Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

Hiroyuki Kobayashi, Hisashi Aso, Nakao Ishida, Hiroshi Maeda, Richard B. Pollard, Fujio Suzuki

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Abstract

The role of suppressor macrophages (S-Mφ) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2 b k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of Mφ from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the Mφ isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic Mφ obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in Mφ isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-Mφ expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.

Original languageEnglish (US)
Pages (from-to)589-602
Number of pages14
JournalCellular Immunology
Volume128
Issue number2
DOIs
StatePublished - 1990

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Cytotoxic T-Lymphocytes
Macrophages
Mixed Lymphocyte Culture Test
Inbred C3H Mouse
Spleen
Immune Sera
Immunization
I-J-antigen
Thymoma
Lymphocyte Activation
Indomethacin
Plastics
Cell Survival
Iron
Lymphocytes

ASJC Scopus subject areas

  • Cell Biology
  • Immunology

Cite this

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes. / Kobayashi, Hiroyuki; Aso, Hisashi; Ishida, Nakao; Maeda, Hiroshi; Pollard, Richard B.; Suzuki, Fujio.

In: Cellular Immunology, Vol. 128, No. 2, 1990, p. 589-602.

Research output: Contribution to journalArticle

Kobayashi, Hiroyuki ; Aso, Hisashi ; Ishida, Nakao ; Maeda, Hiroshi ; Pollard, Richard B. ; Suzuki, Fujio. / Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes. In: Cellular Immunology. 1990 ; Vol. 128, No. 2. pp. 589-602.
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abstract = "The role of suppressor macrophages (S-Mφ) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2 b k). In mice injected with CTL plus EL-4 cells survival rate was 20{\%} compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80{\%} survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of Mφ from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100{\%} survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20{\%} of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the Mφ isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic Mφ obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in Mφ isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80{\%} survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-Mφ expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.",
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AU - Suzuki, Fujio

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