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."     

Bone marrow transplantation across major histocompatability barriers in mice. III. Treatment of donor grafts with monoclonal antibodies directed against Lyt determinants

We studied the effect of eliminating T cells from donor grafts of mice in a system in which bone marrow was transplanted across major histocompatibility barriers. BALB/c bone marrow (added as a source of hematopoietic stem cells) combined with equal volumes of spleen cells (added as a source of GVHD-promoting cells) was pretreated in vitro with monoclonal anti-Lyt-1.2 or Lyt-2.2 plus absorbed rabbit complement before injection into C57BL/6 total-body-irradiated recipients. Functional activity of anti-Lyt monoclonal antibodies was determined in CML assay. Treatment with anti Lyt-1.2 plus C did not have any anti-stem cell activity, as measured by CFU-S assay, and protected recipients from the onset of lethal GVHD. Treatment with Lyt-2.2 plus C also did not reduce CFU-S; however, mice receiving treated marrow did develop GVHD and were all dead by 2 mo, as were untreated control mice. Surviving "anti-Lyt-1.2 + C chimeras" demonstrated a high percentage of donor mononuclear cells in their peripheral blood. Similar results were obtained when C3H/HeN donor BMS was treated with monoclonal anti-Lyt-1.1 plus C and injected into C57BL/6 recipients. These findings show that monoclonal antibodies directed against determinants unrelated to Thy-1 can eliminate T cells in the presence of C and successfully protect transplanted mice from lethal GVHD. They also suggest that these anti-Lyt antibodies may be useful tools in determining subpopulations of T cells that contribute to the development of GVHD.     

Study on the possible factors influencing the expression of H-2 restriction specificity and Ir phenotype of antigen-specific proliferative T cells with various types of radiation chimeras

To better understand the factors described previously as influencing the manifestation of H-2 restriction specificity and Ir phenotype of T cells from radiation bone marrow chimeras, we also examined H-2 restriction specificity (Ir phenotype) of antigen (DNP-OVA, (T, G)-A-L, (H, G)-A-L)-specific proliferative T cells generated in various types of H-2 incompatible radiation chimeras prepared under our specific-pathogen-free (SPF) condition. The results indicated the following: (a) T cells generated in F1----parent bone marrow chimeras preferentially manifested host-type H-2 restriction specificity and Ir phenotype, regardless of the radiation dose (8.70 vs 11.59 Gy); (b) T cells recovered from twice-reconstituted F1----(PA----PB) chimeras manifested primary host (PB)-type Ir phenotype; (c) T cells which were recovered from (B10.Thy-1.1 X B10.BR.Thy-1.1)F1----parent (Thy-1.2) bone marrow chimeras and treated with anti-Thy-1.2 plus complement to deplete host-derived T cells still manifested preferentially the restriction specificity for host-type H-2; (d) PA-derived T cells which had differentiated in a fully allogeneic host (PB) environment of (PA + PB)----PB chimeras manifested fully allogeneic host-type Ir phenotype; (e) T cells from F1----parent chimeras that were prepared with 13-day fetal liver cells also manifested host H-2-restricted Ir phenotype; and (f) host preference for Ir phenotype of antigen-specific proliferative T cells was observed even in the case of F1----parent bone marrow chimeras reconstituted with "intact" bone marrow cells. The data suggest that thymic APCs, surviving host T cells or the source of stem cells (adult bone marrow vs 13-day fetal liver), do not necessarily play a significant role in the manifestation of H-2 restriction specificity and Ir phenotype of T cells generated in H-2 incompatible radiation chimeras.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver (1-3). We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity. However, when combined with the systemic administration of RIL 2, these T cell-depleted, non-lytic LAK cells remained as effective in reducing the number of established pulmonary metastases upon adoptive transfer as untreated or complement-treated lytic LAK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

The expression of theta-like antigen by rat peripheral lymphocytes: serologic and functional studies.

AKR/Cum mice (Thy-1b = thetaC3H) immunized with nucleated cells from WF rat thymus, Peyer's patches, peritoneal exudate, mesenteric lymph nodes, blood, bone marrow, or spleen produced antibodies cytotoxic for ADR/J (Thy-1a = thetaAKR) but not for AKR/Cum thymocytes. The specificity of these antibodies for the Thy-1.1 (theta-AKR) antigen was confirmed by tests using thymocytes from backcross mice segregating at the Thy-1 locus. This result suggested that the rat lymphocyte antgen cross-reactive with Thy-1.1 was expressed by at least some members of each of the rat lymphoid cell populations tested. AKR/Cum mice immunized with killed rat cells also produced anti-Thy-1.1 antibodies; thus indicating that further differentiation of the injected cells was not a prerequisite for the anti-Thy-1.1 response. Unexpectedly, about 9% of unimmunized adult AKR/Cum males were found to be producing antibodies against Thy-1.1. To our knowledge, natural antibodies of this specificity have not been previously reported. Finally, it was found that peritoneal exudate cells taken from WF rats previously immunized with EL-4 mouse leukemia cells were neither killed nor functionally inactivated by treatment with anti-Thy-1.1 antibodies and complement.     

Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors.

Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors.     

Thy-1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors

To determine the role of Thy-1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti-Thy-1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (CFU-GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen-stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony-forming unit (CFU-E) and the macrophage progenitor stimulated by L-cell-conditioned media (LCM), were spared by anti-Thy-1.2 antibody and complement treatment. Here, to separate the effects of anti-Thy-1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU-E and CFU-GM was not restored. To more precisely replace required accessory cells, male complement-treated marrow was cocultured with female anti-Thy-1.2 antibody and complement-treated marrow. Even marrow cells failed to restore female BFU-E and CFU-GM growth. Fluorescent-activated cell sorting (FACS) and immune sheep red cell rosetting with anti-Thy-1.2-labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy-1.2. These techniques revealed enrichment of BFU-E and CFU-GM in the Thy-1.2-positive fraction, demonstrating the presence of Thy-1.2 on early murine hematopoietic progenitors. CFU-E and CFU-M were present in the Thy-1.2-negative fraction following FACS separation. These data demonstrate that Thy-1.2 is a differentiation antigen, present on at least some murine BFU-E and CFU-GM and lost as they mature to CFU-E and CFU-M.     

Effector cells working for rejection of Ad12 tumors in mice

Immune spleen cells (ISC) capable of inhibiting the growth of adenovirus type 12 (Ad12) tumors were raised in C57BL/6 mice by immunization with Ad12, fractionated according to their affinity for plastic and nylon-wool substrates or treated with various antisera plus complement, and subjected to the tumor-neutralization test (Winn) to define the effector cells for the cell species. Full antitumor activity of ISC was recovered in the cell fractions nonadherent to the two substrates; the antitumor activity of ISC was abrogated entirely by anti-Thy-1,2 serum and almost entirely by anti-Lyt-2.2 ascites fluid plus complement. These results clearly indicate that T-lymphocytes, particularly those bearing Lyt-2.2 antigen, are the principal effectors in ISC against Ad12 tumors in animals.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Dysfunction of irradiated thymus for the development of helper T cells

The development of cytotoxic T cells and helper T cells in an intact or irradiated thymus was investigated. C57BL/6 (H-2b, Thy-1.2) mice were whole body-irradiated, or were irradiated with shielding over either the thymus or right leg and tail, and were transferred with 1.5 X 10(7) bone marrow cells from B10.Thy-1.1 mice (H-2b, Thy-1.1). At various days after reconstitution, thymus cells from the recipient mice were harvested and a peanut agglutinin low-binding population was isolated. This population was further treated with anti-Thy-1.2 plus complement to remove host-derived cells and was assayed for the frequency of cytotoxic T cell precursors (CTLp) and for the activity of helper T cells (Th). In the thymus of thymus-shielded and irradiated mice, Th activity reached normal control level by day 25, whereas CTLp frequency remained at a very low level during these days. In the thymus of whole body-irradiated mice, generation of CTLp was highly accelerated while that of Th was retarded, the period required for reconstitution being 25 days and more than 42 days for CTLp and Th, respectively. Preferential development of CTLp was also seen in right leg- and tail-shielded (L-T-shielded) and irradiated recipients. Histological observation indicated that Ia+ nonlymphoid cells were well preserved in the thymus of thymus-shielded and irradiated recipients, whereas in L-T-shielded and irradiated recipients, such cells in the medulla were markedly reduced in number. These results suggest strongly that the generation of Th but not CTLp is dependent on radiosensitive thymic component(s), and that such components may represent Ia+ cells themselves in the medulla or some microenvironment related to Ia+ cells.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Endotoxin-induced interferon-gamma production in culture cells derived from BCG-infected C3H/HeJ mice

The cultured cells prepared from the spleens and peritoneal exudate cells of the C3H/HeJ strain of mice produce very little or no interferon (IFN) by stimulation of bacterial lipopolysaccharide (LPS). However, the cells taken from LPS-non-responder C3H/HeJ mice which had been infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) prior to the experiment were capable of producing IFN in culture in the presence of LPS. The peritoneal exudate cells of BCG-primed C3H/HeJ mice were separated into adherent cell and nonadherent cell populations by their adhesiveness to plastic culture dishes. IFN production required the presence of both these cell populations in the same culture, and the IFN activities produced were mainly IFN-gamma. The cultures with nonadherent cells and fixed adherent cells still produced IFN, but the cell cultures reconstituted with the BCG-primed cell population and unprimed cell population produce little if any IFN-gamma. Moreover, when both of the populations were cultured in Marbrook culture vessels separated by a membrane filter, the cultures produced very little or no IFN-gamma. These results indicate that there is a mechanism of IFN-gamma induction by LPS which requires the direct contact between adherent cells and nonadherent cells without the participation of any soluble factor(s) from the adherent cells. The producer cells were mainly in the nonadherent cell population. Previous treatment of nonadherent cells with anti-Thy-1.2 antibody, anti-Lyt-1.1 antibody, anti-L3T4 antibody, or anti-asialo-GM1 antibody and complement diminished the ability of the cells for LPS-induced IFN production with the help of adherent cells. Therefore, it is concluded that both T cells (presumably L3T4+T cells) and asialo-GM1+ natural killer cells in the BCG-primed C3H/HeJ cell cultures produced IFN-gamma in the presence of LPS, and the production was supported by the function of macrophages.     

Influence of antibody isotype on passive serotherapy of lymphoma

We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.     

Modulation of granulomatous hypersensitivity. VI. T lymphocyte subsets influence mast cell density in liver granulomas of Schistosoma mansoni-infected mice

In murine schistosomiasis mansoni, a complex series of cell-cell interactions involving T cell subclasses regulates the intensity of the inflammatory granulomatous response. Recent evidence suggests that granuloma mast cells also participate in the regulatory process by the release of histamine. The current study was performed to determine factors that affect the number of granuloma mast cells. More mast cells were detected in liver granulomas from chronically (20-wk) as opposed to acutely (8-wk) infected mice. Adoptive transfer of spleen cells from 20-wk-infected donors into acutely (6-wk) infected recipients increased granuloma mast cell density. Treatment of spleen cells with anti-Thy-1.2 or anti-Lyt-1.1 antiserum and complement, but not anti-Lyt-2.1 or normal mouse serum, abrogated adoptive transfer-induced, augmentation of granuloma mast cell density. Treatment of acutely infected animals with cyclophosphamide or cimetidine (H2 antagonist enhanced granuloma mast cell density. These data suggest that granuloma mast cell density is dependent upon subsets of T lymphocytes.     

Combined prophylactic suppression of graft-versus-host and host-versus-graft reactions following treatment of prospective bone marrow recipients with rat IgG 2b anti-mouse T cell antibodies

A new approach to avoid typical complications from bone marrow transplantation into MHC different mice was studied. Rat monoclonal anti-Thy-1 antibodies of the IgG 2b isotype were identified, which inhibit T lymphocytes in vivo so that transplanted donor T cells as well as residual T cells of the conditioned marrow recipient were suppressed. A single injection of these antibodies after irradiation and before marrow transplantation did not only prevent graft-versus-host mortality but suppressed also host-versus-graft reactivity so that the radiation dose necessary for engraftment of donor cells differing in H-2, IA (both haplotypes) major histocompatibility antigens could be reduced to 6.0 Gy. In addition an anti-T leukemic cell effect from the injected monoclonal T cell antibodies was observed.     

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.     

Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences

Information was sought on the phenotype of lymphoid cells causing lethal graft-vs-host disease (GVHD) in irradiated mice expressing whole or partial H-2 differences. In all strain combinations tested, pretreating donor lymph node (LN) cells with anti-Thy-1 monoclonal antibodies (MAb) plus complement (C) abolished mortality. With GVHD directed to class I H-2 differences, pretreating LN cells with anti-Lyt-2 MAb prevented mortality, whereas MAb specific for Ly-1 or L3T4 cell surface determinants caused severe mortality. These data imply that lethal GVHD directed to class I H-2 differences is mediated by L3T4-, Lyt-2+ cells; this subset of T cells was shown previously to control GVHD directed to multiple minor histocompatibility antigens, i.e., antigens seen in the context of self-class I molecules. With whole H-2 differences, GVHD appeared to be controlled largely but not exclusively by L3T4+, Lyt-2-T cells. This T cell subset was also the predominant cause of GVHD directed to class II differences. With class II incompatibilities, depleting donor cells of L3T4+ T cells, either by pretreatment with anti-L3T4 MAb + C or by fluorescence activated cell sorter selection, greatly reduced but did not completely abolish GVHD. These data might imply that L3T4-, Lyt-2+ cells have some capacity to elicit anti-class II GVHD. A more likely possibility, however, is that the residual GVHD to class II differences observed with Lyt-2+-enriched cells reflected minor contamination with L3T4+ cells.