Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81–100 and Mtb lysate. Remarkably, while the recognition of the dominant p81–100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88–107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

“…For comparative purposes we also included ten additional Mtb antigens identified previously (Rv0867, Rv1009, Rv1733, Rv1737, Rv2029, Rv2032, Rv2034, Rv2389, Rv2450, and Rv3353)2324414748495051525354. Diluted whole blood samples were stimulated for six days and the cytokine levels were assessed by a seven-plex assay (data shown only for IFN-γ, IL-17, TNF-α and IP-10 Fig.…”

“…As controls, 5-μg/ml PPD (Statens Serum Institut, Copenhagen, Denmark) and 2-μg/ml PHA (Remel, Oxoid, Haarlem, The Netherlands) were included. After six days, the supernatants were used for cytokine determination as previously described24. Tritium thymidine was added for the last 16 hours of culture after which the cells were harvested and counted on a Microbetaplate counter (Wallac, Turku, Finland).…”

“…In combination with the in vitro immune characterization of human cells from Mtb exposed individuals, this work allowed us to identify the first in vivo expressed TB candidate antigens (IVE-TB). This included Rv2034, which induced poly-functional T cell responses with Mtb- inhibitory activity at the clonal level24 and induced encouraging protection against TB challenge in HLA-DR transgenic mice and in guinea pigs25. Several other strategies have been followed to explore the Mtb anti-genome11, including predictive MHC-peptide binding bioinformatics tools combined with human or mouse based T-cell screening approaches to discover novel HLA-class Ia1126, Ib27 and II11192829 restricted Mtb antigens.…”

Identification of Mycobacterial RplJ/L10 and RpsA/S1 Proteins as Novel Targets for CD4+T Cells

“…Numerous mycobacterial proteins have been recognized as T cell antigens in humans (32,33) and animals infected with M. tuberculosis (34,35). Secreted mycobacterial proteins have been a major focus of previous studies to identify immunogenic molecules (36,37).…”

CD4+ T cells from patients with acquired thrombotic thrombocytopenic purpura recognize CUB2 domain-derived peptides

“…Antigen-specific CD4 1 T cells were identified using the activation marker CD40 ligand (CD40L; CD154). [13][14][15] CD40L expression is induced following binding of the T-cell receptor (TCR) to antigenic peptides presented on MHC-II. 16 In this study, we screened peripheral blood mononuclear cells (PBMCs) from HLA-typed healthy donors and patients affected by acquired TTP for CD4…”