Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

We investigated the cytotoxicity mechanisms of alloantigen-specific human CD4+ and CD8+ cytotoxic T lymphocytes (CTLs) using cells from family members with the Fas gene mutation. Alloantigen-specific CD4+ and CD8+ CTL bulk lines and clones were generated from 2 individuals by stimulation of their peripheral blood lymphocytes with allogeneic Fas−/− or Fas+/− cell lines that were established from B-lymphocytes of a patient with Fas deficiency and her mother, respectively. Both CD4+ and CD8+CTL bulk lines and clones directed against allogeneic HLA antigens exerted cytotoxicity against Fas−/− and Fas+/− cells to almost the same degree. The cytotoxicity of CD4+ and CD8+ CTLs appeared to be Ca2+-dependent and was completely inhibited by concanamycin A, an inhibitor of perforin-mediated cytotoxicity. Messenger RNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B were all detected in these CD4+CTLs with the use of the reverse transcriptase polymerase chain reaction. The majority of CD4+ CTL clones that showed Fas-independent cytotoxicity were TH0, as determined by their cytokine production profile. These data, obtained with the use of a novel experimental system, clearly show that the main pathway of cytotoxicity mediated by alloantigen-specific human CD4+as well as by CD8+ CTLs is granule exocytosis, and not the Fas/Fas ligand system.

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Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

Blood ◽

10.1182/blood.v94.9.3084.421k02_3084_3093 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3084-3093 ◽

Cited By ~ 2

Author(s):

Premlata Shankar ◽

Zhan Xu ◽

Judy Lieberman

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Lymphocytes ◽

Cell Lines ◽

Cytotoxic T Lymphocytes ◽

Cd4 T Cells ◽

Target Cells ◽

Granule Exocytosis ◽

Immunodeficiency Virus ◽

Bystander Cells

Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

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CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes

Blood ◽

10.1182/blood.v95.12.3853.012k37_3853_3858 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3853-3858 ◽

Cited By ~ 1

Author(s):

Peter Chu ◽

William G. Wierda ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Lymphocytic Leukemia ◽

Target Cells ◽

Apoptosis Pathway ◽

Granule Exocytosis ◽

Concanamycin A ◽

Cd40 Activation

Cytotoxic T lymphocytes (CTLs) can kill target cells by the granule/exocytosis pathway or the Fas-mediated apoptosis pathway. The sensitivity of chronic lymphocytic leukemia (CLL) B cells to CTL-mediated apoptosis before and after CD40 activation was examined. Resting or CD40-activated CLL cells were found to be equally sensitive to class I–restricted CTL-mediated killing. Despite expressing CD95, the CD40-activated CLL target cells were found to be resistant to apoptosis induced by CH11, an IgM CD95 monoclonal antibody (mAb). Consistent with this, inhibitors of caspases, which are involved in the Fas-induced apoptotic pathway (eg, N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone [z-VAD-fmk]), were unable to block destruction of CLL target cells by CTL. In addition, preincubation of the effector T cells with the anti-Fas ligand mAb NOK-2 failed to inhibit their subsequent ability to kill CLL target cells. On the other hand, CTL activity was blocked by inhibitors of the granule exocytosis pathway such as ethylene-glyco-tetra-acetic acid or concanamycin A. These results indicate that CD40 activation does not impair the sensitivity of CLL cells to Fas-independent CTL-mediated apoptosis.

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CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes

Blood ◽

10.1182/blood.v95.12.3853 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3853-3858 ◽

Cited By ~ 21

Author(s):

Peter Chu ◽

William G. Wierda ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Lymphocytic Leukemia ◽

Target Cells ◽

Apoptosis Pathway ◽

Granule Exocytosis ◽

Concanamycin A ◽

Cd40 Activation

Abstract Cytotoxic T lymphocytes (CTLs) can kill target cells by the granule/exocytosis pathway or the Fas-mediated apoptosis pathway. The sensitivity of chronic lymphocytic leukemia (CLL) B cells to CTL-mediated apoptosis before and after CD40 activation was examined. Resting or CD40-activated CLL cells were found to be equally sensitive to class I–restricted CTL-mediated killing. Despite expressing CD95, the CD40-activated CLL target cells were found to be resistant to apoptosis induced by CH11, an IgM CD95 monoclonal antibody (mAb). Consistent with this, inhibitors of caspases, which are involved in the Fas-induced apoptotic pathway (eg, N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone [z-VAD-fmk]), were unable to block destruction of CLL target cells by CTL. In addition, preincubation of the effector T cells with the anti-Fas ligand mAb NOK-2 failed to inhibit their subsequent ability to kill CLL target cells. On the other hand, CTL activity was blocked by inhibitors of the granule exocytosis pathway such as ethylene-glyco-tetra-acetic acid or concanamycin A. These results indicate that CD40 activation does not impair the sensitivity of CLL cells to Fas-independent CTL-mediated apoptosis.

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Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans

Blood ◽

10.1182/blood.v95.7.2352 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2352-2355 ◽

Cited By ~ 84

Author(s):

Masaki Yasukawa ◽

Hideki Ohminami ◽

Junko Arai ◽

Yoshihito Kasahara ◽

Yasushi Ishida ◽

...

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Fas Ligand ◽

Hla Antigens ◽

Peripheral Blood Lymphocytes ◽

Messenger Rnas ◽

Granule Exocytosis ◽

Ligand System ◽

Concanamycin A ◽

Main Pathway

Abstract We investigated the cytotoxicity mechanisms of alloantigen-specific human CD4+ and CD8+ cytotoxic T lymphocytes (CTLs) using cells from family members with the Fas gene mutation. Alloantigen-specific CD4+ and CD8+ CTL bulk lines and clones were generated from 2 individuals by stimulation of their peripheral blood lymphocytes with allogeneic Fas−/− or Fas+/− cell lines that were established from B-lymphocytes of a patient with Fas deficiency and her mother, respectively. Both CD4+ and CD8+CTL bulk lines and clones directed against allogeneic HLA antigens exerted cytotoxicity against Fas−/− and Fas+/− cells to almost the same degree. The cytotoxicity of CD4+ and CD8+ CTLs appeared to be Ca2+-dependent and was completely inhibited by concanamycin A, an inhibitor of perforin-mediated cytotoxicity. Messenger RNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B were all detected in these CD4+CTLs with the use of the reverse transcriptase polymerase chain reaction. The majority of CD4+ CTL clones that showed Fas-independent cytotoxicity were TH0, as determined by their cytokine production profile. These data, obtained with the use of a novel experimental system, clearly show that the main pathway of cytotoxicity mediated by alloantigen-specific human CD4+as well as by CD8+ CTLs is granule exocytosis, and not the Fas/Fas ligand system.

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