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.

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.

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.

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.

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.

Expansion of Human Papillomavirus-Specific T Cells in Periphery and Cervix in a Therapeutic Vaccine Recipient Whose Cervical High-Grade Squamous Intraepithelial Lesion Regressed.

Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) ß deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. In order to verify the vaccine-specificity of these clonotypes, T cells with specificity to a region, HPV 16 E6 91-115, previously identified to be vaccine-induced using an interferon-γ enzyme-linked immunospot assay, were sorted and analyzed using single-cell RNA-seq and TCR sequencing. HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR ß bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells not only in the periphery but also in the cervix. Such an approach should be useful as a novel approach to assess vaccine-specific responses in various anatomical areas.

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.

Identification of neoantigen-reactive T lymphocytes in the peripheral blood of a patient with glioblastoma.

The adoptive transfer of naturally occurring T cells that recognize cancer neoantigens has led to durable tumor regressions in select patients with cancer. However, it remains unknown whether such T cells can be isolated from and used to treat patients with glioblastoma, a cancer that is refractory to currently available therapies. To answer this question, we stimulated patient blood-derived memory T cells in vitro using peptides and minigenes that represented point mutations unique to patients' tumors (ie, candidate neoantigens) and then tested their ability to specifically recognize these mutations. In a cohort of five patients with glioblastoma, we found that circulating CD4+ memory T cells from one patient recognized a cancer neoantigen harboring a mutation in the EED gene (EEDH189N) that was unique to that patient's tumor. This finding suggests that neoantigen-reactive T cells could indeed be isolated from patients with glioblastoma, thereby providing a rationale for further efforts to develop neoantigen-directed adoptive T cell therapy for this disease.

Direct identification of neoantigen-specific TCRs from tumor specimens by high-throughput single-cell sequencing.

BACKGROUND: Recognition of neoantigens by T cells plays a major role in cancer immunotherapy. Identification of neoantigen-specific T-cell receptors (TCRs) has become a critical research tool for studying T cell-mediated responses after immunotherapy. In addition, neoantigen-specific TCRs can be used to modify the specificity of T cells for T cell-based therapies targeting tumor-specific mutations. Although several techniques have been developed to identify TCR sequences, these techniques still require a significant amount of labor, making them impractical in the clinical setting. METHODS: Thanks to the availability of high-throughput single-cell sequencing, we developed a new process to isolate neoantigen-specific TCR sequences. This process included the isolation of tumor-infiltrating T cells from a tumor specimen and the stimulation of T cells by neoantigen-loaded dendritic cells, followed by single-cell sequencing for TCR and T-cell activation markers, interferon-γ and interleukin-2. RESULTS: In this study, potential neoantigen-specific TCRs were isolated from three melanoma and three colorectal tumor specimens. These TCRs were then synthesized and transduced into autologous T cells, followed by testing the recognition of neoantigens. A total of 28 neoantigen-specific TCRs were identified by this process. If identical TCR sequences were detected from two or more single cells, this approach was highly reliable (100%, 19 out of 19 TCRs). CONCLUSION: This single-cell approach provides an efficient process to isolate antigen-specific TCRs for research and clinical applications.

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.

Single-Cell Transcriptome Analysis Reveals Gene Signatures Associated with T-cell Persistence Following Adoptive Cell Therapy.

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can mediate responses in some patients with metastatic epithelial cancer. Identifying gene signatures associated with successful ACT might enable the development of improved therapeutic approaches. The persistence of transferred T cells in the peripheral blood is one indication of clinical effectiveness, but many T-cell and host factors may influence T-cell persistence. To limit these variables, we previously studied a patient with metastatic colorectal cancer treated with polyclonal TILs targeting the KRAS(G12D) hotspot mutation, who experienced a partial response for 9 months. Three dominant clonotypes specifically recognizing KRAS(G12D) epitopes were identified, but we found that only two clonotypes persisted 40 days after ACT. Because of these findings, in this study, we performed the single-cell transcriptome analysis of the infused TILs. The analysis revealed a total of 472 genes that were differentially expressed between clonotypes 9.1-NP and 9.2-P single cells, and 528 genes between 9.1-NP and 10-P. Following these clonotypes in the peripheral blood after ACT, the gene expression patterns changed, but IL7R, ITGB1, KLF2, and ZNF683 remained expressed in the persistent 9.2-P and 10-P cells, compared with the nonpersistent 9.1-NP cells. In addition, four autologous TILs, which were used for treatment but persisted poorly 1 month after ACT, did not express the gene profiles associated with persistence. These results suggest that certain TIL populations possess a unique gene expression profile that can lead to the persistence of T cells. Thus, this single-patient study provides insight into how to improve ACT for solid cancer.

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.

Pilot Trial of Adoptive Transfer of Chimeric Antigen Receptor-transduced T Cells Targeting EGFRvIII in Patients With Glioblastoma.

A deletion variant of epidermal growth factor receptor (EGFRvIII) is a known driver mutation in a subset of primary and secondary glioblastoma multiforme. Adoptive transfer of genetically modified chimeric antigen receptor (CAR) lymphocytes has demonstrated efficacy in hematologic malignancies but is still early in development for solid cancers. The surface expression of the truncated extracellular ligand domain created by EGFRvIII makes it an attractive target for a CAR-based cancer treatment. Patients with recurrent glioblastoma expressing EGFRvIII were enrolled in a dose escalation phase I trial, using a third-generation CAR construct derived from a human antibody. Transduced cells were administered after lymphodepleting chemotherapy and supported posttransfer with intravenous interleukin-2. The dose escalation proceeded at half-log increments from 10 to >10 cells. Primary endpoints were safety and progression-free survival. Eighteen patients were treated with final infusion products ranging from 6.3×10 to 2.6×10 anti-EGFRvIII CAR T cells. Median progression-free survival was 1.3 months (interquartile range: 1.1-1.9), with a single outlier of 12.5 months. Two patients experienced severe hypoxia, including one treatment-related mortality after cell administration at the highest dose level. All patients developed expected transient hematologic toxicities from preparative chemotherapy. Median overall survival was 6.9 months (interquartile range: 2.8-10). Two patients survived over 1 year, and a third patient was alive at 59 months. Persistence of CAR cells correlated with cell dose, but there were no objective responses. Administration of anti-EGFRvIII CAR-transduced T cells did not demonstrate clinically meaningful effect in patients with glioblastoma multiforme in this phase I pilot trial.

Immunologic Recognition of a Shared p53 Mutated Neoantigen in a Patient with Metastatic Colorectal Cancer.

Adoptive cell therapy (ACT) with T cells targeting neoantigens can mediate durable responses in patients with metastatic cancer. Cell therapies targeting common shared antigens for epithelial cancers are not yet broadly available. Here, we report the identification and characterization in one patient of T-cell receptors (TCRs) recognizing mutated p53 p.R175H, which is shared among a subset of patients with cancer. Tumor-infiltrating lymphocytes were screened for recognition of mutated neoantigens in a patient with metastatic colorectal cancer. HLA-A*0201-restricted recognition of mutated p53 p.R175H was identified, and the minimal peptide epitope was HMTEVVRHC. Reactive T cells were isolated by tetramer sorting, and three TCRs were identified. These TCRs mediated recognition of commercially available ovarian cancer, uterine carcinoma, and myeloma cell lines, as well as an NIH patient-derived esophageal adenocarcinoma line that endogenously expressed p53 p.R175H and HLA-A*0201. They also mediated recognition of p53 p.R175H+ colon, breast, and leukemia cell lines after transduction with a retrovirus encoding HLA-A*0201. This work demonstrates that common shared mutated epitopes such as those found in p53 can elicit immunogenic responses and that the application of ACT may be extended to patients with any cancer histology that expresses both HLA-A*0201 and the p53 p.R175H mutation.

Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients.

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4+, and CD8+ memory T cells targeting the mutated KRASG12D and KRASG12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.

Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.

Immunotherapy using either checkpoint blockade or the adoptive transfer of antitumor lymphocytes has shown effectiveness in treating cancers with high levels of somatic mutations-such as melanoma, smoking-induced lung cancers and bladder cancer-with little effect in other common epithelial cancers that have lower mutation rates, such as those arising in the gastrointestinal tract, breast and ovary1-7. Adoptive transfer of autologous lymphocytes that specifically target proteins encoded by somatically mutated genes has mediated substantial objective clinical regressions in patients with metastatic bile duct, colon and cervical cancers8-11. We present a patient with chemorefractory hormone receptor (HR)-positive metastatic breast cancer who was treated with tumor-infiltrating lymphocytes (TILs) reactive against mutant versions of four proteins-SLC3A2, KIAA0368, CADPS2 and CTSB. Adoptive transfer of these mutant-protein-specific TILs in conjunction with interleukin (IL)-2 and checkpoint blockade mediated the complete durable regression of metastatic breast cancer, which is now ongoing for >22 months, and it represents a new immunotherapy approach for the treatment of these patients.

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.