Tumors escape from immune surveillance by producing the immunosuppressive cytokine TGF-beta. However, the mechanism by which TGF-beta inhibits T cell-mediated tumor clearance in vivo is unknown. We demonstrate that TGF-beta acts on cytotoxic T lymphocytes (CTLs) to specifically inhibit the expression of five cytolytic gene products-namely, perforin, granzyme A, granzyme B, Fas ligand, and interferon gamma-which are collectively responsible for CTL-mediated tumor cytotoxicity. Repression of granzyme B and interferon-gamma involves binding of TGF-beta-activated Smad and ATF1 transcription factors to their promoter regions, indicating direct and selective regulation by the TGF-beta/Smad pathway. Neutralization of systemic TGF-beta in mice enables tumor clearance with restoration of cytotoxic gene expression in antigen-specific CTLs in vivo. We suggest that TGF-beta suppresses CTL function in vivo through an anticytotoxic program of transcriptional repression.

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Abstract. The role of integrins in muscle differentiation was addressed by ectopic expression of integrin o~ subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human a5 subunit or the chicken et6 subunit produced contrasting phenotypes. The ot5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the or6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail et6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic a subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric a subunits, ct5ex/6cyto and a6ex/5¢yto, produced phenotypes opposite to those observed with ectopic or5 or or6 expression. Myoblasts that express a5ex/6mo show decreased proliferation while differentiation is partially restored. In contrast, the et6ex/5¢yto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human ct5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in a5 regulation of differentiation. Ectopic c~5 and o~6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human et5 subunit differentiate only in the absence of serum while differentiation of untransfected and ot6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to a5-transfected myoblasts results in unique responses that differ from their effects on untransfected ceils. Both bFGF or TGFI3 inhibit the serum-free differentiation of a5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-13 inhibits it. Insulin or TGF-ot promote proliferation and differentiation of ot5-transfected myoblasts; however, insulin alters myotube morphology. TGF-o~ or PDGF-BB enhance muscle ct-actinin organization into myofibrils, which is impaired in differentiated or5 cultures. With the exception of TGF-ot, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic ct5 expression only in the presence of bFGF and insulin while TGF-et and TGF-~ promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin a subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the et subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival dep...

Granzyme B (GzmB) is a component of cytotoxic lymphocyte granules that can rapidly initiate apoptosis in target cells. While several procaspases are cleaved and activated by GzmB, the absolute requirement of caspase activation for GzmB-induced apoptosis is controversial. In this report, we demonstrate that GzmB can initiate apoptosis in the absence of caspase-3 activity by directly cleaving DFF45/ICAD to liberate activated DFF40/CAD. DFF45/ICAD cleavage occurs less efficiently in cells that lack caspase-3 activity, suggesting that the caspases normally amplify the GzmB death signal. DFF45/ICAD-deficient mouse embryo fibroblasts are partially resistant to GzmB-induced death, demonstrating the biological importance of DFF45/ICAD for GzmB-mediated apoptosis.

Granzyme (gzm) B-deficient cytotoxic lymphocytes (CTL) have a severe defect in the rapid induction of target cell apoptosis that is almost completely corrected by prolonged incubation of the CTL effectors and their targets. We show in this report that perforin-dependent, gzmB-independent cytotoxicity is caused by gzmA (or tightly linked genes). CTL deficient for gzmA and gzmB retain normal perforin function, but these CTL have a cytotoxic defect in vivo that is as severe as perforin-deficient CTL. Collectively, these results suggest that perforin provides target cell access and/or trafficking signals for the gzms, and that the gzms themselves deliver the lethal hits. The gzmA pathway appears to function independently from gzmB and may therefore provide a critical "back-up" system when gzmB is inhibited in the target cell.

Granzyme B (GzmB) is a serine protease that is used by activated cytotoxic T lymphocytes to induce target cell apoptosis. Although GzmB directly cleaves the Bcl2 family member BID on target cell entry, Bid -deficient (and Bax, Bak doubly deficient) cells are susceptible to GzmB-induced death, even though they fail to release cytochrome c from mitochondria. GzmB still induces mitochondrial depolarization in Bax, Bak double knockout cells without cytochrome c release or opening of the permeability transition pore. Because GzmB cannot directly cause depolarization of isolated mitochondria, novel intracellular factor(s) may be required for GzmB to depolarize mitochondria in situ . GzmB therefore utilizes two distinct mitochondrial pathways to amplify the proapoptotic signal that it delivers to target cells.

Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette (GzmB−/−/+PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB−/−/ΔPGK-neo) restored the expression of both genes. Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB−/−/+PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB−/−/ΔPGK-neo). Similarly, GzmB−/−/+PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB−/−/ΔPGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo.

In mice deficient in either lymphotoxin α (LT-α) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-α and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-α–deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-α–expressing cells required to establish organized FDC are derived from BM. The role of LT-α in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)–deficient BM cells into LT-α–deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-α–deficient recipient. Thus, expression of LT-α in the BM-derived cells, but not in the non–BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I–deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non–BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I–deficient BM was able to restore FDC organization and GC formation in LT-α–deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-α–expressing BM-derived cells and by TNFR-I-expressing non–BM-derived cells.