Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering

Abstract

CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell-antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.

Figure 1

Parallel downregulation and degradation of CD4 and CD8 together with CD3 in T cell clones stimulated by specific antigen. (A) Time course of CD3 (•) and CD4 (▵) downregulation in clone KS140 stimulated with APCs pulsed with 10 nM TT830-842. (B) CD3 (•) and CD8 (□) downregulation in clone CER22 stimulated with APCs pulsed with 100 nM M58-66. (C) Surface and total levels of CD3 and CD4 as determined by staining before and after permeabilization in KS70 cells conjugated for 5 h with APCs unpulsed (empty bars) or pulsed with 0.1 μM (hatched bars) or 10 μM (filled bars) TT830-842.

Figure 1

Parallel downregulation and degradation of CD4 and CD8 together with CD3 in T cell clones stimulated by specific antigen. (A) Time course of CD3 (•) and CD4 (▵) downregulation in clone KS140 stimulated with APCs pulsed with 10 nM TT830-842. (B) CD3 (•) and CD8 (□) downregulation in clone CER22 stimulated with APCs pulsed with 100 nM M58-66. (C) Surface and total levels of CD3 and CD4 as determined by staining before and after permeabilization in KS70 cells conjugated for 5 h with APCs unpulsed (empty bars) or pulsed with 0.1 μM (hatched bars) or 10 μM (filled bars) TT830-842.

Figure 2

Downregulation of coreceptors can occur in the absence of an interaction with cognate or noncognate MHC molecules. Downregulation of CD3 and coreceptor in CD4⁺ (KS70; A) or CD8⁺ (CER43; B) T cell clones stimulated by specific peptide–MHC (•), superantigens (□), or monovalent anti-CD3 antibodies: w632/T3 (▵), L243/T3 (○). (C) Downregulation of CD3 versus CD4 (▴) and CD8 (○) in alloreactive CD4⁺ CD8⁺ T cell clones stimulated by specific alloantigen (D). Downregulation of CD3 and CD8 in CD8⁺, class II–alloreactive T cell clones stimulated with class I⁻ APCs (.221) expressing the relevant class II alloantigen.

Figure 3

Intracellular association of coreceptor with triggered TCRs. (A) T cell clones were stimulated (+) or not (−) with APCs, lysed, and the CD4 or CD8 immunoprecipitates were subjected to an in vitro kinase assay, followed by reimmunoprecipitation with anti–ZAP-70 antiserum. CD4⁺ clone KS140 conjugated with peptide-pulsed or unpulsed APCs (lanes 1 and 2). CD8⁺ clone MS3 either untreated or conjugated with allogeneic class I⁻ APC (lanes 3 and 4). (B) Downregulation of CD3 (○, •) and CD4 (▵, ▴) in CD4⁻ Jurkat cells transfected with wild-type CD4 (▵, ○) or with mutant CD4.401 (▴, •).

Figure 3

Intracellular association of coreceptor with triggered TCRs. (A) T cell clones were stimulated (+) or not (−) with APCs, lysed, and the CD4 or CD8 immunoprecipitates were subjected to an in vitro kinase assay, followed by reimmunoprecipitation with anti–ZAP-70 antiserum. CD4⁺ clone KS140 conjugated with peptide-pulsed or unpulsed APCs (lanes 1 and 2). CD8⁺ clone MS3 either untreated or conjugated with allogeneic class I⁻ APC (lanes 3 and 4). (B) Downregulation of CD3 (○, •) and CD4 (▵, ▴) in CD4⁻ Jurkat cells transfected with wild-type CD4 (▵, ○) or with mutant CD4.401 (▴, •).

Figure 4

The sensitivity to inhibition by anti-CD4 depends on the nature of the ligand. (A and B) Proliferative response of clone KS70 to various doses of TSST or TT830-843 in the presence (▴) or absence (▵) of anti-CD4. (C) Proliferative response of clone KS164 to TT830-843 (•, ○) or to TT830-843 Ala⁸³⁹-substituted peptide that behaves as a weak agonist (▪, □) in the presence (•, ▪) or absence (○, □) of anti-CD4.

Figure 5

Quantitative contribution of CD4 to TCR triggering and T cell activation. (A) IFN-γ production by T cell clone AL15.1 stimulated by TT830-843 (•, ○) or by TT830-843 Gln⁸³⁹ (▴, ▵) in the presence (•,▴) or absence (○, ▵) of anti-CD4 antibody. (B) CD3 downregulation in the same experiment. (C–E) Levels of IFN-γ (▴), IL-2 (•), TNF-α (▪), and IL-3 (▵) as a function of the number of TCRs downregulated in cultures stimulated by wild-type agonist (C), wild-type agonist + anti-CD4 (D), or weak agonist (E). No cytokine production was observed in cultures stimulated by weak agonist + anti-CD4.