T cell functions in granulocyteymacrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyteymacrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to alloantigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF 1y1 and GM-CSF 2y2 mice. To investigate the responses of CD81 and CD41 T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD81 T cells with specificity for ovalbumin peptide could not be induced in GM-CSF 2y2 mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF 2y2 mice. Purified CD41 T cells from GM-CSF 2y2 mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-g (IFN-g) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD41 T cells from GM-CSF 2y2 mice proliferated as well as those from GM-CSF 1y1 mice and produced high amounts of IFN-g and IL-4. To analyze the rescue effect of DC on CD41 T cells, supernatants from (i) CD41 T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD41 T cells and KLH-pulsed spleen cells from GM-CSF 2y2 mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-g production of CD41 T cells from GM-CSF 2y2 mice. Granulocyteymacrophage colony-stimulating factor (GMCSF) has received much attention since the cloning of mouse and human GM-CSF and the greater availability of the recombinant product. Studies with recombinant GM-CSF (rGM-CSF) substantiated initial observations showing the effect of GM-CSF on the proliferation and maturation of myeloid progenitor cells in vitro and the increase in granulocytes and monocytes in peripheral blood after GM-CSF injection (1, 2). GM-CSF has also been found to be critical for the in vitro differentiation and proliferation of dendritic cells (DC) from hematopoietic precursor cells (3). Although the hematopoietic activities of GM-CSF have received the most attention, there is growing interest in the immunological effects of this cytokine, particularly its use as an immunological adjuvant (4) and its ability to augment immune responses to tumor antigens. In a study of mouse B16 melanoma, Dranoff et al. (5) compared the relative immunogenicity of B16 cells transduced with retroviral vectors coding for various cytokines. In a comparison of seven different cytokines, immunization with GM-CSF producing B16 cells showed the greatest protection against subsequent challenge with parental B16 cells; this immunity was mediated by CD41 and CD81 T cells (5). Tao and Levy (6) reported that immunization with an idiotypeyGM-CSF fusion protein produced higher levels of anti-idiotype antibodies and greater protection against an idiotype positive B cell lymphoma than immunization with idiotype alone, idiotype mixed with GM-CSF, or idiotype with adjuvant. Jäger et al. (7) have recently shown that rGM-CSF can enhance the generation of cytotoxic T lymphocytes (CTL) and development of hypersensitivity after immunization with peptides derived from melanoma differentiation antigens. The availability of GM-CSF deficient mice now makes it possible to define more precisely the role of this cytokine in immune responses. In the present study, we examined T and B cell functions in mice lacking GM-CSF. MATERIALS AND METHODS Mice. GM-CSF-deficient mice (GM-CSF 2y2 mice) on a C57BLy6 3 129 background were generated at the Melbourne Branch of Ludwig Institute for Cancer Research (8), and breeding stocks were transferred to the New York Branch. (C57BLy6 3 129)F2 mice were used as controls for GM-CSF 2y2 mice. BALByc mice were obtained from the breeding facility at Memorial Sloan–Kettering Cancer Center. mAbs. Anti-L3T4 (CD4) and anti-Lyt2.2 (CD8) mAbs were kindly provided by F. Fitch (University of Chicago) and U. Hämmerling (Memorial Sloan–Kettering Cancer Center), respectively. Other mAbs used in this study were purchased from PharMingen. Tumor Cell Lines. EL4 is a chemically induced leukemia cell line of C57BL origin. RL?1 is a BALByc radiation-induced leukemia. Peptide. The ovalbumin (OVA) peptide spanning residues 257–264 (SIINFEKL) was synthesized and purified by BioSynthesis (Lewisville, TX) (9). OVA Peptide Immunization. OVA peptide (5 mg) in TiterMax (CytRx, Norcross, GA) was injected in the hind footpads. In mice receiving rGM-CSF, 5 ng rGM-CSF (PharMingen) was injected in the hind footpads with peptide followed by 10 ng rGM-CSF injected i.p. for 5 days. Keyhole Limpet Hemocyanin (KLH) Immunization. Mice were immunized with 100 mg KLH (Pierce) in complete Freund’s adjuvant (CFA) (Sigma) in the hind footpads. In mice receiving rGM-CSF, 40 ng rGM-CSF was injected in the hind footpads with KLH followed by 10 ng rGM-CSF injected i.p. for 5 days a week until mice were killed. Generation of CTL Specific to OVA Peptide. Details have been described elsewhere (9). Mixed Lymphocyte Reaction. For proliferation assays, 3 3 105 responding spleen cells (H-2b background) were cultured with 2 3 105 mitomycin C (Sigma)-treated BALByc mice The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9412557-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: GM-CSF, granulocyteymacrophage colony-stimulating factor; DC, dendritic cells; APC, antigen-presenting cells; CTL, cytotoxic T lymphocytes; rGM-CSF, recombinant GM-CSF; Con A, concanavalin A; OVA, ovalbumin; KLH, keyhole limpet hemocyanin; CFA, complete Freund’s adjuvant; TCR, T cell receptor; LPS, lipopolysaccharide; IFN-g, interferon-g; IL, interleukin.