Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice

Abstract

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti-CD3-coated beads. However, rapid anti-CD3-induced granule secretion was drastically defective in both CD8(+) and CD4(+) T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor-stimulated interferon-gamma secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.

Figure 1

In vitro cytotoxicity of cytotoxic T and NK lymphocytes from ashen and dilute mice. (A) Activity of secondary CTL from ashen versus C3H control mice on Fas-negative L1210 targets, redirected with anti-CD3xanti-TNP. (B) NK activity of spleen cells from poly I:C–injected ashen versus C3H mice. (C) Activity of secondary CTLs from ashen versus C3H control mice on Fas-transfected L1210 targets, redirected with anti-CD3xanti-TNP. Solid circles are hidden by solid squares symbols in this graph. (D) Activity of primary CTLs from dilute versus C57Bl/6 control mice on Fas-negative L1210 targets, redirected with anti-CD3x–anti-TNP. (E) NK activity of spleen cells from poly I:C–injected dilute versus C57Bl/6 mice.

Figure 2

Expression of granule proteins and Rab27a in purified CD8⁺ CTLs from ashen versus C3H control mice. (A) Granzyme A activity of Triton X-100 lysates of CTLs from ashen versus C3H control mice. Activity levels were determined from the linear portion of plots of activity versus cell number. (B) Western blot analysis of granzyme B, perforin, and Rab 27a from purified CD8⁺ CTLs. (C and D) Immunofluorescent detection of intracellular perforin in ashen and C3H CTL, showing normal appearance of ashen CTL granules. Projected images are shown. Bar, 2.3 μm.

Figure 3

Anti-CD3 induced secretion in activated T cells from ashen versus C3H mice. T cells from 7-d secondary MLR cultures were incubated on wells coated with anti-CD3 or control hamster IgG. Secretion was determined from enzyme activity in supernatants, expressed as a percentage of levels in Triton X-100 cell lysates. (A) Time course of β-hexosaminidase release from ashen versus C3H T cells. (B) Supernatants from the experiment in A were analyzed for γ-interferon. (C) Purified CD4⁺ and CD8⁺ T cells were tested for β-hexosaminidase secretion after 4 h of incubation. (D) Secretion of the granule marker granzyme A in purified CD4⁺ and CD8⁺ after 4 h.

Figure 4

Immunofluorescence detection of Rab 27a and granzyme B in CD8⁺ C3H CTLs. (A) Granzyme B, red. (B) Rab 27a, green. (C) Colocalization of granzyme B and Rab 27a (yellow). Projected images are shown. Bar, 4.6 μm.

Figure 5

Anti-CD3 induced surface CTLA-4 expression in ashen versus C3H control mice. Secondary MLR-activated T cells from ashen and C3H mice were cultured on immobilized anti-CD3 for 4 h, stained for CTLA-4 without permeabilization, and analyzed by flow cytometry. Heavy line, cells stained with biotinylated anti–CTLA-4 and PE-streptavidin. Light line, cells stained with nonbiotinylated anti–CTLA-4 and PE-streptavidin.