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Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice.

Haddad EK, Wu X, Hammer JA, Henkart PA - J. Cell Biol. (2001)

Bottom Line: Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute;, and.

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.

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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.
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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.

Mentions: CTLs from ashen mice were compared with C3H controls for their ability to lyse Fas-negative L1210 target cells using redirected cytotoxicity. Ashen CTLs showed a profound defect in target cell lysis, corresponding to >90% loss of lytic potency as seen by horizontal comparison of the titration curves (Fig. 1 A). A similar deficiency was observed using allospecific EL-4 target cells to measure direct TcR–mediated cytotoxicity (data not shown). NK activity of spleen cells from ashen mice was also decreased ∼10 times compared with controls (Fig. 1 B). These results imply that Rab27a is expressed in both T cell and NK lymphocyte lineages and is required for cytotoxicity via the granule exocytosis cytotoxicity pathway.


Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice.

Haddad EK, Wu X, Hammer JA, Henkart PA - J. Cell Biol. (2001)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195776&req=5

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.
Mentions: CTLs from ashen mice were compared with C3H controls for their ability to lyse Fas-negative L1210 target cells using redirected cytotoxicity. Ashen CTLs showed a profound defect in target cell lysis, corresponding to >90% loss of lytic potency as seen by horizontal comparison of the titration curves (Fig. 1 A). A similar deficiency was observed using allospecific EL-4 target cells to measure direct TcR–mediated cytotoxicity (data not shown). NK activity of spleen cells from ashen mice was also decreased ∼10 times compared with controls (Fig. 1 B). These results imply that Rab27a is expressed in both T cell and NK lymphocyte lineages and is required for cytotoxicity via the granule exocytosis cytotoxicity pathway.

Bottom Line: Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute;, and.

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.

Show MeSH
Related in: MedlinePlus