Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells

Abstract

The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen doseresponse of human naive CD4 + T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 10 6 naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.

Follicular helper T-cells and virus-specific antibody response in primary and reactivated human cytomegalovirus infections of the immunocompetent and immunocompromised transplant patients

“…The CD4 + T-cell response specific for HCMV (IE-1, pp65, gB and gHgLpUL128L), influenza virus (Flu: Matrix protein 1, Nucleoprotein and Neuraminidase) and respiratory syncytial virus (RSV: Fusion protein and Nucleoprotein) antigens was investigated with a novel high throughput method that allows measurement of low frequencies of antigen-specific T-cells in the human T-cell subset repertoire (Geiger et al, 2009 …”

Immunological consequences of intragenus conservation ofMycobacterium tuberculosisT-cell epitopes

“…Interestingly, the memory T cells responsible for the reactivity in HCs have the CCR6 + CXCR3 + phenotype previously described for LTBI and PPD-reactive individuals (22,23,42). The transcriptional signature of these cells shows that they express molecular features, such as ABCB1 (MDR1), KIT (CD117), CCR2, BAFF, and IL12RB2, thought to be relevant for controlling chronic/latent infections (43).…”

“…T-cell library assays were performed as previously described (23). CD4 T cells were isolated from PBMCs by positive selection with microbeads (Miltenyi Biotec).…”

“…Cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 1% (vol/vol) nonessential amino acids, 1% (vol/vol) sodium pyruvate, penicillin (50 U/mL), streptomycin (50 μg/mL) (all from Invitrogen), and 5% (vol/vol) heat-inactivated human serum (Swiss Red Cross). T cells (1,000 cells per well) were stimulated polyclonally with 1 μg/mL PHA (Remel) in the presence of irradiated (45 Gy) allogeneic feeder cells (1.0 × 10 5 cells per well) and IL-2 (500 IU/mL) in a 96-well plate format, and T-cell lines were expanded as previously described (23). Library screening was performed at day 14-21 by culturing extensively washed T cells (∼2.5 × 10 5 cells per well) with irradiated autologous monocytes (2.5 × 10 4 ), either unpulsed or pulsed for 3 h with Mycobacteria whole particles (ratio 1:3).…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…This method, which does not require HLA typing and measures both HCMV-specific CD4 + and CD8 + T cells, is based on the incubation of PBMCs with autologous monocytederived HCMV-infected dendritic cells, followed by determination of multiple membrane/intracellular parameters by cytokine flow cytometry [137]. Recently, the T cell response to four major HCMV proteins (IE-1, pp65, PC and gB) was evaluated using overlapping peptides (15mers) spanning the entire proteins, and libraries of amplified (by the addition of PHA and IL-2) T cells, according to a well-known procedure [138]. The results showed (Fig.…”