Deciphering CD4+ T cell-mediated responses against cancer.

It's been long thought that CD8+ cytotoxic T cells play a major role in T cell-mediated antitumor responses, whereas CD4+ T cells merely provide some assistance to CD8+ T cells as the "helpers." In recent years, numerous studies support the notion that CD4+ T cells play an indispensable role in antitumor responses. Here, we summarize and discuss the current knowledge regarding the roles of CD4+ T cells in antitumor responses and immunotherapy, with a focus on the molecular and cellular mechanisms behind these observations. These new insights on CD4+ T cells may pave the way to further optimize cancer immunotherapy.

Identification of Neoantigen-Reactive Tumor-Infiltrating Lymphocytes in Primary Bladder Cancer.

Immune checkpoint inhibitors are effective in treating a variety of malignancies, including metastatic bladder cancer. A generally accepted hypothesis suggests that immune checkpoint inhibitors induce tumor regressions by reactivating a population of endogenous tumor-infiltrating lymphocytes (TILs) that recognize cancer neoantigens. Although previous studies have identified neoantigen-reactive TILs from several types of cancer, no study to date has shown whether neoantigen-reactive TILs can be found in bladder tumors. To address this, we generated TIL cultures from patients with primary bladder cancer and tested their ability to recognize tumor-specific mutations. We found that CD4+ TILs from one patient recognized mutated C-terminal binding protein 1 in an MHC class II-restricted manner. This finding suggests that neoantigen-reactive TILs reside in bladder cancer, which may help explain the effectiveness of immune checkpoint blockade in this disease and also provides a rationale for the future use of adoptive T cell therapy targeting neoantigens in bladder cancer.

Cancer immunotherapy targeting neoantigens.

Neoantigens are antigens encoded by tumor-specific mutated genes. Studies in the past few years have suggested a key role for neoantigens in cancer immunotherapy. Here we review the discoveries of neoantigens in the past two decades and the current advances in neoantigen identification. We also discuss the potential benefits and obstacles to the development of effective cancer immunotherapies targeting neoantigens.

T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer.

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

An Efficient Single-Cell RNA-Seq Approach to Identify Neoantigen-Specific T Cell Receptors.

The adoptive transfer of neoantigen-reactive tumor-infiltrating lymphocytes (TILs) can result in tumor regression in patients with metastatic cancer. To improve the efficacy of adoptive T cell therapy targeting these tumor-specific mutations, we have proposed a new therapeutic strategy, which involves the genetic modification of autologous T cells with neoantigen-specific T cell receptors (TCRs) and the transfer of these modified T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor intensive, time consuming, and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-transfected or peptide-pulsed autologous antigen-presenting cells (APCs) and the single-cell RNA sequencing (RNA-seq) analysis of T cells to identify paired TCR sequences associated with cells expressing high levels of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Following this new approach, multiple TCRs were identified, synthesized, cloned into a retroviral vector, and then transduced into donor T cells. These transduced T cells were shown to specifically recognize the neoantigens presented by autologous APCs. In conclusion, this approach provides an efficient procedure to isolate neoantigen-specific TCRs for clinical applications, as well as for basic and translational research.

Targeting neoantigens for cancer immunotherapy.

Studies first carried out in the 1980s have demonstrated murine T cells can recognize mutated gene products, known as neoantigens, and that these T cells are capable of mediating tumor rejection. The first human tumor antigens isolated in the early 1990s were the products of non-mutated genes expressed in a tissue-specific manner; subsequent studies have indicated that tumor-infiltrating lymphocytes that are cultured in vitro frequently recognize mutated gene products. In addition, correlative studies indicate that clinical responses to therapies involving the use of antibodies directed against checkpoint inhibitors such as CTLA-4 and PD-1 may be associated with mutational burden, providing indirect evidence that these responses may primarily be mediated by neoantigen-reactive T cells. The importance of neoantigen-reactive T cells may be elucidated by the results of ongoing and future studies aimed at leveraging information gained from mutational profiling to enhance the potency of immunotherapies.

Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression.

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

Levels of peripheral CD4(+)FoxP3(+) regulatory T cells are negatively associated with clinical response to adoptive immunotherapy of human cancer.

CD4(+)FoxP3(+) regulatory T cells (Tregs) have been shown to suppress T cell-mediated host immune responses against self- and nonself-antigens; however, the impact of CD4(+) Tregs on human antitumor immune responses and their influence on cancer treatment are unknown. In the present study, we explored the factors that influence CD4(+) Treg reconstitution in patients receiving adoptive immunotherapy following conditioning regimens designed to enhance T-cell function and evaluated potential associations between CD4(+) Treg levels and clinical responses to therapy. The analysis of 4 trials employing nonmyeloablative chemotherapy with or without total body irradiation (TBI) before adoptive T-cell transfer revealed that the percentage and number of reconstituting CD4(+)FoxP3(+) Tregs observed in the peripheral blood was higher in nonresponders than in responders. The addition of TBI resulted in a further depletion of CD4(+) Tregs, and the degree of depletion was dependent on the TBI dose. The number of administered doses of IL-2 was found to be positively associated with peripheral Treg reconstitution. These observations provide strong evidence that endogenous CD4(+) Tregs have a negative impact on cancer therapy, and suggest that strategies reducing Treg levels may provide clinical benefit to cancer patients. All 5 clinical trials are registered at www.clinicaltrials.gov as NCT00001832, NCT00096382, NCT00335127, NCT00509496, and NCT00513604.

Crucial role for TNF receptor-associated factor 2 (TRAF2) in regulating NFκB2 signaling that contributes to autoimmunity.

TNF receptor-associated factor 2 (TRAF2) is a key intracellular signaling mediator that acts downstream of not only TNFα but also various members of the TNFα superfamily. Here, we report that, despite their lack of TNFα signaling, TRAF2(-/-)TNFα(-/-) mice develop an inflammatory disorder characterized by autoantibody accumulation and organ infiltration by T cells with the phenotypes of activated, effector, and memory cells. RAG1(-/-) mice reconstituted with TRAF2(-/-)TNFα(-/-) bone marrow cells showed increased numbers of hyperactive T cells and rapidly developed progressive and eventually lethal inflammation. No inflammation was observed in RAG1(-/-) mice reconstituted with TRAF2(-/-)TNFα(-/-)T-cell receptor ß(-/-) or TRAF2(-/-)TNFα(-/-)NFκB-induced kinase(+/-) bone marrow cells. The pathogenic TRAF2(-/-)TNFα(-/-) T cells showed constitutive NFκB2p52 activation and produced elevated levels of T-helper 1 and T-helper 17 cytokines. Our results suggest that a regulatory circuit consisting of TRAF2-NFκB-induced kinase-NFκB2p52 is essential for the proper control of effector T-cell polarization and that loss of T-cell TRAF2 function induces constitutive NFκB2p52 activity that drives fatal autoimmune inflammation independently of TNFα signaling. The involvement of this regulatory circuit in controlling autoimmune responses highlights the delicate balance required to avoid paradoxical adverse events when implementing new targeted anti-inflammatory therapies.

C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a.

Complement-derived anaphylatoxins regulate immune and inflammatory responses through G-protein-coupled receptor (GPCR)-mediated signalling. C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence. Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optimizing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling, C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper-responsiveness and inflammation, and are mildly delayed in haematopoietic cell regeneration after gamma-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses.

Oral Shedding of an Oncogenic Virus Alters the Oral Microbiome in HIV+ Patients.

Kaposi's Sarcoma (KS) caused by Kaposi's sarcoma-associated herpesvirus (KSHV) continues to be the most common AIDS-associated tumor. Involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. Numerous types of cancer are associated with the alterations of in components of the microbiome. However, little is known about how KSHV coinfection affects the oral microbiome in HIV+ patients, especially in a "pre-cancer" niche. Using 16S rRNA pyrosequencing, we found that oral shedding of KSHV correlated with altered oral microbiome signatures in HIV+ patients, including a reduction in the microbiota diversity, changing the relative composition of specific phyla and species, and regulating microbial functions. Furthermore, we found that Streptococcus sp., one of the most increased species in the oral cavity of HIV+/KSHV+ patients, induced KSHV lytic reactivation in primary oral cells. Together, these data indicate that oral shedding of KSHV may manipulate the oral microbiome to promote viral pathogenesis and tumorigenesis especially in immunocompromised patients.

TREM-2 mediates dendritic cell-induced NO to suppress Th17 activation and ameliorate chronic kidney diseases.

Chronic kidney disease (CKD) is a global public health issue. CKD is caused by the infiltration of various myeloid cell types into renal tissue, resulting in renal fibrosis and tubular atrophy. Unilateral ureteral obstruction (UUO) surgery in mice is a model of CKD and characterized by high expression of the anti-inflammatory receptor, Triggering receptor expressed on myeloid cells 2 (TREM-2), on myeloid cells in affected kidneys. Here, we show that iNOS expression and nitric oxide (NO) induction were decreased in Trem-2-/- bone marrow-derived DCs (BMDCs) and in Trem-2 knockdown DC2.4 cells stimulated in vitro with LPS. The nitration of RORγt was decreased in T cells co-cultured with LPS-stimulated Trem-2-/- BMDCs, enhancing IL-17 production. UUO-treated Trem-2-/- mice displayed aggravated renal pathogenesis accompanied by greater neutrophil infiltration and enhanced Th17 cells differentiation, phenotypes that could be rescued by the administration of L-arginine (a biological precursor of NO). Our data identify a key mechanism underlying TREM-2-mediated NO to modulate the cellular crosstalk between dendritic cells, Th17, and neutrophils. Furthermore, we also reveal TREM-2 as a potential novel target for the development of anti-inflammatory drugs in CKD treatment. KEY MESSAGES: The expression of TREM-2 is increased in nephritis TREM-2+ DCs maintain NO production to negatively regulate Th17 differentiation The severe pathologies of nephritis can be rescued by L-arginine supplementation.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

c-Rel phenocopies PKCtheta but not Bcl-10 in regulating CD8+ T-cell activation versus tolerance.

Elucidating the signaling events that promote T-cell tolerance versus activation provides important insights for manipulating immunity in vivo. Previous studies have suggested that the absence of PKCtheta results in the induction of anergy and that the balance between the induction of the transcription factors NFAT, AP1 and NF-kappaB plays a key role in determining whether T-cell anergy or activation is induced. Here, we examine whether Bcl-10 and specific family members of NF-kappaB act downstream of PKCtheta to alter CD8(+) T-cell activation and/or anergy. We showed that T cells from mice deficient in c-Rel but not NF-kappaB1 (p50) have increased susceptibility to the induction of anergy, similar to T cells from PKCtheta-deficient mice. Surprisingly T cells from Bcl-10-deficient mice showed a strikingly different phenotype to the PKCtheta-deficient T cells, with a severe block in TCR-mediated activation. Furthermore, we have also shown that survival signals downstream of NF-kappaB, are uncoupled from signals that mediate T-cell anergy. These results suggest that c-Rel plays a critical role downstream of PKCtheta in controlling CD8(+) T-cell anergy induction.

Differential role for c-Rel and C/EBPbeta/delta in TLR-mediated induction of proinflammatory cytokines.

TLR stimulation triggers a signaling pathway via MyD88 and IL-1R-associated kinase 4 that is essential for proinflammatory cytokine induction. Although NF-kappaB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. In this study, we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12p40 induction, but not IL-6 or TNF-alpha. We also investigated other transcription factors and showed that C/EBPbeta and C/EBPdelta expression was limited in MyD88- or IL-1R-associated kinase 4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.

Single-Cell TCR and Transcriptome Analysis: An Indispensable Tool for Studying T-Cell Biology and Cancer Immunotherapy.

T cells have been known to be the driving force for immune response and cancer immunotherapy. Recent advances on single-cell sequencing techniques have empowered scientists to discover new biology at the single-cell level. Here, we review the single-cell techniques used for T-cell studies, including T-cell receptor (TCR) and transcriptome analysis. In addition, we summarize the approaches used for the identification of T-cell neoantigens, an important aspect for T-cell mediated cancer immunotherapy. More importantly, we discuss the applications of single-cell techniques for T-cell studies, including T-cell development and differentiation, as well as the role of T cells in autoimmunity, infectious disease and cancer immunotherapy. Taken together, this powerful tool not only can validate previous observation by conventional approaches, but also can pave the way for new discovery, such as previous unidentified T-cell subpopulations that potentially responsible for clinical outcomes in patients with autoimmunity or cancer.

Neoantigen-Reactive T Cells: The Driving Force behind Successful Melanoma Immunotherapy.

Patients with metastatic cutaneous melanoma have experienced significant clinical responses after checkpoint blockade immunotherapy or adoptive cell therapy. Neoantigens are mutated proteins that arise from tumor-specific mutations. It is hypothesized that the neoantigen recognition by T cells is the critical step for T-cell-mediated anti-tumor responses and subsequent tumor regressions. In addition to describing neoantigens, we review the sentinel and ongoing clinical trials that are helping to shape the current treatments for patients with cutaneous melanoma. We also present the existing evidence that establishes the correlations between neoantigen-reactive T cells and clinical responses in melanoma immunotherapy.

Liquid Biopsy in Hepatocellular Carcinoma: Opportunities and Challenges for Immunotherapy.

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. HCC is often diagnosed at a late stage when the therapeutic options are very limited. However, even at the earlier stages, the best treatment is liver transplantation, surgical resection or ablation. Surgical resection and ablation may carry a high risk of tumor recurrence. The recent introduction of immunotherapies resulted in clinical responses for a subgroup of patients, but there were still no effective predictive markers for response to immunotherapy or for recurrence after surgical therapy. The identification of biomarkers that could correlate and predict response or recurrence would require close monitoring of the patients throughout and after the completion of treatment. However, this would not be performed efficiently by repeated and invasive tissue biopsies. A better approach would be to use liquid biopsies including circulating tumor DNA (ctDNA), circulating RNA (e.g., microRNAs), circulating tumor cells (CTC) and extracellular vesicles (EVs) (e.g., exosomes) for disease monitoring in a non-invasive manner. In this review, we discuss the currently available technology that can enable the use of liquid biopsy as a diagnostic and prognostic tool. Moreover, we discuss the opportunities and challenges of the clinical application of liquid biopsy for immunotherapy of HCC.