Regulation of Ia+ reticulum cell sarcoma (RCS) growth in syngeneic SJL/J mice. I. Inhibition of tumor growth by passive administration of L3T4 monoclonal antibody before or after tumor inoculation

Spontaneously arising reticulum cell sarcoma (RCS) tumors in SJL/J mice stimulate syngeneic host T lymphocytes to proliferate and are dependent on host T cells for maintenance and growth. Tumor-associated Ia antigens have been implicated in the proliferative response both in vivo and in vitro, and the responding T cells are predominantly Lyt-1+2- L3T4+. We hypothesized that elimination or depletion of the responding L3T4 subpopulation in vivo should inhibit growth of transplantable RCS tumors, and continued RCS growth may be dependent on the continued presence of L3T4 cells. This hypothesis was tested experimentally by examining the effect of passive administration of L3T4 monoclonal antibody (mAb) into SJL/J mice either before or at different times after tumor inoculation. The tumor inoculum used killed all mice 15 to 30 days after injection. Administration of a single dose of L3T4 mAb 4 days before tumor inoculation resulted in complete depletion of L3T4 cells and complete inhibition of tumor growth. The antibody-treated mice survived with no sign of tumor growth even after complete recovery of L3T4+ cells. These results demonstrate that initiation of tumor growth is dependent on host L3T4+ cells. Administration of mAb as late as 7 days after tumor inoculation resulted in inhibition of tumor growth, and administration of mAb at day 10 resulted in significant inhibition of tumor growth. Compared with the kinetics of tumor growth in normal control mice, administration of L3T4 after tumor inoculation results in tumor growth arrest. These findings demonstrate that continued tumor growth in vivo is dependent on the presence of L3T4+ cells. In the RCS system, the present studies show that administration of mAb to L3T4+ cells is therapeutic in that it inhibits the induction of tumor growth, and it also prevents tumor growth in tumor-bearing animals.     

The in vivo depletion of V beta 17a+ T cells results in the inhibition of reticulum cell sarcoma growth in SJL/J mice. Evidence for the use of anticlonotypic antibody therapy in the control of malignancy

Previous findings revealed that reticulum cell sarcoma (RCS) of SJL/J mice growth and survival depended on its ability to stimulate a potent host T cell response, by the means of a tumor-associated class II MHC molecule with IE-like specificities. Previously we presented evidence that the V beta 17a TCR+ clonotype of T cell was the predominant T cell involved in the host response to the tumor. We undertook our study to examine whether the depletion of the V beta 17a+ T cells, by the use of the anticlonotypic antibody, KJ23a, resulted in the inhibition of RCS tumor growth in vivo. We present evidence herein that supports this hypothesis. KJ23a-treated mice exhibited a complete reduction in T cells bearing the V beta 17a TCR. These mice exhibited a dramatic reduction in the in vitro proliferative response to RCS. Furthermore, the pretreatment of SJL/J mice with KJ23a mAb resulted in the complete loss in their ability to harbor RCS tumor. When tumor-bearing mice were treated with a single inoculum of KJ23a mAb within the first 7 days after the passage of tumor, the mice showed long term survival with diminishing tumor burden. These results demonstrated that the V beta 17a clonotype of T cells is required for the growth and maintenance of RCS tumor. Within the first 6 wk after tumor inoculation KJ23a-treated mice were capable of transferring tumor to naive syngeneic recipient mice despite the obvious lack of tumor growth in the treated donor animal. These results suggested that RCS tumors in the absence of V beta 17a+ T cells can persist for up to 6 wk in a state of "tumor dormancy." The predominant usage of the V beta 17a gene in RCS-specific T cells suggests that these T cells play an important role in the pathogenesis of RCS tumor. Furthermore, the positive therapeutic course taken by tumor-bearing mice upon the treatment with KJ23a mAb, demonstrates the enormous potential in anticlonotypic antibody therapy in the treatment of T cell-dependent tumors and diseases.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."     

Tim-3+ T-bet+ tumor-specific Th1 cells colocalize with and inhibit development and growth of murine neoplasms

Although T cells infiltrate many types of murine and human neoplasms, in many instances tumor-specific cytotoxicity is not observed. Strategies to stimulate CTL-mediated antitumor immunity have included in vitro stimulation and/or genetic engineering of T cells, followed by adoptive transfer into tumor-bearing hosts. In this model of B cell lymphoma in SJL/J mice, we used Tim-3(+) T-bet(+) Th1 cells to facilitate the development of tumor-specific CTL. Tumor-specific Th1 cell lines were polarized with IL-12 during in vitro stimulation and long term maintenance. As few as 5 million Tim-3(+) T-bet(+) Th1 cells enabled recipients to resist growth of malignant transplantable cells. In addition, similar numbers of Th1 cells injected into 2- to 3-mo-old mice inhibited development of the spontaneous primary lymphomas, which normally arise in 90% of aging mice. CFSE(+) Th1 cells colocalized with injected tumor cells in vivo and formed conjugates with the tumor cells within follicles, whereas in nontumor-challenged recipients the CFSE(+) Th1 cells localized only within the T cell zones of the spleen. These results provide evidence that adoptive immunotherapy with Tim-3(+) T-bet(+) tumor-specific Th1 cells can be used to induce host cytotoxic responses that inhibit the development and growth of neoplastic cells.     

Treatment of murine lupus with F(ab')2 fragments of monoclonal antibody to L3T4. Suppression of autoimmunity does not depend on T helper cell depletion

Treatment with mAb to the L3T4 Ag on Th cells can inhibit autoimmunity in mice. However, the mechanism by which anti-L3T4 inhibits autoimmunity is not known. In these studies, lupus-prone NZB/NZW F1 (B/W) mice were treated with F(ab')2 fragments of mAb to L3T4 to determine whether Th cell depletion is required for the beneficial effects of anti-L3T4. We first showed that treatment of female B/W mice with F(ab')2 anti-L3T4 from age 5 to 9 mo significantly reduced autoantibody production without depleting L3T4+ cells. However, treatment was complicated by the development of a host immune response to the rat mAb fragments. To circumvent this problem, female B/W mice were treated with a single high-dose of intact rat mAb to L3T4 (GK1.5) at age two mo. to induce immune tolerance to the mAb. Then, after recovery of L3T4+ cells, the mice were treated from age four to 14 mo with either F(ab')2 anti-L3T4 (0.5 mg 3 times per wk), intact anti-L3T4, or saline. In mice tolerized by this regimen, neither the F(ab')2 rat mAb nor the intact rat mAb elicited a host response. The mAb fragments bound target Ag but did not deplete the Th cells, whereas intact mAb to L3T4 profoundly depleted the L3T4+ cells. Despite this difference, both therapies had the same substantial beneficial effects on autoimmunity. They significantly decreased anti-DNA Ab production, improved renal function and prolonged survival. The initial tolerizing dose, by itself, did not inhibit autoimmunity. These findings show that anti-L3T4 suppresses autoimmunity by directly altering Th cell function through the L3T4 Ag, and not solely by depleting Th cells. They also document the detrimental effects of the host immune response to therapy with anti-L3T4 mAb, and they demonstrate a new strategy by which this response may be prevented.     

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.     

Effector phenotypes and mechanisms of antitumor immune reactivity of tumor-immunized and tumor-bearing mice in two syngeneic tumors.

By using two different syngeneic tumors, Meth A sarcoma and RL male 1 lymphoma of BALB/c origin, the present study was designed to investigate the subset(s) of T cells mediating in vivo antitumor immune responses and some of the effector mechanisms of in vivo protective immunity in BALB/c mice immunized against tumor or bearing tumor. Spleen cells from the mice immunized against Meth A tumor or bearing Meth A tumor inhibited the growth of Meth A tumor in the Winn assay. In the Meth A-immunized mice, L3T4+ (CD4+) cells played a major role in mediating the inhibitory activity against Meth A tumor growth, whereas in the Meth A-bearing mice, the antitumor protective immunity was mediated by both L3T4+ and Lyt-2+ (CD8+) cells. Spleen cells from the Meth A-immunized or Meth A-bearing mice were not able to generate cytotoxic T lymphocytes (CTL) directed against Meth A tumor after the in vitro restimulation of spleen cells with mitomycin C (MMC)-treated Meth A cells, while fresh spleen cells from the Meth A-immunized or Meth A-bearing mice were able to induce the strong delayed-type hypersensitivity (DTH) responses to Meth A tumor. The DTH response to Meth A tumor was mediated by L3T4+ cells in the Meth A-immunized mice and by both L3T4+ and Lyt-2+ cells in the Meth A-bearing mice. In the similar experiments performed in the RL male 1 lymphoma, the antitumor activity in spleen cells from the RL male 1-immunized or RL male 1-bearing mice depended on Lyt-2+ but not L3T4+ cells in the Winn assay. When spleen cells from the RL male 1-immunized or RL male 1-bearing mice were cultured with MMC-treated RL male 1 cells for 5 days, an appreciable CTL response to RL male 1 tumor was induced. These results suggest that the nature of tumor and/or tumor antigens determines which T cell subset is required to exhibit the protective immunity against tumor and thus the different effector mechanisms could be induced in the different tumor models. Furthermore, these data support the conclusion that antitumor T cell responses are affected by the immune state of host to tumor.     

Self-reactive T cell receptor-reactive CD8+ T cells inhibit T cell lymphoma growth in vivo

Syngenic C57BL/6 mice (H-2(b)) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8(+) T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-gamma and TNF-alpha, but little or no IL-4. By means of TCRbeta-negative variant L12R4 cells, P3.3, and TCR-Vbeta2 cDNA-transfected and TCR-Vbeta2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vbeta12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-K(b), and anti-D(b) mAb. Furthermore, vaccination with Vbeta12 peptide p67-78, which binds to both K(b) and D(b) MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vbeta-specific, K(b)-, or D(b)-restricted CD8(+) T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

CD40 ligand in pathogenesis of autoimmune ovarian disease of day 3-thymectomized mice: implication for CD40 ligand antibody therapy

The blockade of CD40 ligand (CD40L) is effective in autoimmune disease prevention. Recently, a brief period of CD40L mAb treatment was reported to induce tolerance and enhancement of CD4(+)CD25(+) regulatory T cell activity. We therefore determined the efficacy of CD40L mAb treatment in autoimmunity that resulted from CD4(+)CD25(+) regulatory T cell deficiency. Autoimmune ovarian disease (AOD) and oocyte autoantibody response of day 3-thymectomized (d3tx) mice were inhibited by continuous CD40L mAb treatment from day 3, or from days 10-14, whereas CD40L mAb treatment confined to the neonatal week was ineffective. The enhanced expression of memory markers (CD44 and CD62L(low)) on CD4(+) T cells of the d3tx mice was unaffected by CD40L mAb treatment. In contrast, their increased T cell activation markers (CD69 and CD25) were eliminated by CD40L mAb treatment. Moreover, ex vivo activated T cells of d3tx mice expressed elevated intracellular IFN-gamma, and this was also blocked by CD40L mAb. The memory T cells, although nonpathogenic in CD40L mAb-positive environment, transferred severe AOD to CD40L mAb(-) neonatal recipients. Most importantly, CD40L mAb treatment inhibited AOD in recipients of T cells from d3tx donors with severe AOD and led to regression of AOD in d3tx mice documented at 4 wk. Therefore, 1) the continuous presence of CD40L mAb both prevents and causes regression of AOD in the d3tx mice; and 2) the multiple steps of the d3tx autoimmune disease, including T cell activation, cytokine production, T cell-mediated inflammation, and tissue injury, are CD40L dependent.     

L3T4+ T cells regulate Abelson virus-induced lymphomagenesis

To evaluate the role of T cells in regulation of lymphomagenesis, experiments were performed using Abelson murine leukemia virus (AMuLV). In vitro transformation of bone marrow target cells by this B lymphotropic retrovirus was inhibited by peripheral lymph node cells from naive mice. The inhibitory activity depended on Thy-1+ L3T4+ cells but did not require Lyt-2+ cells. In vivo depletion of L3T4+ T cells with a mAb (GK1.5) altered the course of AMuLV-induced lymphoma. L3T4 depletion of naturally resistant C57BL/6 mice resulted in dramatic susceptibility to lymphoma induction. Lymphoma cells from anti-L3T4-treated C57BL/6 mice infected with AMuLV displayed the B lineage transformation marker P1606C3. These studies reveal an important immunologic component of Abelson disease resistance involving L3T4+ T cells.     

Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler's virus

Intracerebral inoculation of mice with Theiler's murine encephalomyelitis virus results in an intense inflammatory response of mononuclear leukocytes which infiltrate into the central nervous system. Resistant strains of mice have the ability to clear virus whereas susceptible strains become infected persistently and are associated with chronic demyelination which is proposed to be immune-mediated. In an attempt to better understand the role of the immune response during demyelination, mononuclear leukocytes were isolated from the central nervous system of infected mice and stained by an immunoperoxidase technique with anti-Thy-1.2, anti-L3T4, anti-Lyt-2 and anti-MAC-1 mAb. Infection of susceptible SJL/J mice resulted in a biphasic immune response which peaked on days 7 and 27 post-infection. In contrast, a single peak (day 7) was observed in resistant C57BL/10SNJ mice. The presence of Thy-1.2, L3T4, and MAC-1+ cells was similar between the two strains. However, although the number of Lyt-2+ cells peaked on day 7 in C57BL/10SNJ mice, they were not detected in SJL/J mice until 14 days post-infection and gradually increased in number over the course of infection. To further study the role of T cells in demyelination, serial frozen sections of brain and spinal cord were stained for the presence of Lyt-2 and L3T4+ cells in the lesions of chronically infected SJL/J mice. L3T4+ cells were observed predominantly in perivascular regions while Lyt-2+ cells were observed infiltrating the parenchyma. These results provide further evidence that Lyt-2+ lymphocytes are important in the mechanism of susceptibility/resistance to Theiler's murine encephalomyelitis virus-induced demyelination.     

Prevention and reversal of experimental autoimmune thyroiditis (EAT) in mice by administration of anti-L3T4 monoclonal antibody at different stages of disease development

Experimental autoimmune thyroiditis (EAT) can be induced in CBA/J mice following the transfer of spleen cells from mouse thyroglobulin (MTg)-sensitized donors that have been activated in vitro with MTg. Since L3T4+ T cells are required to transfer EAT in this model, the present study was undertaken to assess the effectiveness of the anti-L3T4 monoclonal antibody (mAb) GK1.5 in preventing or arresting the development of EAT. Spleen cells from mice given mAb GK1.5 prior to sensitization with MTg and adjuvant could not transfer EAT to normal recipients and cells from these mice did not proliferate in vitro to MTg. Donor mice given GK1.5 before immunization did not develop anti-MTg autoantibody and recipients of cells from such mice also produced little anti-MTg. GK1.5 could also prevent the proliferation and activation of sensitized effector cell precursors when added to in vitro cultures. When a single injection of mAb GK1.5 was given to recipients of in vitro-activated spleen cells, EAT was reduced whether the mAb was given prior to cell transfer or as late as 19 days after cell transfer. Whereas the incidence and severity of EAT was consistently reduced by injecting recipient mice with GK1.5, the same mice generally had no reduction in anti-MTg autoantibody. Since EAT is consistently induced in control recipients by 14-19 days after cell transfer, the ability of mAb GK1.5 to inhibit EAT when injected 14 or 19 days after cell transfer indicates that a single injection of the mAb GK1.5 can cause reversal of the histopathologic lesions of EAT in mice. These studies further establish the important role of L3T4+ T cells in the pathogenesis of EAT in mice and also suggest that therapy with an appropriate mAb may be an effective treatment for certain autoimmune diseases even when the therapy is initiated late in the course of the disease.     

Proliferative responses of T cells from SJL----F1 and F1----SJL bone marrow chimeras to SJL lymphoma cells

RCS tumor cells induce marked proliferation of syngeneic SJL T cells in vivo and in vitro. Certain F1 hybrids of SJL mice give high proliferative responses to gamma-RCS, while other F1 hybrids give low responses. SJL----"non-responder" F1 and "non-responder" F1----SJL semiallogeneic bone marrow chimeras were prepared to study how the host environment affects the ability of T cells to give a proliferative response to gamma-RCS. The results indicate that T cells educated in an SJL host become responsive to RCS cells, while T cells educated in an (SJL X BALB/c)F1 host become unresponsive. This finding applies to both thymus and lymph node T cells. The unresponsiveness in F1 mice is not due to suppressor cells, since added F1 cells do not affect the proliferative response of SJL cells to gamma-RCS. Instead, it appears that RCS-specific T cells are either deleted in (SJL X BALB/c)F1 mice, or expanded in SJL mice as they develop. These findings are discussed in relation to the specificity of the responding T cells, for LPS activated syngeneic B cell blasts as well as RCS cells, and to the presence of a "leaky" thymus barrier in SJL mice for B cells.     

Termination of the B cell lymphoma dormant state in thymectomized AKR mice.

AKR mice are highly susceptible to spontaneous T cell lymphomagenesis and thymus removal at the age of 1 to 3 mo greatly reduces its development. Twelve-mo-old AKR mice thymectomized at young age were shown previously to carry potential lymphoma cells that could be triggered to develop into B cell lymphomas (80 to 100%) after removal from their host "restrictive" environment into young histocompatible hosts. Additional attempts were made to terminate the potential lymphoma cell dormant state in 12-mo-old thymectomized AKR mice. Replenishment of some deficiencies caused by thymectomy at a young age, including a s.c. syngeneic thymus graft or a single injection of the dual tropic recombinant virus isolates DTV-71 or MCF-247 into 12-mo-old thymectomized AKR mice resulted in Ly-1+ pre-B or B cell lymphoma development in 80 to 98% of these treated mice. In vivo elimination of T cell subsets by administration of cyclosporin A or by mAb expressed on Th cells (anti-CD4) or cytotoxic T cells (anti-CD8) stimulated the progression of dormant potential lymphoma cells towards B cell lymphoma development. The most striking results were observed after administration of anti-CD8 mAb: 90 to 100% of these treated mice developed Ly-1+ B cell lymphomas within 80 days. The effect of rIL-2 on dormant PLC was also tested. Administration of rIL-2 to 12-mo-old thymectomized mice terminated tumor dormancy in 94% of the treated mice within 66 days. Tests of the resulting B lymphomas for dual tropic recombinant virus/mink cell focus-inducing virus infection indicated that the breakdown of tumor dormancy did not result from development of pathogenic class I mink cell focus-inducing viruses. These results suggest that T cell subsets and/or their products are involved in the proliferation arrest of potential lymphoma cells present in thymectomized AKR mice.     

Expression of L3T4 molecules on Lyt-2+, class II-restricted antigen-specific T suppressor lymphocytes

Three bovine serum albumin-specific Lyt-2⁺ T suppressor (Ts) cell clones from CBA/J mice have been analyzed with regard to expression of L3T4 molecules. All three Ts-cell clones can be stained with monoclonal antibodies (mAb) to L3T4. Tested for the two clones restricted to recognition of E^k determinants, antigen-specific proliferation on antigen-presenting cells, but not the proliferation induced by conditioned medium can be inhibited by L314-specific mAb. In a similar way, Ts-cell cytolytic effector functions can be blocked by L3T4-specific mAb. Thus L3T4 structures seem to play a role in Ts-cell functions. Furthermore, the data support the view that L3T4 expression can be a property of class II-restricted T cells irrespective of their Lyt phenotype.     

Characterization and growth factor requirements of SJL lymphomas. I. Development of a B cell growth factor-dependent in vitro cell line, cRCS-X

Reticulum cell sarcomas (RCS) of SJL mice are completely dependent on host cells for their growth and therefore fail to grow in vitro. RCS cells induce marked proliferation in SJL Ly-1+2- T cells accompanied by lymphokine production. In an attempt to fully understand the host-tumor cell interaction, an RCS cell line, cRCS-X, was established in vitro from a transplantable tumor by the addition, every 3 wk, of gamma-irradiated syngeneic lymph node (LN) cells to the culture. cRCS-X maintains all of the characteristics of the parent tumor, RCS-X, including cell surface phenotype (Ks and I-As positive, Ds negative and B cell marker 14.8 positive), ability to stimulate host T cells, and ability to grow in nonirradiated but not in gamma-irradiated SJL mice. The growth factor requirements of cRCS-X were examined. It was found that human BCGF can replace gamma-irradiated LN cells in the maintenance of long term in vitro growth of cRCS-X. cRCS-X cells respond to human B cell growth factor (BCGF) or to recombinant murine interleukin (IL)-5 in a short term proliferation assay [( 3H]thymidine incorporation) in a dose-dependent manner in the presence and absence of fetal calf serum. BCGF also promotes colony formation in soft agar by cRCS-X cells. Although both IL-1 and interferon-gamma can synergize with BCGF in the induction of cRCS-X proliferation, these lymphokines, as well as IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, and IL-4 have no effect on cRCS-X growth when added alone. In addition, it was shown that SJL LN cells produce both IL-4 and BCGF II activities as assayed on murine B cells, after stimulation with gamma-irradiated cRCS-X cells. In light of these results it is postulated that IL-5, [corrected] produced by syngeneic T cells [corrected] after stimulation with RCS, is essential for RCS growth, both in vitro and in vivo.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.