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Assessment of functional immune responses of HIV infected individuals without protective HLA-B alleles

Periodic Report Summary 1 - HIV-NOPA (Assessment of functional immune responses of HIV infected individuals without protective HLA-B alleles)

HIV infects CD4+ T cells, the helper T cells of the immune system, and establishes a chronic infection that ultimately leads to death by causing Acquired Immunodeficiency Syndrome (AIDS). Despite extensive research, no vaccine is available to date. Treatment with antiretroviral therapy is efficient in delaying the onset of AIDS, but not in eradicating viral reservoirs [1]. Natural control of the infection is very rare, but approximately 1 out of 300 HIV-infected individuals are capable of controlling the progression of the disease without taking any medications [2]. A genome-wide association study (GWAS) revealed a strong association of natural HIV-control and expression of certain human leukocyte antigen class I B alleles (HLA-B), mainly HLA-B*27 and HLA-B*57 [3]. However, not all individuals expressing those “protective” alleles are able to control progression of the disease and there is also a considerable number of HIV controllers without any protective HLA-B alleles [2] and understanding of their control-mechanism remains elusive. Thus, the project “HIV-NOPA” aimed at characterizing protective mechanisms of HIV-infected individuals lacking protective HLA-B alleles.
The objectives of the project were 1) to assess functional CD8+ T cell phenotypes of HIV elite controllers without protective HLA-B alleles, 2) to compare HIV-specific CD8+ T cell clones from HIV controllers and progressors without protective HLA-B alleles, 3) to analyze interactions between NK cells and T cells in HIV elite controllers without protective HLA-B alleles. The key technology utilized in this project was the analysis of CD8+ T cells by Mass Cytometry/CyTOF.
As a first step to characterize the T-cell phenotypes of HIV-patients without protective alleles, a 40-marker CyTOF panel has been successfully designed and validated. The panel included 12 markers for discrimination of lineages and memory phenotypes, eight exhaustion markers, five activation markers six cytokines or cytotoxic molecules and two transcription factors. To identify HIV-specific CD8+ T cells, the panel utilizes up to six different peptide-MHC tetramers matched to the HLA-class I allele and responses of each patient. CyTOF tetramer staining, which has only been utilized in few studies so far [4, 5], was adapted for this project including extensive testing of different methods to generate HIV-specific MHC-class I tetramers and design of a protocol to multiplex tetramers by using two metal-markers for each individual tetramer.
To identify HIV-specific CD8+ T-cell responses prior to the CyTOF experiments, a flow cytometry-based tetramer pre-screening protocol was also developed during the first phase of the project. In contrast to traditional screening methods like Elispot, this method has the advantage to account for responses more precisely and does only include specific CD8+ T-cell responses against known epitope-HLA combination. Responses of 60 HIV-patients have been mapped this way so far. During phase I of the project, a tetramer library of 42 different specificities (including CMV, EBV and flu epitopes besides HIV) using 20 different HLA-class I alleles was established. This tetramer library allows an unprecedented flexibility to perform flow cytometry based assays, vastly increasing the number of potentially analyzed patients and including rarer HLA-class I alleles, being studied.
The comparison of HIV-specific CD8+ T-cell phenotypes between elite controllers lacking protective alleles, elite controllers with either B*27 or B*57 alleles and chronic progressors revealed similar phenotypes. Most notable, HIV-specific CD8 T cells from all groups exhibited a specific HIV-phenotype with high PD-1 expression and a shift towards CD8 effector memory and terminally differentiated effector cells. Notably, the total population of CD8+ T cells also exhibited an HIV-specific phenotype and the CD8-profile of most HIV elite controllers was comparable to HIV chronic progressors, but differed profoundly from healthy donors that had also been included in the study. CyTOF analysis of this HIV-CD8 profile revealed high expression of exhaustion markers, such as PD-1, CD160, CD244 and TIGIT and a strong decrease in the frequency of naïve CD8 T cells as compared to healthy donors.
Unpublished studies in the laboratory have recently identified a subgroup of HIV elite controllers with diminished HIV-specific antibody responses. Notably, the CD8 profile of those patients much more resembled healthy donors than HIV patients and although regular HIV elite controllers and these ‘super’-elite controllers both have undetectable viral loads in plasma for years and in some cases decades, these two groups showed significant differences in their total CD8+ T-cell phenotypes.
The establishment of CyTOF technology during this project lead to the publication of a standardization and quality control method to monitor quality of individual samples and to facilitate reproducible data analysis. This publication includes a robust data analysis strategy utilizing a specifically validated reference sample and was an important part of analyzing CyTOF data in this project. Additionally, this strategy has sparked interest from other researchers in the CyTOF area and it is anticipated that this publication, which has been vastly supported by the Marie-Curie IOF Fellowship, will have a wider impact on future CyTOF studies by controlling for experiment-to-experiment variations and by facilitating CyTOF data analysis.
Taken together, the work performed so far has shown that it is possible to adapt CyTOF technology to the question of CD8+ T-cell phenotypes and function in human HIV infection and that MHC-class I tetramer technology allows for identification of HIV-specific CD8+ T cells. The results of this answered some of the questions in the initial proposal and revealed that CD8+ T-cell phenotypes of elite controllers largely reflect chronic progressors, except for a small group of elite controllers with decreased HIV-specific responses. The phenotypical differences between HIV controllers with and without protective HLA-B alleles are minimal, however functional responses may be impaired in patients without protective alleles, but the numbers of patients included in these experiments were too low to draw final conclusions. Further detailed analysis of T-cell function and possible alternative control mechanisms was beyond the scope of this project and will have to be addressed by future studies. The results achieved during this Marie-Curie Fellowship indicate, however, that CD8+ T cells of natural HIV-controllers, like other HIV-infected individuals, exhibit an HIV-specific phenotype that vastly differs from uninfected individuals. Functional assays may be necessary in the future to further dissect properties of protective T-cells.