Herpes Virus Infections in Kidney Transplant Patients (HINT) - a prospective observational cohort study.

BACKGROUND: Kidney transplant recipients receive maintenance immunosuppressive therapy to avoid allograft rejection resulting in increased risk of infections and infection-related morbidity and mortality. Approximately 98% of adults are infected with varicella zoster virus, which upon reactivation causes herpes zoster. The incidence of herpes zoster is higher in kidney transplant recipients than in immunocompetent individuals, and kidney transplant recipients are at increased risk of severe herpes zoster-associated disease. Vaccination with adjuvanted recombinant glycoprotein E subunit herpes zoster vaccine (RZV) prevents herpes zoster in older adults with excellent efficacy (90%), and vaccination of kidney transplant candidates is recommended in Danish and international guidelines. However, the robustness and duration of immune responses after RZV vaccination, as well as the optimal timing of vaccination in relation to transplantation remain unanswered questions. Thus, the aim of this study is to characterize the immune response to RZV vaccination in kidney transplant candidates and recipients at different timepoints before and after transplantation. METHODS: The Herpes Virus Infections in Kidney Transplant Patients (HINT) study is a prospective observational cohort study. The study will include kidney transplant candidates on the waiting list for transplantation (n = 375) and kidney transplant recipients transplanted since January 1, 2019 (n = 500) from all Danish kidney transplant centers who are offered a RZV vaccine as routine care. Participants are followed with repeated blood sampling until 12 months after inclusion. In the case of transplantation or herpes zoster disease, additional blood samples will be collected until 12 months after transplantation. The immune response will be characterized by immunophenotyping and functional characterization of varicella zoster virus-specific T cells, by detection of anti-glycoprotein E antibodies, and by measuring cytokine profiles. DISCUSSION: The study will provide new knowledge on the immune response to RZV vaccination in kidney transplant candidates and recipients and the robustness and duration of the response, potentially enhancing preventive strategies against herpes zoster in a population at increased risk. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05604911).

Optimization in Detection of Antigen-Specific T Cells Through Differentially Labeled MHC Multimers.

A large variety of fluorescent molecules are used on a regular basis to tag major histocompatibility complex (MHC) multimers for detection of antigen-specific T cells. We have evaluated the way in which the choice of fluorescent label can impact the detection of MHC multimer binding T cells in an exploratory proficiency panel where detection of MHC multimer binding T cells was assessed across 16 different laboratories. We found that the staining index (SI) of the multimer reagent provided the best direct correlation with the value of a given fluorochrome for T cell detection studies. The SI is dependent on flow cytometer settings and chosen antibody panel; hence, the optimal fluorochrome selection may differ from lab to lab. Consequently, we describe a strategy to evaluate performance of the detection channels and optimize the SI for selected fluorescent molecules. This approach can easily be used to test and optimize fluorescence detection in relation to MHC multimer staining and in general, for antibody-based identification of rare cell populations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens.

Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development.

T-cell Responses in the Microenvironment of Primary Renal Cell Carcinoma-Implications for Adoptive Cell Therapy.

In vitro expansion of large numbers of highly potent tumor-reactive T cells appears a prerequisite for effective adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TIL) as shown in metastatic melanoma (MM). We therefore sought to determine whether renal cell carcinomas (RCC) are infiltrated with tumor-reactive T cells that could be efficiently employed for adoptive transfer immunotherapy. TILs and autologous tumor cell lines (TCL) were successfully generated from 22 (92%) and 17 (77%) of 24 consecutive primary RCC specimens and compared with those generated from metastatic melanoma. Immune recognition of autologous TCLs or fresh tumor digests was observed in CD8+ TILs from 82% of patients (18/22). Cytotoxicity assays confirmed the tumoricidal capacity of RCC-TILs. The overall expansion capacity of RCC-TILs was similar to MM-TILs. However, the magnitude, polyfunctionality, and ability to expand in classical expansion protocols of CD8+ T-cell responses was lower compared with MM-TILs. The RCC-TILs that did react to the tumor were functional, and antigen presentation and processing of RCC tumors was similar to MM-TILs. Direct recognition of tumors with cytokine-induced overexpression of human leukocyte antigen class II was observed from CD4+ T cells (6/12; 50%). Thus, TILs from primary RCC specimens could be isolated, expanded, and could recognize tumors. However, immune responses of expanded CD8+ RCC-TILs were typically weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select, enrich, and expand tumor-reactive polyfunctional T cells may be critical in developing effective ACT with TILs for RCC. In summary, TILs isolated from primary RCC specimens could recognize tumors. However, their immune responses were weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select and expand polyfunctional T cells may improve cell therapy for RCC. Cancer Immunol Res; 6(2); 222-35. ©2018 AACR.

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells.

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore difficult to detect. We used a highly heterogeneous dataset from a recent MHC multimer proficiency panel to assess if MHC multimer-binding CD8+ T cells could be analyzed with computational solutions currently available, and if such analyses would reduce the technical variation across different laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0.01 to 1.5% of lymphocytes within samples from two donors. Experience from this analysis shows that all three programs can be used for the identification of high to intermediate frequency of MHC multimer-binding T cell populations, with results very similar to that of manual gating. For the less frequent populations (<0.1% of live, single lymphocytes), SWIFT outperformed the other tools. As used in this study, none of the algorithms offered a completely automated pipeline for identification of MHC multimer populations, as varying degrees of human interventions were needed to complete the analysis. In this study, we demonstrate the feasibility of using automated analysis pipelines for assessing and identifying even rare populations of antigen-responsive T cells and discuss the main properties, differences, and advantages of the different methods tested.

T-cell responses to oncogenic merkel cell polyomavirus proteins distinguish patients with merkel cell carcinoma from healthy donors.

PURPOSE: Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with strong evidence of viral carcinogenesis. The association of MCC with the Merkel cell polyomavirus (MCPyV) may explain the explicit immunogenicity of MCC. Indeed, MCPyV-encoded proteins are likely targets for cytotoxic immune responses to MCC as they are both foreign to the host and necessary to maintain the oncogenic phenotype. However, to date only a single MCPyV-derived CD8 T-cell epitope has been described, thus impeding specific monitoring of T-cell responses to MCC. METHOD: To overcome this limitation, we scanned the MCPyV oncoprotein large T and small T antigens and the virus capsid protein VP1 for potential T-cell epitopes, and tested for MHC class I affinity. We confirmed the relevance of these epitopes using a high-throughput platform for T-cell enrichment and combinatorial encoding of MHC class I multimers. RESULTS: In peripheral blood from 38 patients with MCC and 30 healthy donors, we identified 53 MCPyV-directed CD8 T-cell responses against 35 different peptide sequences. Strikingly, T-cell responses against oncoproteins were exclusively present in patients with MCC, but not in healthy donors. We further demonstrate both the processing and presentation of the oncoprotein-derived epitopes, as well as the lytic activity of oncoprotein-specific T cells toward MHC-matched MCC cells. Demonstrating the presence of oncoprotein-specific T cells among tumor-infiltrating lymphocytes further substantiated the relevance of the identified epitopes. CONCLUSION: These T-cell epitopes represent ideal targets for antigen-specific immune therapy of MCC, and enable tracking and characterization of MCPyV-specific immune responses.