Multiparameter single-cell profiling of human CD4+FOXP3+ regulatory T-cell populations in homeostatic conditions and during graft-versus-host disease.

Understanding the heterogeneity of human CD4+FOXP3+ regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4+ effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-γ showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4+FOXP3+ T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4+FOXP3+ T cells, explains the remarkable complexity and functional diversity of human Tregs.

Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer.

BACKGROUND AIMS: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies. METHODS: In a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr(51) release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion. RESULTS: TIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8(+) and CD4(+) subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells. CONCLUSION: The procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.

Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a clinical phase 1/2 trial.

Therapeutic dendritic cell (DC) vaccination against cancer is a strategy aimed at activating the immune system to recognize and destroy tumor cells. In this nonrandomized phase 1/2 trial, we investigated the safety, feasibility, induction of T-cell response, and clinical response after treatment with a DC-based vaccine in patients with metastatic renal cell carcinoma. Twenty-seven patients with progressive cytokine-refractory metastatic renal cell carcinoma were vaccinated with DCs loaded with either a cocktail of survivin and telomerase peptides or tumor lysate depending on their HLA-A2 haplotype, and low-dose IL-2 was administered concomitantly. Tumor response, immune response, and serum IL-6 and YKL-40 were measured during treatment. Vaccine generation was successful in all patients and no serious adverse events were observed. None of the patients had an objective response but 13/27 patients obtained disease stabilization (SD) for more than 8 weeks. An antigen-specific immune response was demonstrated in 6/6 patients tested. Furthermore, significant alterations in serum YKL-40 and IL-6 were found during treatment. In conclusion, DC vaccination in our setting is feasible and without severe toxicity. Almost half of the patients obtained SD, and in more than 1/3 of the patients, SD persisted for more than 6 months. However, the evaluation of SD is difficult to interpret in the absence of a randomized trial and, therefore, these results should be interpreted with caution. Antigen-specific immune responses were observed in a subset of the treated patients.

RhoC a new target for therapeutic vaccination against metastatic cancer.

Most cancer deaths are due to the development of metastases. Increased expression of RhoC is linked to enhanced metastatic potential in multiple cancers. Consequently, the RhoC protein is an attractive target for drug design. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The over-expression of RhoC in cancer and the fact that immune escape by down regulation or loss of expression of this protein would reduce the morbidity and mortality of cancer makes RhoC a very attractive target for anti-cancer immunotherapy. Herein, we describe an HLA-A3 restricted epitope from RhoC, which is recognized by cytotoxic T cells. Moreover, RhoC-specific T cells show cytotoxic potential against HLA-matched cancer cells of different origin. Thus, RhoC may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies.

The immunogenicity of the hTERT540-548 peptide in cancer.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is an attractive target antigen for cancer immunotherapy due to its expression in the vast majority of human tumors. The first immunogenic peptide described from hTERT was the HLA-A2-restricted peptide hTERT540 (ILAKFLHWL). However, much discrepancy exists about the processing and presentation of this epitope on the surface of neoplastic cells. Originally, it was described that specific CTL can be generated in vitro and that such cells are able to kill a range of hTERT(+) tumor cell lines and primary tumors in a peptide-specific, HLA-A2-restricted fashion. Furthermore, it was described that vaccination of cancer patients with hTERT540 introduced functional antitumor CD8(+) T cells in patients. More recently, it was described that most patients with cancer have circulating hTERT540-specific CD8(+) T lymphocytes. In contrast, several other studies have concluded that hTERT540 is not presented on the surface of tumor cells and that immunization of cancer patients with hTERT540 leads to the introduction of specific T cells that do not recognize tumor cells in vivo. In the present commentary, we summarize these highly contradictive results about this potentially very important T-cell epitope. Furthermore, we describe novel data showing that naturally occurring immune responses against hTERT540 are, although rare, present in cancer patients and that such hTERT540-specific T cells are able to recognize and kill cancer cells. Hence, our data support the findings that hTERT540 peptide is presented by human tumors and can be a target for CTL-mediated tumor lysis.

Identification of a new hTERT-derived HLA-A*0201 restricted, naturally processed CTL epitope.

By the use of a neural network capable of performing quantitative predictions of peptides binding to HLA-A*0201 molecules, we identified a number of nonamer peptides derived from the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). Five nonimmunogenic peptides with measured binding affinities for HLA-A*0201 ranging from 155 to 1,298 nM were modified at the P1, P2 and P9 positions, respectively, to achieve stronger HLA-A*0201 binding. One peptide, mp30-38 (mp30), with an L to V substitution at position 9 was subsequently found to be immunogenic in mp30 immunized HLA-A*0201/H2K(b) or HHD transgenic mice. The T cell reactivity obtained was directed against both the mp30 and against the unmodified p30. Anti-mp30 specific T cells generated in HLA-A*0201 transgenic mice were dependent on TCR-CD8/MHC-I alpha3 binding and therefore not capable of recognizing mp30-pulsed human HLA-A*0201(+) cells or murine HLA-A*0201 transfectants. In order to show reactivity against naturally processed peptide in human tumor cells, an hTERT positive HLA-A*0201 negative colon carcinoma cell line (CCL220) was transfected with an HLA-A*0201/H2K(b) cDNA construct and used as target in ELISPOT and cytotoxicity assays. The data show that T cells from mp30 immunized HHD transgenic mice react specifically against the CCL220 transfectant indicating that p30 is naturally processed. In conclusion, we have identified a new CTL HLA-A*0201 restricted hTERT epitope, which is now, included in an ongoing phase 2 vaccine trial of patients with disseminated cancer.