Unique and common TCR repertoire features of Ni2+ -, Co2+ -, and Pd2+ -specific human CD154 + CD4+ T cells.

BACKGROUND: Apart from Ni2+ , Co2+ , and Pd2+ ions commonly trigger T cell-mediated allergic contact dermatitis. However, in vitro frequencies of metal-specific T cells and the mechanisms of antigen recognition remain unclear. METHODS: Here, we utilized a CD154 upregulation assay to quantify Ni2+ -, Co2+ -, and Pd2+ -specific CD4+ T cells in peripheral blood mononuclear cells (PBMC). Involved αß T cell receptor (TCR) repertoires were analyzed by high-throughput sequencing. RESULTS: Peripheral blood mononuclear cells incubation with NiSO4 , CoCl2 , and PdCl2 increased frequencies of CD154 + CD4+ memory T cells that peaked at ~400 µM. Activation was TCR-mediated as shown by the metal-specific restimulation of T cell clones. Most abundant were Pd2+ -specific T cells (mean 3.5%, n = 19), followed by Co2+ - and Ni2+ -specific cells (0.6%, n = 18 and 0.3%, n = 20) in both allergic and non-allergic individuals. A strong overrepresentation of the gene segment TRAV9-2 was unique for Ni2+ -specific TCR (28% of TCR) while Co2+ and Pd2+ -specific TCR favorably expressed TRAV2 (8%) and the TRBV4 gene segment family (21%), respectively. As a second, independent mechanism of metal ion recognition, all analyzed metal-specific TCR showed a common overrepresentation of a histidine in the complementarity determining region 3 (CDR3; 15% of α-chains, 34% of ß-chains). The positions of the CDR3 histidine among metal-specific TCR mirrored those in random repertoires and were conserved among cross-reactive clonotypes. CONCLUSIONS: Induced CD154 expression allows a fast and comprehensive detection of Ni2+ -, Co2+ -, and Pd2+ -specific CD4+ T cells. Distinct TCR repertoire features underlie the frequent activation and cross-reactivity of human metal-specific T cells.

Frequencies and TCR Repertoires of Human 2,4,6-Trinitrobenzenesulfonic Acid-specific T Cells.

Allergic contact dermatitis is a widespread T cell-mediated inflammatory skin disease, but in vitro monitoring of chemical-specific T cells remains challenging. We here introduce short-term CD154/CD137 upregulation to monitor human T cell responses to the experimental sensitizer 2,4,6-trinitrobenzenesulfonic acid (TNBS). Peripheral blood mononuclear cells (PBMC) from healthy donor buffy coats were TNBS-modified and incubated with unmodified PBMC. After 5 and 16 h, we detected TNBS-specific activated CD154+CD4+ and CD137+CD8+ T cells by multi-parameter flow cytometry, respectively. Activated cells were sorted for restimulation and bulk T cell receptor (TCR) high-throughput sequencing (HTS). Stimulation with TNBS-modified cells (3 mM) induced CD154 expression on 0.04% of CD4+ and CD137 expression on 0.60% of CD8+ memory T cells, respectively (means, n = 11-17 donors). CD69 co-expression argued for TCR-mediated activation, which was further supported by TNBS-specific restimulation of 10/13 CD154+CD4+ and 11/15 CD137+CD8+ T cell clones and lines. Major histocompatibility complex (MHC) blocking antibodies prevented activation, illustrating MHC restriction. The high frequencies of TNBS-specific T cells were associated with distinct common changes in the TCR ß-chain repertoire. We observed an overrepresentation of tryptophan and lysine in the complementarity determining regions 3 (CDR3) (n = 3-5 donors), indicating a preferential interaction of these amino acids with the TNBS-induced epitopes. In summary, the detection of TNBS-specific T cells by CD154/CD137 upregulation is a fast, comprehensive and quantitative method. Combined with TCR HTS, the mechanisms of chemical allergen recognition that underlie unusually frequent T cell activation can be assessed. In the future, this approach may be adapted to detect T cells activated by additional chemical sensitizers.

Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system.

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering-based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon-derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 3 106 cells/1000 cm2 surface) on CytodexTM type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/ lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD-PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. "Bead-to-bead" transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion.