CD56+ putative natural killer cell lymphomas: production of cytolytic effectors and related proteins mediating tumor cell apoptosis?

Apoptosis is a regulated form of cell death that may be triggered by natural killer (NK) or cytotoxic T cells, which effect target cell lysis by cytolytic effector and related proteins through complex intracellular signals. This study was aimed to investigate whether there is selective expression of these cytolytic markers in the putative NK-cell lymphomas and whether there is correlation with zonal tumor cell death in these tumors. Expression of the cytolytic effectors perforin, granzyme B9, and the granule membrane protein TIA1 were examined in 24 putative NK-cell lymphomas, 18 postthymic T-cell lymphomas (one case CD8+ CD56+ and three anaplastic large cell lymphomas (ALCL), three T-lymphoblastic lymphomas, and 20 B-cell lymphomas. Nineteen (79%) putative NK-cell lymphomas expressed perforin, and all 24 cases expressed granzyme B9 and TIA1. The only CD8+ CD56+ postthymic T-cell lymphoma also expressed all three cytolytic markers, two CD8- ALCL expressed TIA1; other postthymic T-cell, T-lymphoblastic, and B-cell lymphomas were consistently negative. There was strong correlation between percentage perforin-positive cells and zonal tumor cell death. Angioinvasion, in contrast, was present only in a proportion (37%) of these lymphomas despite the frequent presence of zonal tumor cell death (71%). We propose that cytolytic effector and related proteins produced by putative NK and some CD8+ CD56+ postthymic T-cell lymphomas, probably in conjunction with other mechanisms, may effect massive tumor cell apoptosis. The frequent expression of cytolytic effector markers in the CD2+ surface CD3- CD56+ putative NK-cell lymphomas lends further support to their probable NK cell origin.     

Mouse palatal width growth rates as an "at risk" factor in the development of cleft palate induced by hypervitaminosis A

In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86 + C1300, expressing a high level of mouse CD86 on the cell surface. While CD86 + C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86 + C1300-rejecting mice with CD86 + C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86 + C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86 + C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86 + C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors.     

Xenospecific CD8+ cytotoxic T lymphocyte generation: accessory function for CD4+ T cells and natural killer 1.1+ cells

BACKGROUND: Controversy exists as to whether natural killer (NK)1.1+ cells additionally support cytotoxic T lymphocyte (CTL) generation. We have previously demonstrated that mice generate a strong in vitro xenospecific CTL response in local popliteal lymph nodes (LN) to footpad immunizations with large numbers of human tumor cells. METHODS: In vivo depletion of various LN subsets using cytotoxic monoclonal antibodies was used to determine their relative importance in stimulating xenospecific CD8+ CTL responses to human Jurkat tumor cells. Depletion of functional NK cells in vivo was evidenced by the relative lack of NK1.1+ cells and NK activity in the spleens and LN of anti-NK1.1 monoclonal antibody-treated mice. CONCLUSION: Depletion of LN subsets indicated that CD4+ T cells were critical in generating an effective xenospecific CD8+ CTL response, but also suggested that NK1.1+ cells play a significant additional accessory role in the development of mouse anti-human xenospecific CTL.     

A molecular model of myelin oligodendrocyte glycoprotein

We evaluated the mechanism of the antitumor effects of mouse rIFN-gamma-inducing factor/IL-18 protein on the growth of mouse tumor cell lines in vivo. Mice received IL-18 before or after challenge with CL8-1, a mouse melanoma cell line. Both regimens significantly suppressed tumor growth and reduced the number of mice with growth of tumor from 60% (3/5) to 20% (1/5). Furthermore, IL-18 administered before and after tumor inoculation completely abrogated the establishment of CL8-1 in all animals. IL-18 administration also significantly suppressed the growth of MCA205, a sarcoma cell line, even when treatment was delayed to 7 days following tumor inoculation. Although IL-18/IL-12 combination therapy had the most significant and immediate antitumor effects, many mice so treated succumbed with markedly elevated serum IFN-gamma levels. The antitumor effects of IL-18 were abrogated almost completely when NK cells were eliminated using anti-asialo GM1 Ab administration, but only marginally impaired in IFN-gamma or IL-12 gene-disrupted mice. Immunohistochemical staining revealed that the number of the CD8+ T cells, but not CD4+ T cells, found at the tumor site was reduced in animals treated with IL-18. These results indicate that IL-18 has potent antitumor effects mediated by CD4+ T cells and NK cells, but in IFN-gamma- and IL-12-independent pathways.     

Combination chemotherapy and IL-15 administration induce permanent tumor regression in a mouse lung tumor model: NK and T cell-mediated effects antagonized by B cells

Previous studies have demonstrated that IL-15 administration after cyclophosphamide (CY) injection of C57BL/6J mice bearing the i.m. 76-9 rhabdomyosarcoma resulted in a significant prolongation of life. In the present study, we investigated the immune response against the 76-9 experimental lung metastases after CY + IL-15 therapy. Administration of CY + IL-15, but not IL-15 alone, induced prolongation of life and cures in 32% of mice bearing established experimental pulmonary metastases of 76-9 tumor. The CY + IL-15 therapy resulted in increased levels of NK1.1+/LGL-1+ cells, and CD8+/CD44+ T cells in PBL. In vitro cytotoxic assay of PBL indicated the induction of lymphokine-activated killer cell activity, but no evident tumor-specific class I-restricted lytic activity. Survival studies showed that the presence of NK and T lymphocytes is necessary for successful CY + IL-15 therapy. Experiments using knockout mice implied that either alphabeta or gammadelta T cells were required for an antitumor effect induced by CY + IL-15 therapy. However, mice lacking in both alphabeta and gammadelta T cells failed to respond to combination therapy. Cured B6 and alphabeta or gammadelta T cell-deficient mice were immune to rechallenge with 76-9, but not B16LM tumor. B cell-deficient mice showed a significant improvement in the survival rate both after CY and combination CY + IL-15 therapy compared with normal B6 mice. Overall, the data suggest that the interaction of NK cells with tumor-specific alphabeta or gammadelta T lymphocytes is necessary for successful therapy, while B cells appear to suppress the antitumor effects of CY + IL-15 therapy.     

Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes

To elucidate the role of NK1.1+ T cells in the antitumor immune response, we established cloned NK1.1+ T cell lines from tumor-infiltrating lymphocytes (TIL) of B16 melanoma, and examined their mode of action in generating antitumor effector T cells both in vitro and in vivo. An NK1.1+ T cell clone (TM4.2) was phenotypically CD3+ TCR-alphabeta+ CD4- CD8- NK1.1+, and CD28+. The TM4.2 cells suppressed the in vitro generation of anti-B16 melanoma CTLs, but not the effector function of CTLs. The results using a transwell membrane suggested that their suppressive activity was mediated by both soluble factors and a direct cell to cell interaction. As for the soluble factors, the suppressive activity of the culture supernatant of TM4.2 cells was neutralized by anti-TGF-beta mAb, and the TM4.2 cells actually produced a considerable amount of TGF-beta. On the other hand, the TM4.2 cells showed a high level of cytolytic activity against B cell blasts and CD80-transfected P815, and such cytolytic activity was reduced by the addition of anti-CD80 mAb. In addition, NK1.1+ T cells in the freshly isolated TIL were revealed to express CD28. Furthermore, the TM4.2 cells suppressed the in vitro generation of anti-allo CTLs irrespective of the MHC haplotype. Finally, the TM4.2 cells suppressed the in vivo antitumor immune response. Collectively, these findings demonstrate that NK1.1+ T cells in TIL show immunosuppressive activity in the antitumor immune response through the production of TGF-beta and the preferential cytolysis of B7-expressing cells.     

Adoptive immunotherapy of murine lymphosarcoma: comparative study using allo-immune or tumor-immune T cells

The antitumor effect of allosensitization with lymphocytes and skin graft of DBA/2 mice was evaluated using immunogeneic, transplantable Lymphosarcoma (LS-A) syngeneic to Swiss mice. A dose dependent tumor inhibitory effect in terms of tumor free mice was observed in mice sensitized i.p. with lymphocyte doses between 10-100 million per animal. Sensitization with allogeneic primary skin graft was more effective than lymphocyte immunization. The antitumor immunity could be adoptively transferred in syngeneic Swiss mice using either allo-immune or tumor-immune T cells. Analysis of T cell phenotypes using monoclonal antibodies against cell surface markers CD4 and CD8, indicated absolute dependence on the CD4+ T cells subset in tumor cure in case of allo-immune as well as tumor-immune T cells. CD8+ T cell subset was found essential only in case of allo-immune T cell therapy. Immunosuppression of mice with whole body gamma irradiation (4 Gy), 6 hr before transfer of allo-immune or tumor-immune T cells did not abrogate the therapeutic ability of allo-immune or tumor-immune T cells. Our results suggest that allosensitization could be an effective method of generating effector lymphocyte populations that might be used to treat tumors that exhibit detectable immunogenecity.     

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.     

Involvement of B lymphocytes in the growth inhibition of human pulmonary melanoma metastases in athymic nu/nu mice by an antibody-lymphotoxin fusion protein

Antibody-cytokine fusion proteins can target biologically active cytokines to various tumor sites, achieving local concentrations sufficient to induce host immune responses leading to tumor elimination. Here, we demonstrate the therapeutic efficacy of a tumor-specific antibody-lymphotoxin fusion protein (ch225-LT) on xenografted pulmonary metastases of human melanoma. In vitro studies indicated a direct cytotoxic effect of such construacts on melanoma cells via the induction of apoptosis, as demonstrated by cell cycle analysis and DNA fragmentation. However, ch225-LT lacked any therapeutic effect in immune deficient C.B17 scid/beige and scid/scid mice, indicating the insufficiency of this direct mechanism in vivo. In contrast, in athymic nu/nu mice, ch225-LT completely inhibited outgrowth of the xenografted tumor. This therapeutic effect was accompanied by infiltrations of CD45+, Mac-1+, and asialo-GM1+ cells into the tumor; B220+ cells were present in the surrounding tissue and the periphery of the tumor. The functional role of asialo-GM2+ cells was confirmed by in vivo depletion studies. Our data indicate that an antibody-lymphotoxin fusion protein effectively inhibits the growth of disseminated melanoma metastases by mechanisms that function in the absence of mature T cells, but require B, NK, and other asialo-GM1+ cells.     

Antitumor effect of CD40 ligand: elicitation of local and systemic antitumor responses by IL-12 and B7

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 has been implicated in the establishment of cell-mediated immunity as well as humoral immune responses. To examine the role of CD40L in eliciting antitumor immunity, we introduced murine CD40L gene into P815 mastocytoma (CD40L-P815). CD40L-P815 cells underwent prompt rejection when inoculated s.c. into syngenic DBA/2 mice or athymic BALB/c nu/nu mice, which was mediated by NK cells and dependent on endogenous IL-12. The primary rejection of CD40L-P815 cells in DBA/2 mice elicited CD8+ T cell-mediated protective and systemic immunity against parental tumor cells, which was induced by CD4+ T cells and endogenous B7. These results indicated a potent antitumor effect of CD40L that is mediated by potentiation of host Ag-presenting cell functions, and introduction of CD40L will be useful as a new strategy of immuno-gene therapy against tumors.     

Lymphotactin gene-modified bone marrow dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity

Dendritic cells (DC) are regarded as attractive candidates for cancer immunotherapy. Our aim is to improve the therapeutic efficacy of DC-based tumor vaccine by augmenting DC preferential chemotaxis on T cells. Mouse bone marrow-derived DC were transduced with lymphotactin (Lptn) gene by adenovirus vector. The supernatants from Lptn gene-modified DC (Lptn-DC) were capable of attracting CD4+ and CD8+ T cells in a chemotaxis assay, whereas their mock control could not. Lptn expression of Lptn-DC was further confirmed by RT-PCR. Lptn-DC were pulsed with Mut1 peptide and used for vaccination. Immunization with the low dose (1 x 10(4)) of Mut1 peptide-pulsed DC induced weak CTL activity, whereas the same amounts of Mut1 peptide-pulsed Lptn-DC markedly induced specific CTL against 3LL tumor cells. A single immunization with 1 x 10(4) Mut1 peptide-pulsed Lptn-DC could render mice resistant to a 5 x 10(5) 3LL tumor cell challenge completely, but their counterpart could not. The protective immunity induced by Mut1 peptide-pulsed Lptn-DC depends on both CD4+ T cells and CD8+ T cells rather than NK cells in the induction phase and depends on CD8+ T cells rather than CD4+ T cells and NK cells in the effector phase. Moreover, the involvement of CD28/CTLA4 costimulation pathway and IFN-gamma are also necessary. When 3LL tumor-bearing mice were treated with 1 x 10(4) Mut1 peptide-pulsed Lptn-DC, their pulmonary metastases were significantly reduced, whereas the same low dose of Mut1 peptide-pulsed DC had no obvious therapeutic effects. Our data suggest that Lptn-DC are more potent adjuvants for peptide delivery to induce protective and therapeutic antitumor immunity.     

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.     

CD8+ T cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection

BALB/c mice vaccinated with vaccinia virus expressing the major surface glycoprotein G of respiratory syncytial virus (RSV) develop lung eosinophilia during RSV challenge. The G protein is remarkable in that it induces CD4+, but no CD8+ T cells in this mouse strain. Studies using passive T cell transfers show that co-injection of CD8+ T cells greatly reduces the Th2-driven lung eosinophilia caused by G-specific CD4+ T cells. By contrast, vaccination with the fusion protein (F) induces both CD8+ and CD4+ T cells, but not lung eosinophilia during RSV infection. These observations suggest that CD8+ T cells play a crucial role in preventing Th2-driven pathology. We therefore depleted mice with anti-CD8 antibodies in vivo. This treatment allowed lung eosinophilia to develop in F-primed mice. Depletion of interferon (IFN)-gamma had a similar effect, suggesting that secretion of this cytokine is the mechanism by which CD8+ T cells exert their effect. To test whether similar effects occurred in other strains of mice, RSV-infected C57BL/6 mice (which do not develop eosinophilia after sensitization to G) were treated with anti-IFN-gamma. Again, these mice developed eosinophilia. In this strain, genetic deletion of CD8-alpha, beta2-microglobulin or genes coding for the transporter associated with antigen presentation (which in each case eliminates CD8+ T cells) caused lung eosinophilia during RSV infection. These studies show the critical roles that CD8+ T cells and IFN-gamma production play in regulating Th2-driven eosinophilia and provide a unifying explanation for previous studies of lung eosinophilia. We propose that vaccines designed to enhance CD8+ T cell recognition might avoid disease caused by CD4+ Th2 cells.     

Inhibition of murine renal carcinoma pulmonary metastases by systemic administration of interferon gamma: mechanism of action and potential for combination with interleukin 4

We have previously demonstrated that IFN-gamma causes cell growth inhibition and up-regulation of MHC antigens in human renal cell carcinoma cell lines. In this study, we have investigated the therapeutic potential of IFN-gamma for the treatment of 5-day established pulmonary metastases induced by i.v. injection of Renca cells, a murine renal adenocarcinoma. We found that systemic injections of IFN-gamma significantly reduced the number of lung metastases in a dose-dependent manner and increased mouse survival. Histological evaluation of IFN-gamma-treated lungs showed residual small tumor nodules containing extensive necrosis and mononuclear infiltrates. Immunohistochemistry studies on lung sections showed macrophage infiltration into tumor nodules, and in vivo depletion of macrophages partially inhibited IFN-gamma antitumor effect, suggesting a role for the macrophages in tumor destruction. Lymphocyte depletion of either natural killer (NK) cells or CD4+ or CD8+ T-cell subsets or both T-cell subsets did not affect the IFN-gamma effect, whereas depletion of both NK and T cells decreased the antitumor activity of IFN-gamma. These data indicate that neither T cells nor NK cells are essential for this activity but that either lymphocyte population can contribute to the IFN-gamma effect. An optimal dose of IFN-gamma inhibited by 60% the growth of Renca cells treated for 3 days in vitro, but this effect was transient and less pronounced in a long-term colony assay, suggesting that IFN-gamma direct growth inhibition may play a role but may not be sufficient to mediate its antitumor effect in vivo. In vitro, IFN-gamma caused up-regulation of class I MHC antigens and induction of class II antigen expression in Renca cells, an effect that may enhance Renca immunogenicity but may be relevant only when a T-cell response is elicited. A sequential administration of IFN-gamma followed by interleukin 4 was therapeutically better than IFN-gamma alone for the treatment of advanced pulmonary metastases, probably due to different antitumor mechanisms induced by these two cytokines.     

Antitumor responses induced by transgenic expression of CD40 ligand

Because CD40 ligand (CD40L) is a co-stimulator molecule for multiple components of the immune response, we wanted to determine whether transgenic expression of the molecule would increase immune responses against a weakly immunogenic murine tumor, neuro-2a. Tumor cells were transduced with a retroviral construct containing the CD40L gene and co-injected with variable numbers of non-CD40L transduced cells into syngeneic mice. Mice injected with cells that expressed CD40L had a significant reduction in average tumor size as compared to controls (p < 0.0001). In addition, survival of the neuro-2a/CD40L mice was 48 days versus 34 days for the neuro-2a/neo controls (p < 0.02). Expression of CD40L by less than 1.5% of neuro-2a cells was sufficient for significant antitumor effects (p < 0.001). These antitumor effects protected mice from subsequent challenge with parental neuro-2a cells. The protective effects of CD40L were associated with systemic immunomodulation. In vivo depletion of CD8+ cells abrogated the CD40L-mediated antitumor effects. Analysis of spleens from CD40L-protected animals showed increased numbers of CD4+ and CD8+ cells, the majority of which co-expressed the activation marker CD25. In addition, an increased number of antigen-presenting cells (APCs) expressed the co-stimulatory molecule CD86. These experiments illustrate that transducing even a small percentage of tumor cells with CD40 ligand can create a long-lasting systemic immune response capable of impeding growth of unmodified neuroblastoma cells.     

Adoptive transfer of anti-CD3-activated CD4+ T cells plus cyclophosphamide and liposome-encapsulated interleukin-2 cure murine MC-38 and 3LL tumors and establish tumor-specific immunity

The infusion of anti-CD3-activated murine T cells plus interleukin-2 (IL-2) exerts antitumor effects against several tumors in murine immunotherapy models. This study compares the therapeutic efficacy of anti-CD3-activated CD4+ or CD8+ T-cell subsets, when given with cyclophosphamide (Cy) and liposome-encapsulated IL-2 (L-IL2) in a murine model. C57BL/6 mice bearing subcutaneous (S.C.) MC-38 colon adenocarcinoma, 3LL Lewis lung carcinoma, or 38C13 lymphoma for 7 to 14 days were pretreated with low-dose intraperitoneal (I.P.) Cy before intravenous (I.V.) injection of anti-CD3-activated T cells or T-cell subsets. Cell administration was followed by I.P. administration of L-IL2 for 5 days. Mice receiving activated CD4+ T cells showed significantly reduced tumor growth or complete remissions with prolonged disease-free survival in MC-38, 3LL, and 38C13. The timing of Cy doses in relation to adoptive transfer was critical in obtaining the optimal antitumor effect by CD4+ cells. Injecting Cy 4 days before the infusion of CD4+ cells greatly enhanced the antitumor effect of the CD4+ cells and improved survival of the mice compared with other Cy regimens. C57BL/6 mice cured of MC-38 after treatment with CD4+ T cells developed tumor-type immunologic memory as demonstrated by their ability to reject rechallenges with MC-38, but not 3LL. Similarly, mice cured of 3LL tumors rejected rechallenges of 3LL, but not MC-38. The immunologic memory could be transferred with an I.V. injection of splenocytes from mice cured of MC-38 or 3LL. No cytotoxic T-lymphocyte activity was detected in T cells or T-cell subsets from mice cured of MC-38 or 3LL. Increased IL-2 and interferon-gamma (IFN-gamma) production was observed from CD4+ subsets in cured animals when stimulated in vitro with the original tumor, but not with an unrelated syngeneic tumor. These results suggest that tumor-specific immunity can be achieved in vivo with anti-CD3-stimulated CD4+ T cells in this cellular therapy model.     

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.     

Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma

A safe and effective therapeutic combination of moderate doses of Adriamycin (doxorubicin, 4 mg/kg, IV, Days 1 and 8 or only Day 8) plus prolonged administration of moderate doses of interleukin 2 (2 micrograms, b.i.d., Days 9-40) was developed in the syngeneic EL4 (5 x 10(4) cells, IP, Day 0) lymphoma--C57B1/6 mouse model and has been reported in the companion paper. The studies described herein demonstrate that the effectiveness of this combination treatment against EL4 lymphoma growing intraperitoneally in C57B1/6 mice was dependent upon the presence of CD8+ cells. Thus, the induction of long-term survivors (60-80%) by Adriamycin plus interleukin 2 was completely ablated by pretreatment of mice with anti-CD8 monoclonal antibody (MAb), whereas pretreatment with anti-CD4 MAb only partially inhibited the therapeutic effects and anti-NK1.1 MAb had no effect. A close association between survival, an increase in phenotypically identified CD8+ cells, and an increase in specific anti-EL4 cytolytic activity was demonstrated with cells from the tumor site (peritoneum) but not consistently with cells from the spleen. No association was observed between survival and modulations in natural killer (NK), lymphokine-activated killer (LAK), or tumoricidal macrophage activity of spleen or peritoneal cells. Taken together the results indicate that, in this model, the most relevant correlate of a therapeutically effective host antitumor response is the level of specific EL4 tumor killing by cells present at the tumor site. Based on the findings reported herein, it can be predicted that weakly immunogenic tumors may be eradicated by immunologic mechanisms elicited in conjunction with properly designed therapeutic regimens.     

Antimetastatic vaccination against Lewis lung carcinoma with autologous tumor cells modified to express murine interleukin 12

Interleukin 12 (IL-12) is a disulfide-linked heterodimer molecule produced predominantly by professional antigen presenting cells. It promotes the induction of sundry biological effects with significant relevance to antitumor immunity, such as enhancing a T(H)1 helper response, an in vivo antiangiogenic effect, induction of adhesion molecules that assist in lymphocyte homing to sites of tumor growth, and a direct stimulatory effect on both T-cells and NK cells. We tested the efficacy of an antimetastatic vaccine composed of autologous murine D122 cells transfected with both subunits of IL-12 cDNA to express biologically-active IL-12 molecule. Expression of IL-12 by D122 cells significantly reduced their tumorigenicity and metastatic potential in immunocompetent syngeneic hosts. Furthermore, vaccination of mice with 2 x 10(6) irradiated IL-12-transfected D122 cells engendered a protective CTL response which rejected a subsequent challenge with parental D122 cells and eradicated lung micrometastasis in animals whose primary tumors have been surgically removed. The antitumor effects of IL-12 were mediated primarily by its ability to induce gammaIFN expression in vivo. CD8+ T-cells as well as NK cells were crucial in the execution of the antitumor effects of IL-12. These results suggest that autologous tumor cells expressing IL-12 by gene transfer are a potent antitumor vaccine able to induce a systemic immune response against poorly immunogenic and spontaneously metastatic tumors.