Suppression of T and B lymphocyte activation by a Yersinia pseudotuberculosis virulence factor, yopH

Abstract

The acquired immune responses are crucial to the survival of Yersinia-infected animals. Mice lacking T cells are sensitive to Yersinia infection, and a humoral response to Yersinia can be protective. Diverse mechanisms for Yersinia to impair and evade the host innate immune defense have been suggested, but the effects of Yersinia on lymphocytes are not known. Here, we demonstrate that after a transient exposure to Y. pseudotuberculosis, T and B cells are impaired in their ability to be activated through their antigen receptors. T cells are inhibited in their ability to produce cytokines, and B cells are unable to upregulate surface expression of the costimulatory molecule, B7.2, in response to antigenic stimulation. The block of lymphocyte activation results from the inhibition of early phosphorylation events of the antigen receptor signaling complex. Through the use of Y. pseudotuberculosis mutants, we show that the inhibitory effect in both T cells and B cells is dependent on the production of Yersinia outermembrane protein (Yop) H, a tyrosine phosphatase. Our results suggest a mechanism by which the pathogenic bacteria may modulate a wide range of T and B cell-mediated immune responses.

Figure 1

Y. pseudotuberculosis suppresses T cell antigen–specific IL-2 production in a YopH-dependent manner. IL-2 production of T cell activation assays with (A) MCC9 or (B) CH27 preexposed to either wild-type (Wt), YopH mutant (H−), Lcr mutant (Lcr−) Yersinia, or medium alone (T); or with (C) MCC9 T cells activated by plate-bound MCC/I-E^k after the T cells were preexposed to wild-type (Wt), YopH mutant (H−), YopE mutant (E−), YopO/YopJ mutant (A−), pYV⁻ (P−) Yersinia, or medium alone (T). At least three independent experiments were conducted, and representative result is shown.

Figure 1

Y. pseudotuberculosis suppresses T cell antigen–specific IL-2 production in a YopH-dependent manner. IL-2 production of T cell activation assays with (A) MCC9 or (B) CH27 preexposed to either wild-type (Wt), YopH mutant (H−), Lcr mutant (Lcr−) Yersinia, or medium alone (T); or with (C) MCC9 T cells activated by plate-bound MCC/I-E^k after the T cells were preexposed to wild-type (Wt), YopH mutant (H−), YopE mutant (E−), YopO/YopJ mutant (A−), pYV⁻ (P−) Yersinia, or medium alone (T). At least three independent experiments were conducted, and representative result is shown.

Figure 2

The inhibitory effect on T cell activation can be overridden by PMA and ionomycin. IL-2 production from MCC9 T cells exposed to wild-type (Wt), pYV⁻ (P−), or YopH⁻ (H−) Yersinia, or medium alone (T) for 1 h and incubated overnight with PMA and ionomycin (PMA/Iono) or 60 μg/ml of plate-bound MCC/I-E^k.

Figure 3

YopH inhibits tyrosine phosphorylation of the TCR signaling complex. (A) Antiphosphotyrosine blot of resting (−) and OKT3-activated (+) T cell lysates from Jurkat T cells exposed to either wild-type (Wt), YopE⁻ (ΔE), YopH⁻ (ΔH), or pYV⁻ (P−) Y. pseudotuberculosis. Arrows indicate the heavy and light chains of OKT3. (B) Blots were stripped and reprobed for actin. (C) Antiphosphotyrosine blot of immunoprecipitated CD3ζ from extracts.

Figure 4

Yersinia inhibits tyrosine phosphorylation of the BCR signaling complex in a YopH-dependent manner. (A) Antiphosphotyrosine blot of cell extracts from splenic B cells from anti-HEL transgenic mice. B cells were incubated with medium alone (B), wild-type (Wt), YopH⁻ (ΔH), or pYV⁻ (P−) Yersinia for 1 h, then activated with HEL protein (500 ng/ml) for the indicated times. (B) Blots were stripped and reprobed for actin.

Figure 5

YopH suppresses B cells antigen–specific B7.2 upregulation. Splenic B cells from anti-HEL Ig transgenic mice were exposed to either wild-type (A), pYV⁻ (B), or YopH⁻ (C) Y. pseudotuberculosis before the addition of HEL protein. After 12 h, cells were stained with FITC-conjugated anti-B220, PE-conjugated B7.2, or propidium iodide, and analyzed by FACS^®. Only B220⁺ and propidium iodide–negative cells (live) were included in the analysis. Bold lines represent expression of B7.2 on Yersinia-exposed, HEL-activated cells. Thin lines represent B7.2 expression on unexposed, HEL-activated B cells. At least three independent experiments were conducted, and a representative plot is shown.