Cultured human fetal astrocytes can be induced by interferon-gamma to express HLA-DR.

Interferon-gamma (IFN-gamma) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-gamma. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-gamma. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-gamma treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, Drw52, DRw53 and DQwl. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-gamma in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Treatment-dependent loss of polyfunctional CD8+ T-cell responses in HIV-infected kidney transplant recipients is associated with herpesvirus reactivation.

Antiretroviral-therapy has dramatically changed the course of HIV infection and HIV-infected (HIV(+)) individuals are becoming more frequently eligible for solid-organ transplantation. However, only scarce data are available on how immunosuppressive (IS) strategies relate to transplantation outcome and immune function. We determined the impact of transplantation and immune-depleting treatment on CD4+ T-cell counts, HIV-, EBV-, and Cytomegalovirus (CMV)-viral loads and virus-specific T-cell immunity in a 1-year prospective cohort of 27 HIV(+) kidney transplant recipients. While the results show an increasing breadth and magnitude of the herpesvirus-specific cytotoxic T-cell (CTL) response over-time, they also revealed a significant depletion of polyfunctional virus-specific CTL in individuals receiving thymoglobulin as a lymphocyte-depleting treatment. The disappearance of polyfunctional CTL was accompanied by virologic EBV-reactivation events, directly linking the absence of specific polyfunctional CTL to viral reactivation. The data provide first insights into the immune-reserve in HIV+ infected transplant recipients and highlight new immunological effects of thymoglobulin treatment. Long-term studies will be needed to assess the clinical risk associated with thymoglobulin treatment, in particular with regards to EBV-associated lymphoproliferative diseases.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

Clinically-relevant threshold of preformed donor-specific anti-HLA antibodies in kidney transplantation.

BACKGROUND: Pretransplant anti-HLA donor-specific antibodies (DSA) are recognized as a risk factor for acute antibody-mediated rejection (AMR) in kidney transplantation. The predictive value of C4d-fixing capability by DSA or of IgG DSA subclasses for acute AMR in the pretransplant setting has been recently studied. In addition DSA strength assessed by mean fluorescence intensity (MFI) may improve risk stratification. We aimed to analyze the relevance of preformed DSA and of DSA MFI values. METHODS: 280 consecutive patients with negative complement-dependent cytotoxicity crossmatches received a kidney transplant between 01/2008 and 03/2014. Sera were screened for the presence of DSA with a solid-phase assays on a Luminex flow analyzer, and the results were correlated with biopsy-proven acute AMR in the first year and survival. RESULTS: Pretransplant anti-HLA antibodies were present in 72 patients (25.7%) and 24 (8.6%) had DSA. There were 46 (16.4%) acute rejection episodes, 32 (11.4%) being cellular and 14 (5.0%) AMR. The incidence of acute AMR was higher in patients with pretransplant DSA (41.7%) than in those without (1.6%) (p<0.001). The median cumulative MFI (cMFI) of the group DSA+/AMR+ was 5680 vs 2208 in DSA+/AMR- (p=0.058). With univariate logistic regression a threshold value of 5280 cMFI was predictive for acute AMR. DSA cMFI's ability to predict AMR was also explored by ROC analysis. AUC was 0.728 and the best threshold was a cMFI of 4340. Importantly pretransplant DSA>5280 cMFI had a detrimental effect on 5-year graft survival. CONCLUSIONS: Preformed DSA cMFI values were clinically-relevant for the prediction of acute AMR and graft survival in kidney transplantation. A threshold of 4300-5300 cMFI was a significant outcome predictor.