CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma

Abstract

CD4(+) T cells can eliminate tumor cells in vivo in the absence of CD8(+) T cells. We have CD4(+) T cells specific for a MHC class II-restricted, tumor-specific peptide derived from a mutant ribosomal protein expressed by the UV light-induced tumor 6132A-PRO. By using neutralizing mAb specific for murine IFN-gamma and adoptive transfer of CD4(+) T cells into severe combined immunodeficient mice, we show that anti-IFN-gamma treatment abolishes the CD4(+) T cell-mediated rejection of the tumor cells in vivo. The tumor cells were MHC class II negative, and IFN-gamma did not induce MHC class II expression in vitro. Therefore, the tumor-specific antigenic peptide must be presented by host cells and not the tumor cells. Tumor cells transduced to secrete IFN-gamma had a markedly reduced growth rate in severe combined immunodeficient mice, but IFN-gamma did not inhibit the growth of the tumor cells in vitro. Furthermore, tumor cells stably expressing a dominant-negative truncated form of the murine IFN-gamma receptor alpha chain, and therefore insensitive to IFN-gamma, nevertheless were rejected by the adoptively transferred CD4(+) T cells. Thus, host cells, and not tumor cells, seem to be the target of IFN-gamma. Together, these results show that CD4(+) T cells can eliminate IFN-gamma-insensitive, MHC class II-negative cancer cells by an indirect mechanism that depends on IFN-gamma.

Figure 1

Results of MACS of LNC from mL9-immunized mice. LNC from mL9 peptide-immunized mice were restimulated in vitro with 6132A-PRO tumor cell lysate and then incubated with microbead-coupled anti-CD4 antibodies for separation into CD4-positive and -negative fractions by using MACS positive selection columns. After overnight incubation, cell aliquots were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. The numbers in the quadrants indicate the percentage of total cells that the different cell population in each quadrant comprises. Shown is the analysis of experiment 3 shown in Table 2.

Figure 2

6132A-PRO tumor cells expressing a dominant negative IFN-γ receptor (PRO-DNγR) are unresponsive to IFN-γ. 6132A-PRO cells were transfected with an expression vector for the transdominant-negative mutant IFN-γR α chain (20). PRO-DNγR, parental 6132A-PRO cells, and 6132A-PRO cells transfected to express I-E^k (PRO-MHC II, ref. 11) were cultured for 48 hr in the presence or absence of IFN-γ (250 units/ml) and then analyzed for expression of MHC class I and II by flow cytometry (open histograms). Controls (filled histograms) were incubated with second-step anti-Ig alone (control for MHC class I) or without antibody (control for MHC class II).

Figure 3

(Left) IFN-γ-secreting and IFN-γ-insensitive 6132A-PRO tumor cells grow at similar rates in vitro as do unmodified 6132A-PRO parental cells. The in vitro growth of PRO-DNγR cells, PRO-IFN-γ cells, and 6132A-PRO cells (PRO) was analyzed in a [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay by measuring absorbance at 490 nm minus background absorbance at 650 nm. All experiments were done in triplicate, and SEM >0.08 are indicated. (Middle) IFN-γ is neither cytotoxic nor cytostatic for 6132A-PRO cells in vitro. The proliferation of 6132A-PRO tumor cells in vitro in the presence of varying amounts of IFN-γ was measured in an MTS assay by reading the absorbance at 490 nm minus the background absorbance at 650 nm. All experiments were performed in triplicate and SEM >0.08 are indicated. (Right) Growth of IFN-γ-secreting 6132A-PRO tumor cells in vivo. SCID mice were inoculated s.c with 10⁵ 6132A-PRO (PRO) or 3 × 10⁵ IFN-γ-secreting PRO-IFN-γ, and tumor growth was measured every 3–4 days. Two of the five animals challenged with PRO-IFN-γ were treated with anti-IFN-γ antibodies. The SEM were always <0.03 and therefore are not shown. The tumor growth differs across the three groups (P value = 0.036) by Fisher’s Exact test considering rapid growth in the three PRO-challenged mice or the two mice challenged with PRO-IFN-γ cells but treated with anti-IFN-γ versus the three mice challenged with PRO-IFN-γ cells.