Unique molecular surface features of in vivo tolerized T cells

Abstract

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vbeta8.1 T cell antigen receptor (TCR) transgenic mice with a Vbeta8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1(a)) induces tolerance to Mls-1(a) by clonal anergy. CD4 lymph node T cells from Mls-1(a) inoculated transgenic mice enriched for the 6C10(-) phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10(+) CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10(-) T cells remain 3G11(hi) but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

Figure 1

Analysis of changes in 6C10 and 3G11 expression on tolerant CD4 lymph node T cells. Vβ8.1 transgenic mice were tolerized by inoculating with Mls-1^a prior to days indicated, and expression of 6C10 and 3G11 on purified CD4 lymph node T cells was determined by flow cytometry, gating on CD4⁺ and TO-PRO-3⁻ cells. The percent of 6C10⁺ and 3G11^hi CD4 T cells is indicated by the number above the brackets. Representative flow cytometry profiles are shown.

Figure 2

6C10, but not 3G11, down-modulation on CD4 T cells is correlated with duration of tolerance to Mls-1^a. (Upper) Percent of 6C10⁺ (○) and 3G11^hi (•) CD4 T cells as determined by flow cytometry, on the days indicated after Mls-1^a inoculation (day 120 data not shown). (Lower) Proliferation of purified CD4 T cells in response to Mls-1^a in vitro. Results are expressed as stimulation indices of responses to CBA/J stimulators (day 0 T cells, which incorporated approximately 80,000 cpm) divided by responses to syngeneic CBA/Ca stimulators and represent averages ± SEM of four experiments.

Figure 3

Modulation of 6C10 and 3G11 expression on Mls-1^a reactive CD4 lymph node T cells from nontransgenic mice after administering Mls-1^a parallels changes in 6C10 and 3G11 expression in Vβ8.1 transgenic mice. Nontransgenic CBA/CaH mice were inoculated with Mls-1^a prior to the day indicated and percent of Vβ6⁺ (□) and Vβ14⁺ (♦) T cells (Top); Vβ6⁺ and Vβ14⁺ T cells expressing 6C10 (Middle) and 3G11 (Bottom) determined by flow cytometry.

Figure 4

6C10⁻ CD4 lymph node T cells from Mls-1^a inoculated Vβ8.1 transgenic mice are found to be functionally anergic. CD4 T cells from tolerant mice were purified into 6C10 positive and negative populations by magnetic sorting and analyzed for 6C10, 3G11, and Vβ8.1 expression by flow cytometry (A), dose-dependent proliferation to anti-CD3 stimulation [uninoculated mice (□) and inoculated mice unsorted (⋄), purified 6C10⁻ (○), and 6C10⁺ (▵) (B), dose-dependent proliferation to CBA/J stimulators (legend as for B except inoculated unsorted is not shown) (C), and proliferative response to phorbol 12-myristate 13-acetate/ionomycin (D). Representative data from experiments repeated four times are shown.