Anti-CD3-activated killer T cells: Interleukin-6 modulates the induction of major histocompatibility complex-unrestricted cytotoxicity and the expression of genes coding for cytotoxic effector molecules.

We have investigated the role of interleukin-6 (IL-6) in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxicity, as well as granzyme B, perforin, and Fas ligand gene expression, following mouse T lymphocyte activation with anti-CD3 monoclonal antibody (mAb). The generation of anti-CD3-activated killer-T (AK-T) cells was inhibited when anti-IL-6 neutralizing mAb was added at initiation of culture but not 24 h later, indicating that IL-6 is involved at an early stage of AK-T cell development. However, AK-T cell induction in the presence of exogenous IL-6 did not result in enhanced cytotoxicity, suggesting that saturating levels of IL-6 are normally synthesized in AK-T cell cultures. The inhibitory effect of IL-6 neutralization on AK-T cell generation could not be attributed to a defect in AK-T cell proliferation or to an inability of AK-T cells to recognize and adhere to P815 tumor target cells. However, IL-2 synthesis and CD25 expression were downregulated in AK-T cell cultures performed in the presence of anti-IL-6 mAb. In addition, IL-6 neutralization resulted in decreased expression of granzyme B and perforin, but not Fas ligand, mRNA. Exogenous IL-2 (50 U/ml) added at initiation of culture completely reversed the inhibitory effect of anti-IL-6 mAb on AK-T cell development, restoring CD25 expression and tumoricidal activity, as well as granzyme B and perforin mRNA expression, to control levels. We conclude that IL-6 modulates AK-T cell induction through an IL-2-dependent mechanism.

Greene AL ，Makrigiannis AP ，Fitzpatrick L ，Hoskin DW ... -  《JOURNAL OF INTERFERON AND CYTOKINE RESEARCH》

Anti-CD3-activated killer T cells: interferon-gamma and interleukin-10 cross-regulate granzyme B expression and the induction of major histocompatibility complex-unrestricted cytotoxicity.

Fitzpatrick L ，Makrigiannis AP ，Kaiser M ，Hoskin DW ... -  《JOURNAL OF INTERFERON AND CYTOKINE RESEARCH》

Inhibition of CTL induction by rapamycin: IL-2 rescues granzyme B and perforin expression but only partially restores cytotoxic activity.

Rapamycin (RAP) is a potent inhibitor of CTL induction. Since RAP is known to block IL-2 signaling through the IL-2R, we hypothesized that RAP may interfere with CTL generation by inhibiting IL-2-induced expression of granzyme (Gzm) B, perforin, and/or Fas ligand (FasL). MHC-unrestricted mouse CTL induced in vitro with anti-CD3 mAb in the presence of RAP (1 ng/ml) exhibited dramatically reduced cellular proliferation and cytotoxicity against P815 tumor target cells. Gzm B mRNA expression and enzymatic activity in RAP-treated CTL were greatly reduced compared with those in control CTL. Perforin mRNA expression was also reduced by RAP. In contrast, RAP failed to inhibit FasL mRNA expression. RAP, therefore, inhibits induction of the perforin/Gzm B cytolytic pathway but spares Fas/FasL-mediated cytotoxicity. To determine whether RAP exerts a total blockade of the IL-2R signaling pathway, we induced CTL in the presence of both RAP and exogenous rIL-2 (100 U/ml). Under these conditions, Gzm B and perforin mRNA and protein expression as well as cellular proliferation were restored to at least control levels. Surprisingly, inhibition of cytotoxicity was only partially alleviated when CTL were induced in the presence of RAP plus rIL-2, even though CTL conjugated normally with target cells and had an intact granule secretory pathway. We conclude that 1) the inhibitory effect of RAP at the level of the IL-2R is incomplete; and 2) the suppressive effect of RAP on CTL induction is only partly due to inhibition of Gzm B and perforin gene expression.

Makrigiannis AP ，Hoskin DW 《JOURNAL OF IMMUNOLOGY》

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.

Musgrave BL ，Watson CL ，Hoskin DW 《JOURNAL OF INTERFERON AND CYTOKINE RESEARCH》

IL-4 regulation of perforin gene expression and BLT-esterase production in alpha CD3-induced activated killer cells.

Ting CC ，Liang SM ，Wang J ，Chen YY ，Hargrove M ，Xu N ... -  《-》