A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans

Abstract

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50–75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive “megapool” of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

“…By the use of this protocol pools of hundreds of epitopes can be formulated to maintain solubility of each component at sufficiently high concentrations, enabling testing in low and non-toxic solvent concentrations. The present results validate previous observations in the case of dust mite allergens, DENV and tuberculosis infection that have shown that the megapool approach allows detecting responses directly ex vivo [24, 33, 37, 38]. …”

“…We recently reported a strategy to generate pools of large numbers of potential epitopes for use in cellular assays and avoid toxicity associated with high amount of DMSO required to achieve solubility of such large numbers of peptides [24, 33]. Herein we applied this “megapool” strategy to the detection of TT specific responses.…”

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis

“…Although Ag85B is much larger than ESAT-6 (325 amino acids vs. 95aa), significantly higher frequencies of T cells responded to ESAT-6 than to Ag85B (Figure 1C). Thus, like mice, humans exhibited higher frequencies of T cells recognizing ESAT-6 than Ag85B during chronic Mtb infection, consistent with another recent report (Lindestam Arlehamn et al, 2016). …”

Characterization of Mycobacterium tuberculosis–Specific Cells Using MHC Class II Tetramers Reveals Phenotypic Differences Related to HIV Infection and Tuberculosis Disease

“…Thus, we compared the ability of cells to secrete IFN-γ, IL-2 and TNF-α in response to CFP-10 or ESAT-6 peptide stimulation in the four clinical groups (Figure 4A). Of note, as IL-17 expression is rarely detectable in response to peptide stimulation (23, 24), this antibody was not included in our panel. Overall, despite the different phenotype observed in ex vivo Mtb-specific tetramer+CD4+ T cells in HIV or active TB, the functional capacities of Mtb-specific CD4+ T cells were comparable between the different clinical groups (Figure 4B).…”