CD95-mediated tumor recognition by CD4+ effector cells in a murine mammary model.

The authors examined cellular mechanisms involved in anti-tumor reactivity induced by the murine MT-9G1 mammary tumor line, which was transduced to secrete granulocyte macrophage-colony-stimulating factor (GM-CSF). Compared with the parental MT-901 tumor, MT-9G1 subcutaneous tumors elicited an influx of CD4+ cells and dendritic cells. Secondary in vitro activation of tumor-draining lymph node cells with anti-CD3 and interleukin-2 resulted in effector cells that can mediate regression of established pulmonary metastases after adoptive transfer. In vivo depletion of T-cell subsets showed that tumor regression required CD4+ tumor-draining lymph node cells rather than CD8+ cells. The activated CD4+ cells expressed CD95L and mediated lysis of CD95+ MT-901 tumor cells, which were major histocompatibility complex class II negative. The CD4+ cells also released GM-CSF in response to tumor stimulation. A Fas fusion protein inhibited tumor lysis and GM-CSF release by the CD4+ cells. These studies document an alternate pathway by which CD4+ immune cells may recognize major histocompatibility complex class II-deficient tumors in which CD95L-bearing T cells induced an anti-tumor response mediated via CD95L:CD95.

Enhanced adjuvant effect of granulocyte-macrophage colony-stimulating factor plus interleukin-12 compared with either alone in vaccine-induced tumor immunity.

Using the poorly immunogenic D5 murine melanoma, we examined the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) secretion by gene-modified tumor cells inoculated as a vaccine to prime tumor-draining lymph nodes (TDLNs). D5 transfectants that secreted IL-12 or GM-CSF alone were compared with a double transfectant that secreted equivalent amounts of both cytokines. TDLN cells harvested 9-10 days after subcutaneous tumor inoculation were cultured sequentially in anti-CD3 and IL-2 and assessed for antitumor reactivity against wild-type D5 tumor. The double transfectant-induced TDLN effector cells had greater cytotoxicity in a long-term assay than TDLN cells primed by single transfectants. In adoptive immunotherapy, the TDLN cells primed by the double transfectant were significantly better at mediating the regression of established tumors compared with the TDLN cells elicited by the single transfectants. Both IL-12 and GM-CSF had adjuvant effects in promoting tumor-reactive TDLN cells, but the combination was better than either alone. These observations suggest that the immunomodulation roles of IL-12 and GM-CSF are different and complementary.

Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.

Interferon-gamma-inducing factor elicits antitumor immunity in association with interferon-gamma production.

Interferon-gamma-inducing factor (IGIF) is a novel cytokine that stimulates T-cell proliferation, augments natural killer (NK) cell lytic activity, and induces interferon-gamma (IFN-gamma) production in established type 1 T-helper (Th1) cells in the presence of anti-CD3 antibody. The in vitro induction of IFN-gamma by recombinant murine IGIF in these cells was more potent than that induced by murine interleukin-12 (IL-12) and occurred apparently independent of murine IL-12. Here we report that subcutaneous injection into mice of tumor cells transfected with murine IGIF complementary DNA (cDNA) resulted in > or = 10-fold increase of mitogen-stimulated IFN-gamma production in cultured splenocytes. In addition, IGIF-transfected Renca and K1735 tumor cells can be rejected in vivo. The IGIF antitumor effect was abrogated in mice that were sublethally irradiated or depleted of both CD4+ and CD8+ T cells but not in mice depleted of either subpopulation alone. The antitumor effect mediated by IGIF appears to be dependent on IFN-gamma production, because in vivo neutralization of IFN-gamma was accompanied by growth of IGIF-transfected tumors in 100% of the animals. Taken together, our results show that murine IGIF can elicit T-cell-dependent antitumor immunity associated with IFN-gamma induction.

Recombinant interleukin 12 enhances cellular immune responses to vaccination only after a period of suppression.

Adjuvant use of recombinant murine IL-12 (rmIL-12) was examined in mice vaccinated with irradiated syngeneic tumor or allogeneic cells. rmIL-12 given to A/J mice vaccinated with irradiated SCK tumor cells engineered to secrete granulocyte/macrophage-colony-stimulating factor resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose dependent and manifested as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. Transient immune suppression was also seen with rmIL-12 given during vaccination of C3H/HeN mice with irradiated K1735 melanoma cells and of C57BL/6 mice with irradiated allogeneic HKB cells. The period of suppression coincided with transiently reduced splenic T-cell mitogenic responses to concanavalin A and IL-2, suggesting that they may be causally related. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, which is actually enhanced as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. Demonstration that rmIL-12, as it is frequently used, induces a transient period of impaired immune response that can compromise host protection suggests that the unquestioned effectiveness of rmIL-12 against murine tumors is primarily due to activation of mechanisms other than antigen-specific tumor immunity (e.g., antiangiogenic effects) and that use of human IL-12 should be monitored for similar effects.

Reduced efficacy of allogeneic versus syngeneic fibroblasts modified to secrete cytokines as a tumor vaccine adjuvant.

We examined the relative efficacy of allogeneic versus syngeneic fibroblasts admixed with tumor cells as a vaccine to induce antitumor T-cell reactivity. Allogeneic (3T3) or syngeneic (BLK) fibroblasts transfected to secrete equivalent amounts of GM-CSF were admixed with either D5 melanoma or MCA 207 sarcoma and inoculated s.c. into the flanks of C57BL/6 mice. Vaccine-primed lymph node (LN) cells were examined for in vivo antitumor reactivity in an adoptive transfer model. At fibroblast: tumor cell ratios of < or=1, allogeneic and syngeneic granulocyte macrophage colony-stimulating factor-secreting fibroblasts enhanced T-cell reactivity to tumor cells. However, at ratios of 2.4, the adjuvant effect induced by granulocyte macrophage colony-stimulating factor was not evident. Instead, we observed increased alloreactivity of primed LN cells against 3T3 targets as assessed by cytotoxicity and cytokine release assays, which was not observed with syngeneic fibroblasts. Moreover, with increasing numbers of allogeneic fibroblasts, there was a skewing of the T-cell Vbeta repertoire. These latter cells responded to tumor stimulation with the release of greater amounts of interleukin 10, which may account for the diminished antitumor reactivity observed in vivo. Allogeneic fibroblasts transduced to secrete interleukin 2 or IFN-gamma also induced diminished tumor reactivity of primed LN cells. Syngeneic fibroblasts are superior to allogeneic fibroblasts as vehicles to deliver cytokines in tumor vaccines.

Type 1 versus type 2 cytokine release by Vbeta T cell subpopulations determines in vivo antitumor reactivity: IL-10 mediates a suppressive role.

We have previously reported that CD8+ tumor-draining lymph node (TDLN) cells activated with anti-CD3 and IL-2-mediated tumor regression in adoptive immunotherapy. In this study, we examined the TCR Vbeta repertoire usage of TDLN cells with respect to cytokine release profiles and therapeutic efficacy in vivo. The majority of the whole population of TDLN cells after activation with anti-CD3 were composed of Vbeta3+, -5+, -7+, -8+, and -11+ cells. Enrichment of Vbeta subsets of TDLN cells by in vitro activation with anti-Vbeta mAb revealed Vbeta8+ cells released high amounts of IFN-gamma and granulocyte/macrophage-CSF (GM-CSF) with minimal amounts of IL-10 in response to tumor and mediated tumor regression in vivo. In contrast, enriched populations of Vbeta5+, Vbeta7+, and Vbeta11+ cells released low amounts of IFN-gamma and GM-CSF with high levels of IL-10 and had no in vivo antitumor reactivity. In vitro depletion of specific Vbeta subsets from the whole TDLN pool confirmed that the profile of cytokines released correlated with in vivo antitumor function. Therapeutic efficacy mediated by TDLN cells required the release of IFN-gamma and GM-CSF since in vivo neutralization of both cytokines inhibited tumor regression. The administration of anti-IL-10 mAb abrogated the suppressed antitumor response manifested by adoptively transferred TDLN cells, which elaborated increased levels of IL-10. Our study documents that type 1 cytokine release (i.e., IFN-gamma and GM-CSF) promotes in vivo tumor Ag recognition, in contrast to type 2 release (i.e., IL-10), which suppresses this interaction, and discriminates the functional activity of Vbeta subpopulations of effector cells.

Immune responsiveness to a murine mammary carcinoma modified to express B7-1, interleukin-12, or GM-CSF.

This report characterizes the immunological host response to a syngeneic murine mammary carcinoma along with variants genetically modified to express B7-1 or secrete GM-CSF and interleukin-12 (IL-12). MT-901 is a subline of a mammary adenocarcinoma that was chemically induced in the Balb/c host. It was found to be weakly immunogenic by immunization/ challenge experiments, and it induced tumor-specific T-cell responses in lymph nodes (LN) draining progressive subcutaneous tumors. Tumor clones expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity without completely abrogating tumor growth, whereas IL-12 elaboration lead to complete tumor growth inhibition. In vivo subcutaneous inoculation of a transgenic cell clone secreting GM-CSF (240 ng/10(6) cells/24 hours) resulted in significantly enhanced T-cell reactivity of tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. This finding was obtained from observations assessed by several different methods, including: 1) in vitro cytotoxicity, 2) in vitro interferon-gamma release, and 3) adoptive transfer in mice with established tumor. Moreover, the transfer of activated LN cells derived from mice inoculated with GM-CSF-secreting tumor cells resulted in the prolonged survival of animals with macroscopic metastatic disease, which was not evident utilizing LN cells from mice inoculated with wild-type tumor. By contrast, clones that expressed B7-1 or IL-12 (4 ng/10(6) cells/24 hours) did not elicit enhanced tumor-reactive TDLN cells compared with wild-type tumor when assessed in the adoptive transfer model. The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to this weakly immunogenic tumor.

Different cytokine profiles released by CD4+ and CD8+ tumor-draining lymph node cells involved in mediating tumor regression.

We have previously demonstrated that the growth of weakly immunogenic murine sarcomas leads to the induction of immunologically specific pre-effector cells in tumor-draining lymph nodes (TDLN). The in vitro activation of TDLN cells with anti-CD3 monoclonal antibodies (mAbs) and interleukin-2 (IL-2) resulted in the acquisition of effector function as measured by tumor regression in the adoptive immunotherapy of pulmonary metastases. Further studies were performed to characterize the mechanisms associated with in vivo tumor reactivity mediated by activated TDLN cells. By positive selection, CD4+ and CD8+ T cells were purified and activated by the anti-CD3/IL-2 method. CD8+, but not CD4+, cells manifested tumor-specific granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) release in vitro, and elicited tumor regression in vivo. By contrast, only activated CD4+ were found to release significant amounts of IL-2 in response to tumor antigen but did not mediate tumor regression in vivo. Mixing the two purified populations enhanced the antitumor activity of the CD8+ T cells. In culture, IL-2 was found to augment the relative amount of tumor-specific release of GM-CSF and IFN-gamma by activated TDLN cells. We found that the tumor-specific release of GM-CSF and IFN-gamma by activated lymphocytes was strongly associated with the in vivo therapeutic efficacy of these cells. Evidence in support of this included the following: (1) cytokine release of TDLN derived after different durations of tumor growth correlated with tumor reactivity in adoptive transfer studies, (2) cytokine release of T cells derived from different lymphoid organs corresponded with tumor reactivity in adoptive transfer, and (3) in vivo administration of neutralizing mAb to IFN-gamma and GM-CSF significantly inhibited the antitumor reactivity of TDLN cells. These studies document the contributory roles of IFN-gamma, GM-CSF, and IL-2 released by activated CD4+ and CD8+ T cells involved in tumor regression.

Macromolecules that are colocalized with deposits of beta 2-microglobulin in hemodialysis-associated amyloidosis.

BACKGROUND: Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein. EXPERIMENTAL DESIGN: Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin. RESULTS: Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000. CONCLUSIONS: These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.