Convergent and divergent effects of costimulatory molecules in conventional and regulatory CD4+ T cells

Abstract

Costimulatory molecules of the CD28 family on T lymphocytes integrate cues from innate immune system sensors and modulate activation responses in conventional CD4(+) T cells (Tconv) and their FoxP3(+) regulatory counterparts (Treg). To better understand how costimulatory and coinhibitory signals might be integrated, we profiled the changes in gene expression elicited in the hours and days after engagement of Treg and Tconv by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BLA, or -CD80. In Tconv, a shared "main response" was induced by CD28, ICOS, and, surprisingly, BTLA and CD80, with very limited CD28-specific (primarily Il2) or ICOS-specific elements (including Th1 and Th2 but not the follicular T signature). CTLA4 and PD1 had a very subtle impact in this system, similarly inhibiting the response to anti-CD3. Treg responded to the same costimulatory hierarchy and to the same extent as Tconv, but inducing different clusters of genes. In this reductionist system, costimulatory or coinhibitory engagement mainly elicits generic responses, suggesting that the variability of their effects in vivo result from temporal or anatomical differences in their engagement, rather than from inherently different wiring.

Fig. 1.

Impact of costim engagement on T-cell proliferation. CD4+ splenocytes were labeled with CFSE, and stimulated for 66 h in vitro with beads conjugated with anti-CD3 alone or with anti-costim mAbs, and cell division assessed by flow cytometry measure of residual CFSE in CD4⁺CD3⁺ cells. *P < 0.05; **P < 0.005 from anti-CD3 alone.

Fig. 2.

Transcriptional impact of costim coengagement . CD4⁺ splenocytes from B6.Foxp3^fgfp mice were stimulated in vitro with bead-conjugated anti-CD3 alone or with anti-costim antibodies, and CD4⁺CD3⁺GFP⁻ Tconv and GFP⁺ Treg were sorted for gene expression profiling. (A) Heatmap representation of the ratio of expression levels for each costim coengagement relative to CD3 alone, for Tconv and Treg cells (for transcripts that change by > twofold with CD3 or any of the costims); order by hierarchical clustering. (B) Fold change/fold change (FC/FC) plot comparing the effect in Tconv cells of each costim (y axis) to that of CD28 (x axis).

Fig. 3.

Subtle differences between CD28 and ICOS engagement on activated Tconv. (A). FC/FC plot comparing the late effect of CD28 (x axis) and ICOS (y axis) over CD3 engagement alone. Red and blue dots denote genes preferentially responding to CD28 or ICOS, respectively. (B) Same plot, highlighting genes over- or under-expressed in different Th signatures (numbers along the axes indicate the number of signature genes over- or underexpressed in response to the corresponding costim). Genes in the Th1/2/17 signatures per ref. ; the Tfh signature per ref. .

Fig. 4.

Costim effects on TCR-induced responses. (A) Transcripts most strongly induced or repressed by CD3 engagement in Tconv cells were selected and ranked according to this ratio (black dots), and their change (relative to Unstimulated) in response to anti-CD3 plus each costim were plotted on the same scale (blue dots). (B) FC/FC plots comparing the response to CD3 alone vs. 3+CD28 (Left) or 3+ICOS (Right). (C) Inhibitory effects of CTLA4 or PD1: The effect of coinibitory engagement was calculated as a ratio of Fold changes [as ((CD3+CTLA4)/Unstim)/(CD3/Unstim)] and significance estimated as a t test. Transcripts with P value <0.05 for either CTLA4 or PD1 are shown.

Fig. 5.

Comparative effects of costims in Tconv and Treg cells. (A) Effect of CD28. The ratio of expression after CD3+CD28 engagement, relative to CD3 alone, was calculated in Tconv (x axis) and Treg (y axis) at early (Left) and late (Right) times. Genes induced specifically (>twofold differential) in Treg or Tconv are highlighted (blue and red, respectively); genes repressed in both are shown in green. (B) Disposition of the genes identified and color-coded in A, in plots that compare the effect of each costim in Tconv (x axis) vs. Treg (y axis) cells.