Late Expression of Granulysin by Microbicidal CD4+ T Cells Requires PI3K- and STAT5-Dependent Expression of IL-2Rβ That Is Defective in HIV-Infected Patients

Abstract

Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4+ T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4+ T cells is defective during HIV infection. This is because CD4+ T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rβ, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rβ precedes granulysin, and preventing the increased expression of IL-2Rβ using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rβ, blocking subsequent signaling by IL-2 using IL-2Rβ-specific blocking Abs abrogates expression of granulysin. Finally, CD4+ T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rβ and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rβ, which in turn is required for production of granulysin. These results provide a mechanism to explain the “late” production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4+ T cells in HIV-infected patients.

γ c deficiency precludes CD8 + T cell memory despite formation of potent T cell effectors

“…In the absence of γ c , "spurious" CD4 + T cells are generated that show an activated phenotype (CD44 hi CD62L lo ) and migrate to the skin but are unable to elicit graft rejection secondary to severe deficiencies in cytotoxic effector molecules and cytokine production capacities (18). The γ c -dependent signals involved in programming cytotoxic CD4 + T cells engage both STAT5-and PI3K-dependent pathways (19). Based on these results, we hypothesized an analogous role for γ c cytokines in the differentiation of CD8 + T cells.…”

Granulysin Induces Cathepsin B Release from Lysosomes of Target Tumor Cells to Attack Mitochondria through Processing of Bid Leading to Necroptosis

“…ϩ T cells and CD4 ϩ T cells use GNLY, rather than perforin, to kill Cryptococcus neoformans (10,11).…”

Granulysin Production and Anticryptococcal Activity Is Dependent upon a Far Upstream Enhancer That Binds STAT5 in Human Peripheral Blood CD4+ T Cells

“…Whereas g c cytokines share use of the MAPK, Jak/STAT, or the PI3K/AKT pathways for signal transduction, the canonical pathway involves Jak1/3 and STAT3/5 (22). We have demonstrated that granulysin production by IL-2-treated CD4 + T cells is dependent on activation of the Jak3 and STAT5 signaling pathways (23). Moreover, defects in this pathway lead to impaired anticryptococcal activity in HIV-infected patients (12).…”

“…However, the genetic regulation of granulysin expression, especially in human CTLs, is still elusive. Previous studies have demonstrated that IL-2 activated human peripheral blood CD4 + T cell signals via Jak3/STAT5, downstream of the IL-2/IL-2R signaling pathway, leading to production of granulysin (12,23). Because the signaling pathway and the resultant microbicidal activity is defective in HIV-infected patients, the mechanism by which STAT5 leads to granulysin expression is of great interest.…”

“…PBMCs were prepared immediately after collection as previously described (12,23). The isolation and culture of primary CD4 + T cells was also performed as previously described (12,23).…”