Mediation of in vivo tumor-neutralizing activity by Lyt-2+ as well as L3T4+ T cell subsets

The present study reexamines the cell surface nature of T cells mediating in vivo protective tumor immunity with the use of anti-L3T4 and -Lyt-2 antibodies. C3H/HeN mice hyperimmune against syngeneic MH134 hepatoma or MCH-l-Al fibrosarcoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated challenges with viable tumor cells. Spleen cells from these mice were fractionated into L3T4+ or Lyt-2+ T cell subset by treatment with anti-Lyt-2 or -L3T4 antibody plus complement (C). Winn assays performed by utilizing such fractionated T cells have revealed that both L3T4+ and Lyt-2+ T cell subsets from hyperimmune mice produced complete tumor protection. Flow microfluorometry study illustrated that the treatment with anti-L3T4 or -Lyt-2 antibody plus C resulted in the complete isolation of L3T4- Lyt-2+ (Lyt-2+) or L3T4+ Lyt-2- (L3T4+) T cell subset, respectively. This contrasted with the failure of treatment with anti-Lyt-1 antibody plus C to isolate all T cells expressing Lyt-2 marker. It was further demonstrated that each subset of T cells exerted its anti-tumor effect in a tumor-specific way and without a requirement for the other alternative subpopulation of unprimed T cells. These results indicate that Lyt-2+ T cell subset can be successfully isolated by treatment with anti-L3T4 but not with anti-Lyt-1 antibody plus C, and that each single subset of Lyt-2+ and L3T4+ T cells can function as in vivo effector T cells.     

Selective suppression of the generation of anti-tumor L3T4+ but not of Lyt-2+ T cell-mediated immunity in the tumor-bearing state

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2+ and L3T4+ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2+ and L3T4+ T subsets from hyperimmune mice, only the Lyt-2+ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4+ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4+ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2+ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4+ but not of Lyt-2+ T cell function in the tumor-bearing state.     

Selective Suppression of the Generation of Anti-tumor L3T4+ but Not of Lyt-2+ T Cell-mediated Immunity in the Tumor-hearing State

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2⁺ and L3T4⁺ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2⁺ and L3T4⁺ T subsets from hyperimmune mice, only the Lyt-2⁺ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4⁺ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4⁺ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2⁺ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4⁺ but not of Lyt-2⁺ T cell function in the tumor-hearing state.     

Cyclophosphamide-induced immunologically mediated regression of a cyclophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells

It was shown that it is possible to use cyclophosphamide (Cy) to cause immunologically mediated regression of the immunogenic, Cy-resistant L5178Y lymphoma in syngeneic and semisyngeneic mice. In order to cause tumor regression it was necessary to give Cy shortly before or shortly after tumor implantation. However, regardless of whether Cy was given before or after tumor implantation, tumor regression did not commence until 10 days of progressive tumor growth, by which time the tumor was 1 cm in diameter. Tumor regression was associated with the presence in the spleen of an increased number of Lyt-2+ T-cells capable of passively transferring immunity to tumor-bearing recipients. This augmented level of immunity was sustained throughout the period of tumor regression. In contrast, a lower level of concomitant immunity generated by control tumor bearers decayed after Day 12 of tumor growth. Because the therapeutic effect of Cy could be inhibited by passive transfer of L3T4+ T-cells from normal donor mice it is apparent that the therapeutic effect of Cy is based on its ability to preferentially destroy L3T4+ suppressor T-cells. These putative precursor suppressor T-cells were regenerated 4 days after being destroyed by Cy. Taken together the results represent a striking example of the negative regulatory influence of suppressor T-cells on the immune response to an immunogenic tumor.     

Some characteristics of the cyclophosphamide-induced immunopotentiating cells in the spleen of mice bearing a large MOPC-315 tumor

We have shown previously (Ye, Q-W., and Mokyr, M. B. Cancer Res., 44: 3873-3879, 1984) that, following low-dose cyclophosphamide (CY) therapy (15 mg/kg) of mice bearing a large s.c. MOPC-315 tumor and extensive metastases, T-cell-dependent immunopotentiating activity appears in their hitherto immunosuppressive Sephadex G-10-adherent spleen cell population. Here we show that the CY-induced immunopotentiating T-cells express the Lyt 1, Lyt 2, and L3T4 phenotypes. The phenotype of the immunopotentiating T-cells was deduced from our observations that depletion of Lyt 1+, Lyt 2+, or L3T4+ cells from the Sephadex G-10-adherent spleen cell population of CY-treated tumor bearers abolished the ability of the adherent cells to enhance the generation of antitumor cytotoxicity when added to the in vitro immunization culture of normal spleen cells. Moreover, admixture of a Sephadex G-10-adherent cell population depleted of Lyt 2+ cells with a Sephadex G-10-adherent cell population depleted of L3T4+ cells failed to restore the immunopotentiating activity, indicating that T-cells that are apparently expressing simultaneously the Lyt 2 and L3T4 antigens are required for the exertion of the CY-induced immunopotentiating activity. The CY-induced immunopotentiating T-cells from MOPC-315 tumor bearers brought about the appearance of enhanced antitumor cytotoxicity not only against the MOPC-315 tumor cells, but also against two other syngeneic plasmacytomas, with surface immunoglobulin of a different class and antigenic specificity than the MOPC-315 tumor cells, as well as against a variant MOPC-315 tumor line which lacks surface immunoglobulin. The CY-induced immunopotentiating T-cells did not enhance the appearance of antitumor cytotoxicity against a syngeneic (WEHI 22.1) or an allogeneic (EL4) tumor of T-cell origin nor against the natural killer-sensitive YAC-1 cells. Thus, L3T4+, Lyt2+ T-cells from CY-treated MOPC-315 tumor bearers enhance the generation of antitumor cytotoxicity that is directed against plasmacytoma shared antigens other than immunoglobulins.     

Mediation of in vivo Tumor-neutralizing Activity by Lyt-2+ as Well as L3T4+ T Cell Subsets*1

The present study reexamines the cell surface nature of T cells mediating in vivo protective tumor immunity with the use of anti-L3T4 and -Lyt-2 antibodies. C3H/HeN mice hyperimmune against syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated challenges with viable tumor cells. Spleen cells from these mice were fractionated into L3T4⁺ or Lyt-2⁺ T cell subset by treatment with anti-Lyt-2 or -L3T4 antibody plus complement (C). Winn assays performed by utilizing such fractionated T cells have revealed that both L3T4⁺ and Lyt-2⁺ T cell subsets from hyperimmune mice produced complete tumor protection. Flow microfluorometry study illustrated that the treatment with anti-L3T4 or -Lyt-2 antibody plus C resulted in the complete isolation of L3T4^? Lyt-2⁺ (Lyt-2⁺) or L3T4⁺ Lyt-2^? (L3T4⁺) T cell subset, respectively. This contrasted with the failure of treatment with anti-Lyt-1 antibody plus C to isolate all T cells expressing Lyt-2 marker. It was further demonstrated that each subset of T cells exerted its anti-tumor effect in a tumor-specific way and without a requirement for the other alternative subpopulation of unprimed T cells. These results indicate that Lyt-2⁺ T cell subset can be successfully isolated by treatment with anti-L3T4 but not with anti-Lyt-I antibody plus C, and that each single subset of Lyt-2⁺ and L3T4⁺ T cells can function as in vivo effector T cells.     

In vitro differentiation of T-cells capable of mediating the regression of established syngeneic tumors in mice

Previous studies have shown that successful adoptive immunotherapy of a newly induced, weakly immunogenic murine sarcoma, MCA 105, can be achieved either with fresh noncultured immune spleen cells or with immune cells after in vitro stimulation and expansion. In this study, we utilized in vivo and in vitro depletions with monoclonal antibodies (mAb) of T-cell subpopulations expressing the L3T4 or Lyt-2 antigens to investigate the phenotype of the T-cells that mediate in vivo tumor regression. The efficiency of depletion was assessed by flow microfluorometric analysis and by the ability of specifically treated spleen cell populations to generate allogeneic cytotoxic T-lymphocytes. The therapeutic efficacy of adoptively transferred fresh noncultured MCA 105 immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb to mice bearing 3-day established pulmonary metastases. In vitro treatment of fresh noncultured MCA 105 immune cells with either L3T4 or Lyt-2 mAb and complement also abrogated their antitumor efficacy confirming the initial findings. However, mixing L3T4 and Lyt-2 mAb and complement-treated MCA 105 immune cells reconstituted the antitumor efficacy indicating that cellular cooperation between these two lymphoid subpopulations was essential for the regression of established tumors. Unlike fresh noncultured immune cells, the antitumor efficacy of in vitro sensitized and expanded immune cells was abrogated by in vivo treatment with Lyt-2 but not with L3T4 mAb indicating Lyt-2+ cells alone played a major role in mediating the regression of tumors. These findings provide evidence for an in vitro-induced differentiation of therapeutic T-lymphocytes. Our results thus suggest that the antitumor activities expressed by the two types of cells may represent T-cells at different stages of immunological differentiation.     

Importance of Lyt-2+ T-cells in the resistance of melphalan-cured MOPC-315 tumor bearers to a challenge with MOPC-315 tumor cells

We have previously shown that mice bearing a large MOPC-315 tumor can be cured by a low dose of melphalan (L-phenylalanine mustard; L-PAM) if T-cell-dependent antitumor immunity also aids in tumor eradication (S. Ben-Efraim et al., Cancer Immunol. Immunother., 15: 101-107, 1983). Here we describe the phenotype of the T-cells that are responsible for the potent antitumor immunity exhibited by the L-PAM cured MOPC-315 tumor bearers. Initially, we identified the subset of T-cells responsible for the ability of spleen cells from L-PAM-cured MOPC-315 tumor bearers to neutralize tumor growth in the local adoptive transfer assay. The tumor-neutralizing activity of spleen cells from L-PAM-cured tumor bearers was drastically reduced when the spleen cells were depleted either in vitro or in vivo of Lyt-2+ cells. On the other hand, the tumor-neutralizing activity of spleen cells from L-PAM-cured MOPC-315 tumor bearers was not reduced, but actually enhanced, when the spleen cells were depleted either in vitro or in vivo of L3T4+ cells. The Lyt-2+ T-cells, and not the L3T4+ T-cells, were also found to be important for the ability of the intact L-PAM-cured MOPC-315 tumor bearers to reject a challenge with MOPC-315 tumor cells. Specifically, treatment of L-PAM-cured MOPC-315 tumor bearers with monoclonal anti-Lyt-2.2 antibody drastically reduced the ability of the mice to reject the tumor challenge. In contrast, treatment of the L-PAM-cured MOPC-315 tumor bearers with monoclonal anti-L3T4 antibody did not interfere with the ability of the mice to reject the tumor challenge, yet the same protocol of anti-L3T4 antibody treatment abolished the ability of splenic T-cells to provide help for the generation of a primary T-cell-dependent antibody response. The resistance of the L-PAM-cured MOPC-315 tumor bearers to challenge with MOPC-315 tumor cells was also dependent on the participation of carrageenan-sensitive effector cells, most likely macrophages, in tumor eradication. However, the immunity mediated by the T-cells, independent of carrageenan-sensitive effector cells, was extremely powerful and sufficient for the rejection of a tumor challenge with at least 300-fold, and possibly even 3000-fold, the minimal lethal dose of MOPC-315 tumor cells for 100% of normal mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of high-grade tumor-specific immunity in a host using a cytotoxic T-lymphocyte clone specific for a stable tumor antigen on murine leukemia L1210

T-cell clone K4L, the cell surface phenotypes of which were Thy-1+, Lyt-1-, Lyt-2+, and L3T4-, was established from the spleen cells of a murine leukemia L1210-immune mouse. Clone K4L was specific for antigen B on L1210, and this antigen was different from antigen A for which the previously reported T-cell clone K7L was specific. K4L possessed cytotoxicity and tumor growth-inhibitory activity against both L1210 and antigen A loss variant, L1210-K7L-, but not against syngeneic tumor P388 or L5178Y. Previously we showed that antigen A was lost frequently for generation of antigen loss variants. In contrast, antigen B was barely found to be lost. When mice were inoculated with L1210 plus a moderate dose of K4L, the tumor grew after initial suppression but this newly emerging tumor was K4L sensitive and was ultimately rejected. The mice initially given L1210 plus K4L attained a high-grade tumor-specific immunity for rejecting the subsequently challenged high-dose (10(7) cells) L1210. This immunity did not involve any bystander antitumor activity against the third party P388 lymphoma that was injected together with L1210 but accompanied the increase in the L1210-specific cytotoxic T-lymphocyte activity. Evidence was provided that the live L1210, the outgrowth of which was inhibited by K4L, induced an effective immune response of radiation-sensitive host lymphocytes including L3T4+ helper T-cells. Taken together, our results show a novel strategy for inducing high-grade host-dependent antitumor immunity by use of a cytotoxic T-lymphocyte clone specific for a stable tumor-specific transplantation antigen.     

Phenotype analyses and cellular mechanisms of the pre-effector T-lymphocyte response to a progressive syngeneic murine sarcoma

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully functional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an established in vitro sensitization (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 (CD4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM1 (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 metastases. Depletion of Lyt-2+ cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of L3T4+ cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4-, and ASGM-1+. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4+ helper cells. To approach this, mice were depleted of L3T4+, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treatment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of pre-effector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4+ helper cells.     

Mechanisms for recognition of tumor antigens and mediation of anti-tumor effect by noncytolytic Lyt-2+ T cell subset

The mode of anti-tumor function in vivo of noncytolytic Lyt-2+ T cells from C3H/He mice hyperimmune to syngeneic MH134 hepatoma was investigated in a double diffusion chamber system which was recently established in our laboratory. C3H/He mice were implanted intraperitoneally with the double diffusion chamber unit in which each chamber contained either L3T4+ T cell-depleted MH134-hyperimmune spleen cells plus mitomycin C-treated MH134 tumor cells or other syngeneic X5563 viable tumor cells plus normal spleen cells as a source of macrophages. Inclusion of anti-MH134 Lyt-2+ T cells together with MH134 tumor cells in one chamber resulted in comparable growth inhibition of viable X5563 tumor cells in the other chamber to that obtained by unfractionated MH134-hyperimmune spleen cells. The induction in the Lyt-2+ T cell-containing chamber of anti-tumor effect to be delivered into the other chamber was dependent on the co-existence of Ia-positive adherent cells along with Lyt-2+ T cells. Although adherent cell-depleted Lyt-2+ T cells regained the inducibility of anti-tumor immunity when supplemented with splenic adherent cells, the addition of adherent cells pretreated with chloroquine failed to restore the ability of Lyt-2+ T cells to induce their anti-tumor effect. In addition, paraformaldehyde-treated MH134 tumor cells instead of untreated tumor cells were not capable of activating Lyt-2+ T cells. These results indicate that a portion of Lyt-2+ T cells exerts their anti-tumor effect by a mechanism distinct from direct tumor cell lysis and that their activation for mediation of this type of tumor immunity requires the recognition of tumor antigens processed and presented by Ia-positive adherent cells.     

Changes in lymphocyte subsets following multiple administration of recombinant interleukin-2 plus recombinant interferon-beta or -gamma in tumor-bearing mice

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-beta) or -gamma (rIFN-gamma) showed a significant antitumor effect against sc adenocarcinoma 755 in mice, although treatment with either one alone had almost no effect. The combination of rHIL-2 and rIFN-beta caused regression of the tumor but the combination of rHIL-2 and rIFN-gamma did not. Injection of tumor-bearing mice with the combinations of rHIL-2 and rIFN resulted in marked increases in the total number of peritoneal lymphocytes, and the frequency of Lyt-2+ cells was more markedly increased by the combination of rHIL-2 and rIFN-beta than by the combination of rHIL-2 and rIFN-gamma. In Winn assay, elimination of the Lyt-2+ population abolished the protective capacity of the peritoneal cells. The subsets of thymocytes were drastically changed when mice were bearing a tumor or were treated with cytokines. In particular, Lyt-2+/L3T4+ cells were decreased in tumor-bearing mice, but many Lyt-2+/L3T4+ cells were maintained in the thymus by treatment with a cytokine alone. When treated with rHIL-2 and rIFN-beta, the Lyt-2+/L3T4+ cells were markedly decreased, while Lyt-2+/L3T4- T-cells were increased, but these subsets were little changed by treatment with rHIL-2 plus rIFN-gamma. Thus, injections of rHIL-2 and rIFN-beta into tumor-bearing mice resulted in a high frequency of Lyt-2+/L3T4- cells in the peritoneal cavity, together with changes in the T-cell subsets in the thymus. These results suggest that maturation of T-cells in the thymus may be an important step in the pathway by which cytokine treatment brings about regression of tumors.     

T-cell subsets, IFN-gamma production and efferent specificity in anti-parental tumor immunity induced by mouse sensitization with xenogenized variant cells

In addition to previous evidence for a role of L3T4+ T cells in the protective anti-parental tumor immunity induced by xenogenized variant cells of a murine lymphoma (L5178Y/DTIC), we have investigated the possible participation in this effect of L5178Y tumor-specific lymphocytes of the Lyt-2+ T cell subset. Spleen cells from L5178Y/DTIC tumor-immunized mice produced high levels of IFN-gamma in vitro in response to parental antigens, and this activity was only abolished by treating the responder population with anti-Thy-1.2 antibody or a combination of anti-L3T4 and anti-Lyt-2.2 monoclonal antibodies (MAbs) plus complement. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-tumor cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class-II and anti-class-I H-2 antibody to the cultures. Administration of anti-IFN-gamma MAb in vivo significantly impaired the resistance of L5178Y/DTIC-immune mice to challenge with parental cells, as manifested by survival criteria and increased tumor-cell proliferation in the spleens of antibody-treated mice. Although anti-parental tumor protection in vivo and T-cell activation in vitro for IFN-gamma production were strictly antigen-specific, bystander tumor inhibition was observed when antigenically irrelevant cells were inoculated with the L5178Y lymphoma. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in the protective anti-parental tumor immunity induced by xenogenized cells, and that their activity may involve IFN-gamma-mediated stimulation of non-specific tumoricidal mechanisms.     

N-methyl-N-nitrosourea-induced and spontaneous AKR/J thymic lymphomas express distinct differentiation antigen phenotypes

Spontaneous and N-methyl-N-nitrosourea (MNU)-induced AKR/J thymic lymphomas were characterized for expression of several lymphocyte differentiation antigens. The majority (53%) of spontaneous lymphomas expressed both Lyt-2 and L3T4 antigens, similar to the predominant normal thymocyte subset. In contrast, 63% of the thymic lymphomas induced by the chemical carcinogen MNU, expressed an Lyt-2+ L3T4- antigenic profile. Although this profile suggested that MNU-induced lymphomas are phenotypically similar to a mature thymocyte subset, the presence of ThB antigen on Lyt-2+ L3T4- lymphomas did not support this notion. Diagonal gel electrophoresis of 125I-labeled membrane extracts and immunoprecipitates revealed that 17 of 29 Lyt-2+ L3T4-MNU-induced lymphomas expressed cell surface T-cell receptor heterodimer components. Northern blot analyses confirmed that the T-cell receptor material was composed of alpha and beta polypeptide chains. The results from this study indicate a distinct origin or differentiation potential of the target cells involved in viral and chemical induced lymphomagenesis of AKR/J mice.     

Changes in Lymphocyte Subsets Following Multiple Administration of Recombinant Interleukin-2 plus Recombinant Interferon-beta or -gamma in Tumor-bearing Mice

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-β) or -gamma (rIFN-γ) showed a significant antitumor effect against sc adenocarcinoma 755 in mice, although treatment with either one alone had almost no effect. The combination of rHIL-2 and rIFN-β caused regression of the tumor but the combination of rHIL-2 and rIFN-γ did not. Injection of tumor-bearing mice with the combinations of rHIL-2 and rIFN resulted in marked increases in the total number of peritoneal lymphocytes, and the frequency of Lyt-2⁺ cells was more markedly increased by the combination of rHIL-2 and rIFN-β than by the combination of rHIL-2 and rIFN-γ. In Winn assay, elimination of the Lyt-2⁺ population abolished the protective capacity of the peritoneal cells. The subsets of thymocytes were drastically changed when mice were bearing a tumor or were treated with cytokines. In particular, Lyt-2⁺/L3T4⁺ cells were decreased in tumor-bearing mice, but many Lyt-2⁺/L3T4⁺ cells were maintained in the thymus by treatment with a cytokine alone. When treated with rHIL-2 and rIFN-β the Lyt-2⁺/L3T4⁺ cells were markedly decreased, while Lyt-2^-/L3T4^- T-cells were increased, but these subsets were little changed by treatment with rHIL-2 plus rIFN-γ. Thus, injections of rHIL-2 and rIFN-β into tumor-bearing mice resulted in a high frequency of Lyt-2⁺/L3T4^- cells in the peritoneal cavity, together with changes in the T-cell subsets in the thymus. These results suggest that maturation of T-cells in the thymus may be an important step in the pathway by which cytokine treatment brings about regression of tumors.     

Induction of "innocent bystander" cytotoxicity in nonimmune mice by adoptive transfer of L3T4+ Lyt-1+2- mammary tumor immune T-cells

Spleen cells from BALB/c mice, bearing a syngeneic mammary adenocarcinoma, nonspecifically lyse xenogeneic target cells following in vitro reexposure to mammary tumor-associated antigens. The effectors of this reaction have been shown to be Thy-1+ Lyt-1+2+ nylon-adherent cells. Tumor-immune spleen cells are also able to induce nonimmune spleen cells to express nonspecific cytotoxicity. Studies were undertaken to determine whether this inducer activity is mediated by the effector population or by a functionally distinct subset. Cell separation studies demonstrated that the inducers and effectors of innocent bystander cytotoxicity can be separated based upon the property of adherence to nylon wool. Further examination of the nylon-nonadherent inducer population indicated that the phenotype is L3T4 + Lyt-1+2-. By flow cytometry the inducer subset was shown to express a higher density of Thy 1 antigen than that expressed by the cytotoxic effectors. When adult thymectomized mice were implanted with mammary tumors, nonspecific effectors were not generated. In contrast, spleen cells from the same experimental animals were able to induce nonspecific cytotoxicity in normal mice following adoptive transfer of their spleen cells. Thus, these data support the conclusion that during the course of mammary tumor growth, at least two functionally and phenotypically distinct cell populations interact for the expression of "innocent bystander" cytotoxicity.     

T8 and T3 surface glycoproteins in human T-cell mediation of leukocyte adherence inhibition to extracts of autologous cancer

Monoclonal antibodies (MAb's) [anti-Leu-1, anti-Leu-2a (T8), anti-Leu-3a (T4), and anti-Leu-4 (T3)] were used to elucidate the type of T-cell mediating leukocyte adherence inhibition (LAI) and the role of T-cell surface glycoproteins in LAI. T8+ (Leu-2a+) and T4+ (Leu-3a+) subtypes were isolated by panning. T8+ (Leu-2a+) cells showed LAI to extracts of autologous cancer, whereas the T4+ (Leu-3a+) subset had no LAI response. Moreover, MAb to the T8+ (Leu-2a+) glycoprotein negated T-cell LAI, but MAb to Leu-1+ or to T4+ (Leu-3a+) did not negate T-cell LAI to autologous cancer extracts. The T3+ (Leu-4+) differentiation also was essential for T-cell LAI to autologous cancer extracts since anti-Leu-4 (T3) negated the response. Since LAI to autologous cancer extracts depends ultimately on leukotriene and other oxidative metabolites of arachidonic acid generated by the T-cell binding tumor antigen, the effect of MAb on LAI induced by leukotriene B4 isomer III. 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4) was examined. Authentic LTB4 induced nonadherence of 34% of adherent T-cells, and this effect was not negated by anti-Leu-1, anti-T8 (Leu-2a), or anti-T4 (Leu-3a). However, anti-T3 (Leu-4) abrogated LTB4-induced LAI of pure T-cells without any effect on the basic adherence properties of T-cells. The present findings indicated that LAI to autologous cancer extracts was mediated by T-cells of the T8+ phenotype when they recognize tumor antigen and polymorphic major histocompatibility complex determinants on autologous cancer membranes. Moreover, differentiation glycoproteins T8+ (Leu-2a+) and T3+ (Leu-4+) on the surface of the responding effector T-cells performed distinct biologic functions that enabled the tumor antigen to trigger T-cell LAI.     

Effects of interleukin-2 and interferon-alpha A/D treatment on lymphocytes from tumour-bearing mice

The in vivo antitumour activities of recombinant human interleukin-2 (rHIL-2) and recombinant human hybrid interferon alpha A/D (rIFN-alpha A/D) were tested in relation to adenocarcinoma 755. The tumour growth, following s.c. inoculation of tumour cells, was inhibited to a greater extent in mice treated with the combination of cytokines than in mice treated with either one alone. Pretreatment with these cytokines did not affect the tumour growth. Injection of tumour-bearing mice with a combination of these cytokines resulted in a marked increase in the total number of lymphocytes in the peritoneal cavity. Among them, Lyt-2+/L3T4- and asialo GM1+ cells were markedly enhanced by the combination of cytokines, and the frequencies of these marker cells were closely correlated with the antitumour activity. In tumour-bearing mice, the size of the thymus was decreased while that of the spleen was increased compared to non-tumour-bearing (normal) mice. Treatment with rHIL-2 caused the thymus, spleen and liver to be larger compared to untreated tumour-bearing mice, but when treated with a combination of rHIL-2 and rIFN-alpha A/D these organs were smaller than when rHIL-2 was administered alone. Thymocytes were drastically changed when mice were bearing a tumour or were treated with a cytokine. Especially immature T-cells, Lyt-2+/L3T4+, were drastically decreased in tumour-bearing mice, but were maintained following administration of rHIL-2 or rIFN-alpha A/D. When treated with rHIL-2 plus rIFN-alpha A/D, Lyt-2+/L3T4+ T-cells were decreased while Lyt-2+/L3T4- T-cells were increased. Frequency of immature T-cells, Lyt-2-/L3T4-, was not changed. On the other hand, T-cell subsets of splenocytes were markedly decreased in tumour-bearing mice compared to normal mice, but all the subsets of splenocytes were almost unchanged even when tumour-bearing mice were treated with rHIL-2 plus rIFN-alpha A/D. Thus, injection of rHIL-2 and rIFN-alpha A/D to tumour-bearing mice resulted in induction of Lyt-2+/L3T4- and asialo GM1+ cells in the peritoneal cavity, and the frequencies correlated with the observed antitumour activity in vivo in this murine model. The increase in Lyt-2+/L3T4- T-cells in the peritoneal cavity may be related to changes in the T-cells in thymus.     

Immune surveillance: both CD3+ CD4+ and CD3+ CD8+ T cells control in vivo growth of P815 mastocytoma.

The aim of this study was to examine whether a spontaneous immune response controls neoplastic growth in P815-bearing DBA/2 mice, and to characterize the cells involved in tumor resistance in vivo. Several cell lineages such as T-cell-receptor (TcR)-bearing T cells, NK cells and macrophages mediate some anti-tumor activity in vitro. P815 was chosen as a model because it is weakly immunogenic and is a good target both for tumor-specific, MHC-restricted CTL-mediated lysis and for MHC-unrestricted lysis exerted by long-term cultured lymphocytes or activated macrophages. Since most "NK-like activity" in freshly isolated populations appears to be associated with CD3- cells, whereas antigen-specific, MHC-restricted T cells mostly express CD3 determinants, CD3 was a good marker for evaluating the role of T cells and "NK" cells in tumor resistance in vivo. The survival of anti-CD3-treated animals that were inoculated with tumor cells was strongly reduced (mean survival time: 17 days vs. 40 days for the control group) and was associated with increased tumor growth rate. We followed the same approach to define the T-cell subset(s) that mediate(s) this immune response. Both CD4+ and CD8+ T cells were required for induction of immune control on neoplastic growth. The approach used has revealed the important role of CD4+ T cells in immune responses that control in vivo growth of a class-I-positive, class-II-negative tumor and suggests that these cells may play a central role in tumor resistance. Since CD4+ cells are activated by soluble, exogenous proteins, this finding may have important implications for immunotherapy.