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.     

L3T4-positive T cells participate in the induction of graft-vs-host disease in response to minor histocompatibility antigens

The phenotype of T cells that initiate graft-vs-host disease (GVHD) in response to minor histocompatibility antigens (minor HA) was determined in three H-2 compatible strain combinations by using negative selection with monoclonal antibodies to Lyt-2 and L3T4 antigens to test the hypothesis that Lyt-2-positive T cells alone initiate GVHD. The phenotype of T cells required to initiate GVHD was different in each of the three strain combinations studied. Both Lyt-2+ and L3T4+ LP spleen cells were necessary to cause lethal GVHD in C57BL/6 recipients. In the reciprocal transplant, Lyt-2+, but not L3T4+ C57BL/6 spleen cells were sufficient to initiate GVHD in LP recipients. In contrast, L3T4+, but not Lyt-2+ B10.D2 spleen cells were found to initiate GVHD in BALB/c recipients. The optimal response to minor HA requires both Lyt-2+ and L3T4+ T cells because a mixture of the two subsets of spleen cells resulted in a more severe form of GVHD than either subset alone in all three strain combinations studied. This study demonstrates that L3T4+ cells participate in the initiation of GVHD in response to minor HA. The dominant T cell subset that initiates GVHD varies with the specific strain combination tested. The specific minor HA expressed in the transplant recipient, the H-2 type, and possibly non-major histocompatibility complex immune response genes of the donor strain appear to determine the phenotype of the initiator T cells.     

Autoimmune thyroiditis induced in mice depleted of particular T cell subsets. I. Requirement of Lyt-1 dull L3T4 bright normal T cells for the induction of thyroiditis

T cell-depleted C3H/He or (C57BL/6xC3H/He)F1 (B6C3F1) mice were prepared by adult thymectomy and injection of antithymocyte serum, followed 3 wk later by lethal x-irradiation and bone marrow reconstitution. When these T cell-depleted mice were not injected or injected i.v. with normal spleen and lymph node cells treated with either anti-Thy-1, -L3T4 or -Lyt-2 antibody plus C or C alone, none of the groups of mice developed thyroiditis. In contrast, the adoptive transfer of normal cells treated with anti-Lyt-1 plus C resulted in high incidence of the production of antithyroglobulin antibody and the induction of typical thyroiditis lesion. The thyroid was the sole organ involved, because neither typical inflammatory lesion in other organs nor autoantibody such as anti-DNA antibody was detected in mice that exhibited thyroiditis. Analyses of surface phenotypes of cells required for inducing thyroiditis by the adoptive transfer revealed that an appreciable percentage of Lyt-1 dull T cells remained after the treatment of normal lymphoid cells with anti-Lyt-1 plus C. Almost all of these Lyt-1 dull T cells expressed magnitudes of L3T4 or Lyt-2 Ag comparable to those detected on Lyt-1 bright T cells. More important, the induction of thyroiditis was almost completely prevented by either in vitro or in vivo elimination of Lyt-1 dull L3T4+(bright) but not of Lyt-1 dull Lyt-2+(bright) T cells. These results indicate that Lyt-1 dull L3T4+ T cells existing in normal healthy individuals have potential to induce typical thyroiditis which is associated with the production of antithyroglobulin autoantibody, and that the activation and/or function of this T cell subset is regulated by the Lyt-1 bright T cell population coexisting in normal lymphoid cell population.     

Lethal graft-vs-host disease across major histocompatibility barriers: requirement for leucyl-leucine methyl ester sensitive cytotoxic T cells

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is selectively toxic for human natural killer (NK) cells and cytotoxic T lymphocytes (CTL) at both the precursor and effector stage of differentiation. The present studies explored the effects of Leu-Leu-OMe on murine spleen cell function. Leu-Leu-OMe exposure removed NK function from murine spleen cells but spared their capacity to proliferate in response to lipopolysaccharide and Con A. The capacity to generate CTL from both L3T4 (+) and Lyt-2 (+) precursors was lost after Leu-Leu-OMe treatment, whereas alloantigen-induced proliferation and interleukin 2 (IL 2) production by L3T4 (+) T helper cells remained intact. Lethal graft vs host disease (GVHD), which developed in irradiated (C57BL/6 X DBA/2)F1 recipients of C57BL/6 bone marrow and spleen cells was completely prevented by Leu-Leu-OMe treatment of donor cells. In contrast depletion of Lyt-2 positive cells from the donor inoculum did not prevent acute GVHD in this fully major histo-compatibility complex (MHC) incompatible strain combination. However, Leu-Leu-OMe treatment of the Lyt-2 depleted inoculum completely prevented lethal GVHD, although the treated cells retained the capacity to proliferate and secrete IL 2 normally after in vitro stimulation with (C57BL/6 X DFA/2)F1 spleen cells. These findings indicate that L3T4 (+) T helper cells alone are unable to initiate lethal GVHD in this H-2 incompatible strain combination. Rather, lethal GVHD requires the transfer of a Leu-Leu-OMe sensitive T cell subset, likely to be thymus educated pre-CTL. Leu-Leu-OMe treatment should provide a useful way to delineate subpopulations of cells involved in the production of lethal GVHD and an approach to preventing this complication of bone marrow transplantation.     

Clonal analysis of murine graft-vs-host disease. I. Phenotypic and functional analysis of T lymphocyte clones

Acute and chronic graft-vs-host disease (GVHD) due to non-MHC histocompatibility differences differ histopathologically. Acute GVHD is characterized by the cytotoxic destruction of recipient tissues, whereas chronic GVHD is characterized by increased collagen deposition. In an attempt to determine if acute and chronic GVHD represent two phases of the same pathophysiologic process or two distinct processes, the T lymphocytes from the C57BL/6 (B6) recipients of LP spleen cells (non-H-2 GVHD) have been cloned and compared to clones from immune mice (LP anti-B6). Acute GVHD (G) clones were established on day 10-14 posttransplant and chronic GVHD (CG) clones on day 50 from animals with clinical chronic GVHD. Immune (I) clones were established 10 to 14 days after immunization. All I clones exhibited B6-specific blastogenesis and cytotoxicity and had a Thy-1.2+, Lyt-2.2+, L3T4- phenotype. All CG clones were noncytotoxic, had I-Ab-specific blastogenesis, and had a Thy-1.2+, Lyt-2.2-, L3T4+ phenotype. The acute GVHD (G) clones were heterogeneous. Fourteen of 23 clones exhibited B6-specific blastogenesis and had a Lyt-2.2+, L3T4- phenotype (B6-G clones). Seven of 9 B6-G clones were cytotoxic for B6 targets. Nine of 23 G clones exhibited I-Ab-specific blastogenesis, and all but one clone had a Lyt-2.2-, L3T4+ phenotype as the did CG clones. Thus, the principal clonogenic T lymphocytes from mice with acute and chronic GVHD differ in terms of 1) their antigenic specificity, 2) their cytotoxic capacity, and 3) their surface phenotype. The presence of I-Ab-specific T lymphocytes with a phenotype identical to CG clones early after transplantation suggests that the immunologic events that result in chronic GVHD begin soon after transplantation. These results indicate that acute GVHD is due primarily to recipient-specific cytotoxic donor T lymphocytes, whereas chronic GVHD is due to autoreactive helper T lymphocytes.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice

Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti-T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection.     

Role of T cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H-2 differences. II. Protective effects of L3T4+ cells in anti-class II GVHD

Detailed information was sought on the capacity of purified B6 L3T4+ cells to elicit lethal graft-versus-host disease (GVHD) in irradiated class II-different class I-identical (C57BL/6 (B6) x bm 12)F1 hosts. When B6 L3T4+ cells were transferred in small doses (10(5) to 10(6) together with donor bone marrow (BM) cells, the recipients all developed acute lethal GVHD and most of the mice died within 2 wk, probably from gut damage; this syndrome was conspicuous only in mice treated with very heavy irradiation, i.e., 1000 rad. In marked contrast to L3T4+ cells given in small doses, transfer of large doses of B6 L3T4+ cells to heavily irradiated (B6 x bm 12)F1 hosts paradoxically resulted in only limited mortality: most of the recipients survived for greater than 6 mo and manifested little or no evidence of ill health. It is suggested that the capacity of large doses of L3T4+ cells to protect mice against lethal GVHD is a reflection of T helper function: the cellular immunity provided by the donor L3T4+ cells enables the host to repel pathogens entering through damaged mucosal surfaces, with the result that GVHD becomes sublethal. The protective function of L3T4+ cells in the B6----bm 12 combination was only seen in hosts given donor BM. With transfer of donor L3T4+ cells plus host BM, even lightly irradiated recipients died rapidly from hemopoietic failure. Because this syndrome failed to occur in mice given a mixture of donor and host BM, it would appear that L3T4+ cells destroyed host lymphohemopoietic cells by direct cytotoxicity rather than via a bystander effect.     

Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.     

Lyt phenotype analysis of the effector cells responsible for evoking lymphocyte-induced angiogenesis (LIA)

Lymphocyte-induced angiogenesis (LIA) is a vascular response observed when allogeneic or semiallogeneic immunocompetent lymphocytes are inoculated intradermally into immunosuppressed or irradiated host mice. The reported experiments were carried out to characterize the effector cell population(s) responsible for causing LIA. Lyt 1.2, Lyt 2.2, and monoclonal Thy 1.2 antisera were used for negative selection with complement (C′) to investigate the ability of selected subsets of lymphocytes to evoke angiogenesis. Treatment of C57BL/6 spleen cells with either anti-Lyt 1.2 or anti-Thy 1.2 and C′ resulted in an almost complete abrogation of the LIA reaction. In contrast, depletion of Lyt 2+ cells, under conditions which fully abrogated their ability to generate cell-mediated cytotoxicity in allogeneic mixed leukocyte cultures, resulted only in a partial (45%) reduction in the induced vascular response. Synergistic interaction between cell preparations treated separately with either anti-Lyt 1.2 or anti-Lyt 2.2 serum was not observed. We conclude that (i) Lyt 1 + 2?T lymphocytes can induce a significant LIA reaction; (ii) lymphocytes resistant to negative selection with anti-Lyt-1.2 serum are incapable of inducing such a reaction; and (iii) Lyt 1 + 2+ cells directly or indirectly play an additional role in generating a maximal LIA response.     

Enterically induced regulation of systemic immune responses. II. Suppression of proliferating T cells by an Lyt-1+, 2- T effector cell

Peripheral lymph node cells from C3H mice that were fed and injected with bovine serum albumin (REG cells) demonstrate an impaired proliferative response to antigenic stimulation in vitro compared to cells from mice only injected with BSA. To determine whether suppressor cells contributed to this enterically induced impairment of systemic T cell responses, REG cells were pretreated with various monoclonal antibodies and complement (C), and were then co-cultured with antigen-reactive indicator T cells (IND) from parenterally immunized mice. Proliferation of IND cells [( 3H]thymidine uptake) was suppressed only if REG cells were treated with anti-Lyt-2 and C before co-culture. The ability of anti-Lyt-1 plus anti-Lyt-2 and C treatment to abrogate suppression suggested that the suppressor effect was due to an Lyt-1+, 2- REG cell. Suppression was independent of Lyt-2+ IND cells, and was observed at different antigen concentrations, cultivation times, and cell densities. The cells responsible for suppressor activity were radiosensitive, nylon wool nonadherent, and antigen specific. These data suggest that an Lyt-1+, 2- T cell could be an important component in mediating enterically induced regulation of systemic T cell responses.     

A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants

We studied engraftment in a murine model of allogeneic bone marrow (BM) transplantation. Recipient C57BL/6 (H-2b) mice were conditioned with single-dose (9 or 7.5 Gy) total body irradiation (TBI), fractionated (4 X 3.3 Gy) TBI, hyperfractionated (8 X 1.65 Gy) TBI, 2 X 120 mg/kg cyclophosphamide (CY) followed by 7.5 Gy TBI, or 300 mg/kg CY followed by 9 Gy total lymphoid irradiation (TLI). Conditioned mice were transplanted with BALB/c (H-2d) BM supplemented with splenocytes (BMS) to facilitate graft-vs-host disease (GVHD). Ex vivo T cell depletion of the BMS with anti-Thy-1.2 antibody and complement protected recipients from lethal GVHD. Engraftment was measured in transplanted animals by serotyping peripheral blood mononuclear cells with anti-H-2-specific antibodies and complement. Mice that were given a T cell-depleted BMS transplant after conditioning with 9 Gy TBI, fractionated TBI, or CY plus TBI showed a 99 to 100% incidence of engraftment. However, if the T cell-depleted graft was given to mice conditioned with hyperfractionated TBI, 7.5 Gy TBI, or CY plus TLI, only 3 to 32% of the animals engrafted. BM which was not T cell-depleted engrafted in 63 to 100% of the mice regardless of the conditioning used. Nonengrafted mice tested with anti-host type antibody demonstrated autologous recovery. We conclude that engraftment or failure/rejection of BM in transplanted mice is determined in part by a dynamic equilibrium between T cells present in the donor graft and the surviving hemopoietic cells in the conditioned recipient. More intensive conditioning of the recipient allows engraftment of T cell-depleted, mismatched BMS. Such conditioning is not limited to a single modality, but can be achieved with single-dose TBI, fractionated TBI, or with TBI combined with CY. These findings have timely and important implications for the current understanding of engraftment in human allogeneic BM transplantation following T cell depletion.     

Feedback suppression of the immune response in vivo. III. Lyt-1+ B cells are suppressor-inducer cells

We previously demonstrated that injection of a high dose (4 X 10(9] of sheep erythrocytes (SRBC) into C57BL/6 mice results in the generation of splenic B cells (plastic nonadherent, Thy-1- and Ig+) which, when transferred to normal syngeneic recipients, subsequently induce antigen-specific suppressor T cells to suppress the recipient's plaque-forming cell (PFC) responses to SRBC. In the present study we characterized the suppressor-inducer B cells phenotypically. Cytotoxic treatment of the donor's immune spleen cells with anti-Lyt-1 antibody plus complement (C'), but not with anti-Lyt-2 antibody plus C', relieved the suppression of PFC responses in recipients. The FcRr+ population separated by EA-rosette formation showed enriched suppressor-inducing activity, whereas the FcRr- population showed no activity. Our findings, taken together with the previous ones, suggest that suppressor-inducer cells are Thy-1-, Lyt-1+, Lyt-2-, FcRr+, and Ig+.     

A role for Lyt-2+ T cells in resistance to cutaneous leishmaniasis in immunized mice

The role of Lyt-2+ T cells in immunologic resistance to cutaneous leishmaniasis was analyzed by comparing infection patterns in resistant C57BL/6 mice and susceptible BALB/c mice induced to heal their infections after sub-lethal irradiation or i.v. immunization, with similar mice treated in vivo with anti-Lyt-2 antibodies. Administration of anti-Lyt-2 mAb resulted in a dramatic reduction in the number of lymphoid cells expressing the Lyt-2+ phenotype. Such treatment led to enhanced disease in both resistant C57BL/6 and irradiated BALB/c mice, as assessed by lesion size, but did not affect the capacity of these mice to ultimately resolve their infections. In contrast, anti-Lyt-2 treatment totally blocked the induction of resistance in i.v. immunized mice. These results suggest, that Lyt-2+ T cells may play a role in immunity to a Leishmania major infection and that their relative importance to resistance may depend on how resistance is induced.     

Immunotherapy of lymphoid and nonlymphoid tumors with monoclonal anti-Lyt-1 antibodies

Monoclonal antibodies against the T cell differentiation antigen Lyt-1 were effective in the therapy of transplanted mouse tumors. A possible mechanism whereby anti-Lyt-1 antibodies directly bind and affect the tumor cells was excluded by the following findings: a) growth of lymphoid and nonlymphoid tumors (which lack Lyt-1 antigen) was affected by anti-Lyt-1 antibodies; and b) the curative effect of passively administered anti-Lyt-1 anti-bodies was abrogated in mice depleted of T cells, supporting a mechanism whereby host Lyt-1+ cells were involved in tumor therapy. Treatment with anti-Lyt-1 antibodies was not accompanied by depletion of Lyt-1+ cells from lymphoid organs, indicating that the administered antibodies altered Lyt-1+ cell functions without affecting their frequency. In view of the in vitro enhancing effects of anti-Lyt-1 antibodies on a variety of immune responses (including lymphokine secretion and generation of cytotoxic T cell), it is suggested that the potentiation of Lyt-1+ cell activity by passively administered anti-Lyt-1 antibodies results in tumor rejection.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Characterization of effector cells of graft vs leukemia following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia

Summary It is now widely accepted that immunocompetent lymphocytes in allogeneic bone marrow grafts exert an antileukemic effect that contributes to the cure of leukemia. Graft vs leukemia (GVL) effects independent of graft vs host disease were investigated in allogeneic bone marrow chimeras tolerant of host and donor alloantigens. The role of Thy1.2, L3T4 and Lyt2 T lymphocytes as effector cells of GVL were investigated in (BALB/c × C57BL/6)F1 mice inoculated with murine B-cell leukemia and subsequently conditioned with total lymphoid irradiation and cyclophosphamide (200 mg/kg). Mice were reconstituted with C57BL/6 bone marrow cells depleted of well-defined T-cell subsets or enriched for stem cells by the soybean agglutination method. Detection of residual tumor cells, an indicator for efficacy of GVL, was carried out by adoptive transfer of peripheral blood or spleen cells obtained from treated chimeras into secondary naive BALB/c recipients at different time intervals following bone marrow transplantation. Treatment of the primary marrow inoculum with monoclonal anti-Thy 1.2 or anti-Lyt2 abolished the GVL effects and all secondary BALB/c recipients developed leukemia within 60 days. On the other hand, the treatment with monoclonal anti-L3T4 did not influence the effect of GVL and all treated recipients remained without leukemia. The data suggest that T cells may mediate GVL effects in the absence of graft vs host disease and in circumstances where tolerance to conventional alloantigens is elicited. Effector cells of GVL across the major histocompatibility complex (MHC) in the murine B-cell leukemia tumor model system appear to be Thy 1.2⁺ Lyt2⁺ L3T4—. Induction of GVL effects by allogeneic cells tolerant of host MHC suggests that these effects may be independent of graft vs host disease.     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10(-6) or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1:2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of the Lyt-2 and Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearing Lyt-2a and Lyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b congenic with AKR/J and bearing the Lyt-2b and Lyt-3b alleles of Balb/cJ.     

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

The Lyt phenotype of the T cells responsible for in vivo tumor rejection in syngeneic mice

Summary Spleen cells of BALB/c mice hyperimmunized with a transplantable methylcholanthrene-induced sarcoma Meth A (Meth A-Im-SPL) inhibited the growth of Meth A tumor in vivo in a tumor neutralizing test. Meth A-Im-SPL did not neutralize another antienically distinct sarcoma, Meth 1, indicating that the antitumor activity is tumor specific. Lyt-1⁺2^– cells of Meth A-Im-SPL (Im-Lyt-1⁺2^–) were the effectors since in vitro treatment of Meth A-Im-SPL with anti-Thy 1.2 or anti-Lyt 1.2 antibody plus complement completely abrogated their neutralizing activity, whereas treatment with anti-Lyt 2.2 plus complement did not. To further confirm the effector activity of Im-Lyt-1⁺2^– cells, T cell subpopulations were separated from Meth A-Im-SPL by the panning method. The purified Im-Lyt-1⁺2^–, but not Im-Lyt-1⁺2⁺ cells neutralized the tumor in athymic nu/nu mice as efficiently as in +/+ mice, suggesting that the donor Im-Lyt-1⁺2^– cells but not recipient T cells were primarily responsible for neutralizing the tumor. The present study, however, did not exclude the possible contribution of recipient T cells to the tumor neutralization and this is open to further investigation.Abbreviations Meth A-Im-SPL Meth A-immune mouse spleen cells - Meth 1-Im-SPL Meth 1-immune mouse spleen cells - sIg⁺ cells surface immunoglobulin positive cells - moAb monoclonal antibody