Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity.

Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.     

The augmentation of tumor-specific immunity by virus help

Summary The role of vaccinia virus-reactive helper T cells (Th) in augmenting in vivo generation of antitumor protective immunity and the Ly phenotype mediating the enhanced in vivo tumor immunity were investigated. C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate vaccinia virus-reactive Th activity. The mice were subsequently immunized i.p. with virus-infected syngeneic X5563 and MH134 tumor cells, and spleen cells from these mice were tested for in vivo tumor neutralizing activity. Immunization of virus-primed mice with virus-uninfected tumor cells and of virus-unprimed mice with virus-infected tumor cells failed to result in in vivo protective immunity. In contrast, spleen cells from mice immunized with virus-infected tumor cells subsequent to virus-priming exhibited potent tumor-specific neutralizing activities. Such an augmented generation of in vivo protective immunity was accompanied by enhanced induction of tumor-specific cytotoxic T lymphocyte (CTL) and antibody activities in X5563 and MH134 tumor systems, respectively. However, analysis of the effector cell type responsible for in vivo tumor neutralization revealed that enhanced in vivo immunity was mediated by Lyt-1⁺2^– T cells in both tumor systems. Moreover, the Lyt-1⁺2^– T cells exerted their function in vivo under conditions in which anti-X5563 tumor-specific CTL or anti-MH134 tumor-specific antibody activity was not detected in recipient mice. These results indicate that augmenting the generation of a tumor-specific Lyt-1⁺2^– T cell population is essential for enhanced tumor-specific immunity in vivo.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

The mechanism of tumor growth inhibition by tumor-specific Lyt-1+2-T cells. I. Antitumor effect of Lyt-1+2-T cells depends on the existence of adherent cells

In the present study we establish an assay system of tumor growth inhibition with the use of a diffusion chamber and investigate the mechanism by which tumor-specific Lyt-1+2-T cells exhibit their inhibiting effect on tumor cell growth. When a diffusion chamber containing X5563 plasmacytoma cells together with normal syngeneic C3H/HeN spleen cells was implanted in the peritoneal cavity of C3H/HeN mice, these tumor cells continued to proliferate at least 7 to 9 days. In contrast, spleen cells from C3H/HeN mice that had acquired X5563-specific immunity by intradermal (i.d.) inoculation of viable tumor cells, followed by surgical resection of the tumor, exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed in the chamber. This antitumor effect was mediated by Lyt-1+2-T cells and was tumor-specific, because the growth of X5563 or another syngeneic MH134 hepatoma cells was inhibited by spleen cells from C3H/HeN mice immunized to the respective tumor cell types. Most important, these tumor-specific Lyt-1+2-T cells lost their antitumor activity by depleting an adherent cell population contained in spleen cells, indicating that adherent cells are required for the Lyt-1+2-T cell-mediated antitumor effect. This was substantiated by the fact that immune spleen cells depleted of adherent cells could regain their tumor-inhibiting effect when normal spleen cells were added back as an adherent cell source, or more directly by adding back a splenic or peritoneal resident adherent cell population. These results indicate that tumor-specific Lyt-1+2-T cells mediate the tumor growth inhibition and that their antitumor effect depends on the coexistence of an adherent cell population.     

Evidence for the functional binding in vivo of tumor rejection antigens to antigen-presenting cells in tumor-bearing hosts

Spleen cells of BALB/c mice bearing a syngeneic CSA1M fibrosarcoma were treated with anti-Thy-1.2 antibody plus C, yielding a T cell-depleted, APC-containing fraction. The APC-containing fraction was first tested for its capacity to present exogenous modified-self or another tumor (Meth A) Ag after in vitro pulsing. The results showed comparable Ag-presenting capacities to those obtained by APC-containing fraction from normal spleen cells, indicating that APC function is not affected in tumor-bearing mice. We next examined whether APC from CSA1M-bearing mice bind endogenously generated CSA1M tumor Ag onto its surfaces to stimulate tumor-specific T cells. Five rounds of inoculation of APC-containing fraction from CSA1M-bearing mice without further in vitro pulsing resulted in the induction of potent anti-CSA1M immune resistance. The involvement of anti-CSA1M T cells in the induction of anti-CSA1M immunity was excluded by the fact that the in vivo immunity was excluded by the fact that the in vivo immunity was delivered by Thy-1+ cell-depleted, but not by Thy-1+ cell-enriched fractions of spleen cells from CSA1M-bearing mice. Moreover, the failure of Sephadex G10-passed spleen cells to deliver anti-CSA1M resistance demonstrated the absolute requirement of APC for inducing the in vivo immunity. Finally, this in vivo resistance was found to be tumor specific, because APC fractions from CSA1M-bearing and Meth A-bearing BALB/c mice induced immune resistance selective against the corresponding tumor cell challenge. These results indicate that APC from tumor-bearing hosts can not only exert unaffected APC function against exogenous Ag, but also function to present tumor Ag generated endogenously in the tumor-bearing state and to produce tumor-specific immunity in vivo.     

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

The role of tumor-specific Lyt-1+2− T cells in eradicating tumor cells in vivo

Summary The present study investigates some of mechanisms for tumor-specific Lyt-1⁺2^– T cell-mediated tumor cell eradication in vivo through analyses of tumor specificity in the afferent tumor recognition and efferent rejection phases. When C3H/He mice which had acquired immunity against syngeneic MH134 hepatoma were challenged with other syngeneic X5563 plasmacytoma cells, these mice failed to exhibit any inhibitory effect on the growth of X5563 tumor cells. However, the inoculation of X5563 tumor cells into the MH134-immune C3H/He mice together with the MH134 tumor cells resulted in appreciable growth inhibition of antigenically distinct (bystander) X5563 tumor cells. Although the growth of X5563 cells was inhibited in an antigen-nonspecific way in mice immunized to antigenically unrelated tumor cells (bystander effect), the activation of Lyt-1⁺2^– T cells leading to this effect was strictly antigen-specific. Such a bystander growth inhibition also required the admixed inoculation of the bystander (X5563) and specific target (MH134) tumor cells into a single site in mice immunized against the relevant MH134 tumor cells. Furthermore, the results demonstrated that Lyt-1⁺2^– T cells specific to MH134 tumor cells were responsible for mediating the growth inhibition of antigenically irrelevant (bystander) and relevant tumor cells. These results are discussed in the context of cellular and molecular mechanisms involved in the Lyt-1⁺2^– T cell-initiated bystander phenomenon.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Changing patterns of lymphocyte proliferation, IL-2 production and utilization, and IL-2 receptor expression in mice infected with Schistosoma mansoni

In murine schistosomiasis mansoni the soluble egg Ag (SEA)-induced L3T4+ T cell-mediated circumoval granulomatous response peaks at the acute stage (8w) and undergoes Lyt-1+, Lyt-2+ suppressor cell mediated down-modulation at the chronic stage (20w) of the infection. In the present study lymphoproliferation, IL-2 production, utilization, and IL-2R display were examined in splenic lymphocytes of infected mice. The L3T4+ subset was the major SEA-specific proliferative population at both stages of the infection. Chronic infection spleen cells or the L3T4+ subset proliferated less compared with their acute infection counterparts. Elimination of the Lyt-2+ subset did not improve diminished proliferation. Chronic infection cells and the Lyt-2+ subset stimulated with SEA and exogenous rIL-2 regained their proliferative ability to a level comparable with acute infection cells. Ag-stimulated acute infection T cells produced 40 to 50 chronic infection cells less than 5 U/ml IL-2. Elimination of L3T4+ T cells diminished, and the Lyt-2+ T cells left unchanged IL-2 production in the acute infection cells. Elimination of Lyt-2+ subset from the chronic infection population did not enhance IL-2 production. Exogenously added rIL-2 was equally utilized by acute or chronic infection T cells. Scatchard plots of [125I]IL-2 binding showed similar numbers of high affinity receptors on acute (189) and chronic infection cells (118), but the number of low affinity receptors was higher on the acute (2229) vs the chronic infection lymphocytes (578). Analysis of IL-2R expression by two-color fluorescence and flow cytometry revealed that 30 to 40% of the acute, chronic infection L3T4+ cells displayed receptor. Receptor expression increased by added SEA, or SEA + rIL-2. R display among Lyt-2+ cells was only 12 to 18%. SEA stimulation enhanced receptor display more among the acute, SEA + rIL-2 stimuli raised receptor expression only among chronic infection lymphocytes. These data do not show inherent defect in IL-2R display, or utilization in chronic infection T cells. Diminished IL-2 production appears to be the cause of decreased T cell responsiveness.     

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.     

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.     

Production of colony-stimulating factor by tumor cells and the factor-mediated induction of suppressor cells

Spleen mononuclear cells of C3H/HeN mice were cultivated with mitomycin C-treated tumor cells, X5563, MH134, MM48, MM46, and FM3A/R, all of which were of syngeneic origin, in a medium containing normal syngeneic mouse serum but not FCS. There was a proliferative response to X5563, MH134, and MM48, but not to the two other tumor cells, MM46 and FM3A/R. The responder spleen cells were found to be nonadherent cells with a phenotype of Thy-1-L3T4-Lyt2-Ig-Macl-, which were neither mature T and B cells nor mature macrophage/granulocytes. It was also found that the proliferation of these nonadherent no-marker cells was mediated by tumor cell-derived soluble factors but not by direct stimulation with tumor cells. The responsible factor was a molecule(s) with a Mr of 23 to 25 kDa, which had a CSF activity inducing granulocyte (G)-, macrophage (M)- and G + M-colonies in the bone marrow cells. Neutralization tests of this factor-induced proliferation of spleen cells revealed that a major part of the factor may be GM-CSF or a molecule closely related to it. Incubation of spleen mononuclear cells with these GM-CSF-like tumor cell factors resulted in induction of myeloblastic/promyelocytic cells with a phenotype of Mac-1+2+Ia+ Thy-1-L3T4-Lyt2-Ig- in the spleen cell cultures, which could suppress mitogenic responses of the spleen cells to T and B cell mitogens. GM-CSF-like activity could also be detected in the serum of mice bearing X5563, MH134, and MM48, but not in those bearing MM46 and FM3A/R. Subcutaneous inoculation of C3H/HeN mice with these X5563, MH134, and MM48 tumor cells generated massive metastasis in the lung and lymph nodes, whereas MM46 and FM3A/R produced no macroscopic tumor cell metastasis. These results strongly suggest the possibility that in some tumor cell-host systems, a GM-CSF-like factor(s) produced constitutively by the tumor cells may play an important role in the development of tumor metastasis, mediating through suppression of lymphoid tissues of the host.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Cellular interactions and the role of interleukin 2 in the expression and induction of immunity against a syngeneic murine sarcoma

We have previously demonstrated that following the adoptive transfer of immune cells, the regression of established pulmonary metastases from a weakly immunogenic sarcoma, MCA 105, required the collaboration of two T cell subsets. In this study, we found that the critical role played by L3T4+ immune cells was to provide a helper function since tumor regression proceeded in the absence of L3T4+ immune cells if exogenous interleukin 2 (IL-2) was administered. To extend these observations, we analyzed the events leading to the induction and generation of L3T4+ and Lyt-2+ immune T cells after immunization of mice with viable tumor cells admixed with Corynebacterium parvum. The basic protocol involved immunization, surgical excision of the immunization site on day 7, and challenge with viable tumor cells on day 21. The ability of mice to reject tumor challenge provided a means to evaluate the occurrence of a systemic antitumor immunity. With the use of this experimental protocol, we have found that depletion of T cell subsets in vivo with either L3T4 or Lyt-2 monoclonal antibodies after active immunization abrogated the development of antitumor immunity. Mice immunized and depleted of L3T4+ but not Lyt-2+ T cells were able to reject tumor challenge if exogenous IL-2 was given for 7 days. However, the rejection of tumor challenge required 3 days of additional exogenous IL-2 administration. These results indicate that the induction of Lyt-2+ immune T cells depended on the helper function of L3T4+ T cells via the secretion of IL-2. In the absence of L3T4+ immune lymphocytes, the expression of antitumor immunity by Lyt-2+ immune cells could be facilitated by in vivo administration of exogenous IL-2. The induction of L3T4+ immune T cells, on the other hand, occurred independently of the Lyt-2+ T cell response because the transfer of spleen cells from Lyt-2+ cell-depleted, immunized animals was able to restore antitumor reactivity in L3T4+ cell-depleted, immunized mice. These results demonstrate the intricate cellular interactions leading to the induction as well as the expression of antitumor immunity.     

Property of class I H-2 alloantigen-reactive Lyt-2+ helper T cell subset. Abrogation of its proliferative and IL-2-producing capacities by intravenous injection of class I H-2-disparate allogeneic cells

The present study investigates the distinctiveness of Class I H-2 alloantigen-reactive Lyt-2+ helper/proliferative T cell subset in the aspect of tolerance induction. Primary mixed lymphocyte reactions (MLR) revealed that Lyt-2+ and L3T4+ T cell subsets from C57BL/6 (B6) mice were exclusively capable of responding to class I H-2 [B6-C-H-2bm1 (bm1)]- and class II H-2 [B6-C-H-2bm12 (bm12)]-alloantigens, respectively. Anti-bm12 MLR was not affected by i.v. injection of bm12 spleen cells into recipient B6 mice. In contrast, a single i.v. administration of bm1 spleen cells into B6 mice resulted in the abrogation of the capacity of recipient B6 spleen and lymph node cells to give anti-bm1 MLR. This suppression was bm1 alloantigen-specific, since lymphoid cells from B6 mice i.v. presensitized with bm1 cells exhibited comparable anti-bm12 primary MLR to that obtained by normal B6 lymphoid cells. Such tolerance was rapidly (24 h after the i.v. injection of bm1 cells) inducible and lasting for at shortest 3 wk. Addition of lymphoid cells from anti-bm1-tolerant B6 mice to cultures of normal B6 lymphoid cells did not suppress the proliferative responses of the latter cells, indicating that the tolerance is not due to the induction of suppressor cells but attributed to the elimination or functional impairment of anti-bm1 proliferative clones. The tolerance was also demonstrated by the failure of tolerant lymphoid cells to produce IL-2. It was, however, found that anti-bm1 CTL responses were generated by tolerant lymphoid cells which were unable to induce the anti-bm1 MLR nor to produce detectable level of IL-2. These results demonstrate that class I H-2 alloantigen-reactive Lyt-2+ Th cell subset exhibits a distinct property which is expressed by neither Lyt-2+ CTL directed to class I H-2 nor L3T4+ Th cells to class II H-2 alloantigens.     

Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells

Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Characterization of tumor-infiltrating CD4+ T cells as Th1 cells based on lymphokine secretion and functional properties

Recent studies have subdivided the Th cells into mutually exclusive Th1 subset producing IL-2 and IFN-gamma and Th2 cells producing IL-4 and IL-5. The relative role played by these two subsets in the antitumor immunity is not clear. We earlier demonstrated that treatment of C57BL/6 mice bearing a syngeneic Ia- T cell lymphoma, LSA, with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) resulted in 90 to 100% survival of the mice. Furthermore, host's T cell responses were critical for successful BCNU-mediated cures. In our study we observed that immediately after BCNU treatment, there was a dramatic increase in the percentage of CD4+ T cells at the site of tumor growth in the peritoneal cavity. The percentage of CD4+ T cells increased from approximately 3 to 4% found in normal or tumor-bearing mice to approximately 41% in BCNU-treated tumor-bearing mice. The percentage of CD8+ T cells also increased although to a lesser degree. Also, these alterations were primarily restricted to the site of tumor-growth inasmuch as they were not seen in the thymus and were less pronounced in the spleen. The tumor-infiltrating CD4+ T cells obtained after BCNU-treatment, when further characterized, were found to secrete only IL-2 and IFN-gamma but not IL-4, after tumor-specific stimulation. Furthermore, the supernatants from LSA-activated CD4+ T cell cultures failed to provide help to the B cells but were able to activate the macrophages to inhibit the tumor cell proliferation. The CD4+ T cells when adoptively transferred could also protect the nude mice from LSA tumor challenge and induced tumor-specific delayed-type hypersensitivity reaction. Together our data suggest that in the LSA tumor model, the tumor-infiltrating CD4+ T cells have the properties of Th1 cells and these cells can mediate tumor-rejection independent of the CD8+ T cells by activating the macrophages.     

Altered interleukin production during Friend leukemia virus infection

Spleen cells from BALB/c mice, infected 14 to 28 days earlier with Friend leukemia virus (FLV), were shown to be inhibited in their ability to produce interleukin 2 (IL-2) when stimulated with mitogen. Likewise, these spleen cell populations failed to respond following mitogenic stimulation or exogenous addition of recombinant IL-2. By contrast, the FLV-infected spleen cell populations produced normal levels of interleukin 1 (IL-1) and thymocytes from FLV-infected mice responded normally to addition of exogenous IL-1. This suggests that FLV infection selectively affects the ability of spleen cells to produce cytokines. Spleen cell populations enriched for T lymphocytes and depleted of tumor cells by density gradient centrifugation in Ficoll were unable to produce IL-2. This indicates that the failure to detect IL-2 in cells from FLV-infected mice was not due to a dilution of T lymphocytes by tumor cells but was a functional inability to produce IL-2. Furthermore, enriched T lymphocytes from FLV-infected mice failed to respond blastogenically to exogenous IL-2. Additional studies indicate that tumor cells, but not macrophages or T lymphocytes from FLV-infected spleens, suppressed the blastogenic response to mitogens and IL-2 production by normal splenic T lymphocytes.