed anti-drug antibodies (ADA) within a month of the treatment initiation and therefore did not maintain arginine depletion. Further, an IFNg-dependent inflammatory signature was observed in metastatic lesions in patients pre-treated with ICB compared with patients with no pretreatment. Multiplex immunohistochemistry demonstrated variable presence of tumor infiltrating CD8 lymphocytes and PD-L1 expression at the baseline in metastases.

BackgroundImmune checkpoint blockade (ICB) has revolutionized the treatment of many cancer types; however, many patients do not benefit from these therapies due to inherent or acquired resistance. Preferential engagement in glycolysis is a hallmark of cancer cells and contributes to the progression and metastasis of many tumor types, including melanoma and triple-negative breast cancer (TNBC). Tumor reliance on glycolysis is emerging as a mechanism of resistance to immunotherapy, due in part to lactate-mediated acidification and competition for glucose in the tumor microenvironment. We recently found that knocking down the glycolytic enzyme lactate dehydrogenase A (LDHA) in 4T1 TNBC results in improved and long-lasting anti-tumor responses to CTLA-4 blockade in mice. These LDHA-defective tumors consume less glucose than wild-type tumors, leaving more glucose available in the TME for effector T cells to exert their tumoricidal function directly and via lineage destabilization of regulatory T cells. However, it remains to be determined whether systemic pharmacologic targeting of LDH can improve the efficacy of immunotherapy.MethodsLactate production and glucose consumption was quantified by YSI enzymatic analysis of metabolites. LDHA was detected by immunoblot in tumor and T cells. A clonogenic killing assay was used to assess T-cell killing. B16-F10-bearing mice were treated daily with LDH inhibitor GNE-140 and/or biweekly with anti-CTLA-4.ResultsSince activated T cells rely on glycolysis, we first determined that glycolytic cancers overexpress LDH compared to immune cells by analyzing single-cell transcripts from patient melanoma biopsies. LDHA gene expression is significantly higher in malignant cells than infiltrating CD8+ T cells, and we replicated these findings at the protein level in whole cell lysate from B16-F10 melanoma and 4T1 TNBC tumor cells vs. activated tumor-antigen specific T cells in vitro. We showed that the LDH inhibitor GNE-140 reduces tumor lactate production and glucose consumption without inhibiting anti-tumor T-cell killing in vitro. Daily treatment with GNE-140 results in reduced growth in immunocompetent but not immune deficient mice. Additionally, our preliminary findings indicate that targeting LDHA in combination with CTLA-4 blockade is more effective in slowing B16-F10 growth compared with CTLA-4 blockade alone.ConclusionsThese results suggest that targeting LDH with GNE-140 is a safe, efficacious method for relieving tumor glycolysis-mediated immunosuppression within the TME, without adversely affecting immune cell function within the tumor. Our long-term goal is to determine optimal combinations of metabolic inhibitors with ICB to alleviate the metabolic competition between tumor and infiltrating immune cells and better potentiate anti-tumor immune responses.

BackgroundMultiple suppressive mechanisms within the tumor microenvironment are capable of blunting anti-tumor T cell responses, including the engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8+ T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).1 2 While immune checkpoint blockade therapies aimed at reinvigorating T cell effector function have demonstrated their clinical effectiveness,3 4 not all patients demonstrate long-term disease control.5 The refractory nature of terminally differentiated, exhausted CD8+ T cells to be reinvigorated by PD-1 blockade is one potential cause.6–8 This limitation warrants the need to explore modulatory pathways that potentially program T cells toward exhaustion.MethodsSingle cell-RNA sequencing (scRNA-seq) data derived from the tumor-infiltrating lymphocytes (TILs) of melanoma patients9 were used for transcriptomic analysis and flow cytometry results were used to quantify protein levels in TILs. Murine B16-F10 (B16) melanoma model was used for both in vitro and in vivo studies. TCR-transgenic Pmel-1 and OT-1 transgenic mice, as well as CD47-/- (knockout, KO) mice were purchased from the Jackson Laboratory to generate CD47+/+ (wild-type, WT), CD47± (heterozygote, HET) mice with Pmel-1 or OT-1 background. For T cell co-transfer studies, Rag-deficient mice or C57BL/6j mice with sub-lethal irradiation (600cGy) were used as recipients. Naïve TCR-transgenic CD47-WT and CD47-HET CD8+ T cells were labelled, mixed in a 1:1 ratio for co-transfer experiments.ResultsFlow cytometry analysis of human melanoma TILs found a strong upregulation of CD47 expression in tumor-associated, exhausted CD8+ T cells. We confirmed that CD47 transcription is significantly elevated among CD8+ T cells with a phenotype consistent with exhaustion using scRNA-seq results of TILs derived from melanoma patients.9 Our study in murine B16 melanoma model confirms our finding in melanoma patients. To specifically address the role of CD47 in anti-tumor CD8 effector function, we conducted T cell co-transfer studies and found that CD8+ T cells with lower copy number of CD47 (CD47-HET) significantly outnumber the co-transferred CD47-WT CD8+ T cells within the tumor, exhibiting an enhanced effector function and less exhausted phenotype. Our study demonstrates a potentially novel role for CD47 in mediating CD8+ T cell exhaustion.ConclusionsCD47 expression in CD8+ T cells programs T cells toward exhaustion.Ethics ApprovalAll mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the MSKCC Institutional Animal Care and Use Committee (IACUC).ReferencesWherry EJ and M Kurachi. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15(8): p. 486–99.Thommen DS and Schumacher TN. T Cell Dysfunction in Cancer. Cancer Cell 2018;33(4): p. 547–562.Ribas A and Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 2018. 359(6382): p. 1350–1355.Sharma P and Allison JP. The future of immune checkpoint therapy. Science 2015; 48(6230): p. 56–61.Sharma P, et al. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 2017. 168(4): p. 707–723.Schietinger, A., et al., Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 2016;45(2): p. 389–401.Pauken KE, et al., Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354(6316): p. 1160–1165.Philip M, et al., Chromatin states define tumour-specific T cell dysfunction and reprogramming. Nature 2017;545(7655): p. 452–456.Sade-Feldman M, et al., Defining T Cell States associated with response to checkpoint immunotherapy in melanoma. Cell 2018;175(4): p. 998–1013e20.

BackgroundCD4 and CD8 T cells are genetically and functionally distinct cell subsets of the adaptive immune system that play pivotal roles in immune surveillance and disease control. During development in the thymus, transcription factors ThPOK and Runx3 regulate the differentiation and maturation of these two lineages into single positive T cells that enter the periphery with mutually exclusive expression of either the CD4 or CD8 co-receptor.1–2 Despite our expectation that these two cell fates are fixed, mature CD4+CD8+ double positive (DP) T cells have been described in the context of numerous immunological responses, including cancer, but their molecular and functional properties and therapeutic relevance remain controversial and largely unknown.3–5 MethodsOur lab has identified and characterized a heterogenous DP T cell population in murine and human melanoma tumors comprised of CD4 and CD8 T cells re-expressing the opposite co-receptor and a parallel uptake in the opposite cell type’s phenotype and function. Using CD4 (Trp1) and CD8 (Pmel) transgenic TCR T cells specific to B16 melanoma antigens gp75 and gp100 respectively, we demonstrate the re-expression of the opposite co-receptor following adoptive T cell transfer in B16 melanoma tumor bearing mice.ResultsSpecifically, up to 50% of transferred CD4 Trp1 T cells will re-express CD8 to become a DP T cell in the tumor microenvironment. Further, these CD4 derived DP T cells upregulate CD8 lineage regulator Runx3 and cytolytic genes Gzmb, Gzmk, and Prf1 to become potent cytotoxic T cells. Alternatively, a subset of CD8 Pmel T cells differentiate into DP T cells characterized by the increased expression of CD4, ThPOK, and regulatory marker FoxP3 (figure 1). In addition, we utilized 10x single cell and ATAC sequencing to further characterize these divergent DP T cell populations among open repertoire T cells isolated from murine and human melanoma tumors.ConclusionsOur findings highlight the capability of single positive T cells to differentiate in response to antigen and local stimuli into novel T cell subsets with polyfunctional characteristics. The resulting cell subsets will potentially affect the tumor microenvironment in distinct ways. Our studies may inform therapeutic approaches to identify antigen specific T cells as well as innovative signaling pathways to target when genetically engineering T cells to optimize cytotoxic function in the setting of adoptive cell therapy.Ethics ApprovalThe human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107ReferencesEllmeier W, Haust L & Tschismarov R. Transcriptional control of CD4 and CD8 coreceptor expression during T cell development. Cell Mol Life Sci 2013;70:4537–4553.Luckey MA, et al. The transcription factor ThPOK suppresses Runx3 and imposes CD4+ lineage fate by inducing the SOCS suppressors of cytokine signaling. Nature Immunology 2014; 15, 638–645.Bohner P, et al. Double positive CD4(+)CD8(+) T Cells are enriched in urological cancers and favor T Helper-2 polarization. Front Immunol 2019; 10, 622.Nascimbeni M, Shin E-C, Chiriboga L, Kleiner DE & Rehermann B. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood 2004;104:478–486.Nishida K, et al. Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma. Int Immunol 2020;32:347–357.