Characteristics of TCR/CD3 complex CD3ɛ chains of regulatory CD4+ T (Treg) lymphocytes: role in Treg differentiation in vitro and impact on Treg in vivo

Abstract

Tregs are anergic CD4(+)CD25(+)Foxp3(+) T lymphocytes exerting active suppression to control immune and autoimmune responses. However, the factors in TCR recognition underlying Treg differentiation are unclear. Based on our previous data, we hypothesized that Treg TCR/CD3 antigen receptor complexes might differ from those of CD4(+)CD25(-) Tconv. Expression levels of TCR/CD3, CD3ε,ζ chains, or other molecules involved in antigen signaling and the characteristics of CD3ε chains were analyzed in thymus or spleen Treg cells from normal mice. Tregs had quantitative and qualitatively distinct TCR/CD3 complexes and CD3ε chains. They expressed significantly lower levels of the TCR/CD3 antigen receptor, CD3ε chains, TCR-ζ chain, or the CD4 coreceptor than Tconv. Levels of kinases, adaptor molecules involved in TCR signaling, and early downstream activation pathways were also lower in Tregs than in Tconv. Furthermore, TCR/CD3 complexes in Tregs were enriched in CD3ε chains conserving their N-terminal, negatively charged amino acid residues; this trait is linked to a higher activation threshold. Transfection of mutant CD3ε chains lacking these residues inhibited the differentiation of mature CD4(+)Foxp3(-) T lymphocytes into CD4(+)Foxp3(+) Tregs, and differences in CD3ε chain recognition by antibodies could be used to enrich for Tregs in vivo. Our results show quantitative and qualitative differences in the TCR/CD3 complex, supporting the hyporesponsive phenotype of Tregs concerning TCR/CD3 signals. These differences might reconcile avidity and flexible threshold models of Treg differentiation and be used to implement therapeutic approaches involving Treg manipulation.

Heterogeneous CD3 Expression Levels in Differing T Cell Subsets Correlate with the In Vivo Anti-CD3–Mediated T Cell Modulation

“…Notably, the same group reported that CD4 + Foxp3 + T cells isolated from C57BL/6 mice have more undegraded CD3ε and less N-terminal-degraded CD3ε isoforms than do CD4 + Foxp3 2 T cells. This feature accounts for the difference in the avidity to two anti-CD3 (namely, the YCD3-1 and 500A2 clones) (17,35). However, neither of the previously cited studies demonstrated either the ability of nonmitogenic tolerogenic anti-CD3 (i.e., the 145-2C11 clone used in our study) to discriminate the two different isoforms, or the expression of differing CD3ε isoforms in T cells from NOD mice (17,35).…”

“…During the last 10 y, Lambert and Genin (15) and El Hentati et al (16) (17). The present study sought to: 1) define, by means of unsupervised analysis of flow cytometry data, whether CD3 heterogeneous expression levels occur in T cell subsets isolated from mice and humans; and 2) evaluate the biological impact of CD3 expression heterogeneity on anti-CD3 treatment in vivo.…”

The Induction and Maintenance of Transplant Tolerance Engages Both Regulatory and Anergic CD4+ T cells

“…Consequently, their proportion increased in both lymphoid organs and the transplanted islets at the end of treatment. The molecular mechanisms underlying such Treg resistance to apoptosis are not fully understood, but may include a reduced expression and activation of signaling molecules downstream of the TCR/CD3 complex as well as a decreased expression of the CD3 molecules at the cell surface compared to CD4 + Foxp3 − T lymphocytes (21–23). …”

Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin

“…It was suggested that the differential impact of anti-CD3 mAbs may be due to low-level expression of CD3 in CD4 + CD25 + T cells compared to CD4 + CD25 - T cells [30]. Other authors reported that the TCR signalling is attenuated in Foxp3-expressing cells, possibly due to impaired TCR-mediated activation of the Akt pathway [31].…”