anti-L3T4 Monoclonal Antibody Research Articles

Effects of immunotherapy with anti-L3T4 monoclonal antibody on autoimmune cardiomyopathy: experiment with mice

To evaluate the effects of immunotherapy with anti-L3T4 monoclonal antibody (McAb) on autoimmune cardiomyopathy. 24 male Balb/c mice were immunized with mitochondria ADP/ATP carrier peptides so as to establish a model of autoimmune cardiomyopathy, and then randomly divided into 4 equal groups: cardiomyopathy control group, early-treated group injected with anti-L3T4 McAb on the day before immunization, the very day of immunization, and 1 day after the immunization, mid-term-treated group, administrated with anti-L3T4 McAb on the 89th, 90th and 91st days after immunization, and sham-immunization control group, injected with solution without anti-L3T4 McAb. Six months after blood samples were collected. The serum antibodies against ADP/ATP carrier were examined with ELISA, and the expression of IFN-gamma and IL-4, T cell cytokines, in T cells were detected by 3-color flow cytometry. The myocardium underwent pathological examination by using light and electron microscopes. The bulk percentages of cardiac collagen fiber were calculated with computer. Antibodies against ADP/ATP carrier were negative in the early-treated group and the sham-immunization control group and positive in the mid-term-treated and cardiomyopathy groups with a transient decline in the mid-term-treated group. By the end of 6 month, there were no cardiac morphological and histopathological changes like those in dilated cardiomyopathy (DCM) in the early-treated group and sham-immunization group, while they were found in the mid-term-treated and cardiomyopathy groups with severer myocardial impairments in the cardiomyopathy group. The percentage of IFN-gamma/IL-4 of the early-treated group was 6.56% +/- 0.21%/2.54% +/- 0.20%, not significantly different from that of the control group (5.87% +/- 0.19%/2.16% +/- 0.10%), however, the values of these 2 groups were significantly higher than those of the mid-term-treated group (8.91% +/- 0.33%/5.15% +/- 0.13%, both P < 0.01) and the cardiomyopathy group (7.85% +/- 1.42%/9.45% +/- 1.70%, both P < 0.01). Anti-L3T4 McAb administration is effective on protection from autoimmune cardiomyopathy induced by mitochondria ADP/ATP carrier peptides, especially being used at the initial phase of this disease.

Infectious Tolerance to ADP/ATP Carrier Peptides Induced by Anti-L3T4 Monoclonal Antibody in Dilated Cardiomyopathy Mice

CD4 T cells are suspected to play an important role in the pathogenesis of dilated cardiomyopathy (DCM). This study sought to evaluate whether anti-L3T4 monoclonal antibody (McAb) could induce the infectious tolerance to the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier peptides to protect mice from DCM. BALB/c mice (n = 16) were immunized with the peptides derived from human ADP/ATP carrier on the 1st, 14th, 28th, 49th, and 79th days, and some of them (n = 6) were also injected with anti-L3T4 McAb on the -1st, 0, and 1st days. On the 180th day, the splenocytes (SC) from the McAb-treated group were transferred into the syngeneic recipients (n = 6) who were also immunized with the peptides in the same manner. The sham-immunized mice were taken as the controls (n = 10). Results showed that the serum antibody against the ADP/ATP carrier examined with ELISA was positive in all mice only immunized with the peptides (DCM group), while negative in the McAb-treated, the SC-transferred, and the Control groups. The mRNA expression of IFN-gamma, IL-2, and IL-4, especially IL-4 in T cells investigated using real-time quantitative PCR and the percentages of T helper 1 (Th1) and Th2 subsets, especially Th2 subset detected with Flow Cytometry were all increased in DCM group, accompanied by the cardiac histopathological changes like those in DCM. Such findings were not seen in the other three groups. It concluded that anti-L3T4 McAb could inhibit the occurrence of DCM induced by the ADP/ATP carrier peptides in mice, and this immune tolerance could be transferred to the syngeneic recipients.

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody.

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.

Antigen-Induced Recruitment of Eosinophils: Importance of CD4 + T Cells, IL5, and Mast Cells

Eosinophils of sensitized mice readily recruit to the site of antigen challenge. In the present study, experiments were performed to determine the involvement of different cell types in the antigen-induced recruitment of eosinophils. We demonstrated that a single treatment with anti-L3T4 monoclonal antibody (mAb) on the day of allergen challenge significantly decreased antigen-induced recruitment of eosinophils. Treatments with anti-L3T4 mAb during the sensitization period also caused a substantial reduction in the migration of eosinophils into the site of challenge with antigen. Thus, it appears that both stages of eosinophil recruitment, sensitization and antigen-challenge, are dependent upon the presence of L3T4 + T cells. Moreover, while treatments with anti-IL5 mAb blocked eosinophil migration, anti-IL2 mAb failed to alter the antigen-induced recruitment of eosinophils. In addition, significant numbers of eosinophils from the mast-cell-deficient mice were found to migrate into the peritoneal cavities upon allergen challenge. Eosinophil migration was also observed in several mouse strains of different H-2 haplotypes. The present findings suggest that CD4 + T cells and IL5 but not IL2 may play important roles in modulating the recruitment of eosinophils. Moreover, the involvement of mast cells does not appear to be essential for eosinophil migration. Finally, the development of antigen-induced recruitment of eosinophils is probably not under the immunogenetic regulation by genes within the H-2 complex.

Role of CD4+ T cells in the rat to mouse cardiac xenotransplantation.

T cell subsets involved in rejection of xenografts were analyzed using a rat to mouse cardiac xenotransplant model. Proliferating response and interleulin-2 (IL-2) production in recipients' spleen cells were almost completely abrogated by elimination of L3T4+ T cells, but not by elimination of Lyt2.1+ T cells. Cytotoxic T lymphocyte (CTL) activities were mediated by both L3T4+ and Lyt2.1+ T cells with the help of IL-2-producing L3T4+ T cells. Administration of anti-L3T4 monoclonal antibody (mAb) into recipient mice resulted in a significant prolongation of graft survival (mean graft survival was 29.2 days). Moreover, anti-L3T4 mAb treatment plus thymectomy led to indefinite graft survival. Anti-rat endothelial cell (EC) antibody production in the grafted mice was remarkably suppressed by anti-L3T4 mAb treatment. In contrast, Lyt2.1 mAb treatment did not prolong the graft survival and did not suppress anti-EC antibody production. These results indicated the absolute requirement of L3T4+ T cells in the rejection of rat to mouse cardiac xenografts.

Role of CD4+ T lymphocytes and interleukin-5 in antigen-induced eosinophil recruitment into the site of cutaneous late-phase reaction in mice.

Previous studies suggested that the eosinophil recruitment into the site of cutaneous late-phase reaction (LPR) was dependent on IgE antibody and mast cells. In this study, we determined the role of CD4+ T cells and CD8+ T cells in causing antigen-induced eosinophil recruitment of LPR in mouse skin. Eosinophil infiltration into the subcutaneous tissue of ovalbumin (OVA)-sensitized BALB/c mice was biphasic, reaching the first peak at 6 h after the subcutaneous challenge with OVA and the second peak at 24 to 48 h. The in vivo depletion of CD4+ T cells by pretreatment with anti-L3T4 monoclonal antibody (mAb) significantly decreased the second peak (at 24 h and 48 h), but not the first peak (at 6 h), of OVA-induced eosinophil infiltration into the skin of OVA-sensitized mice. However, the depletion of CD8+ T cells by pretreatment with anti-Lyt-2 mAb had no significant effect on either the first peak or second peak of OVA-induced cutaneous eosinophilia. Pretreatment with anti-murine interleukin-5 (IL-5) mAb also decreased the second peak, but not the first peak, of OVA-induced cutaneous eosinophilia. In contrast to the inhibitory effects of depletion of CD4+ T cells and of anti-IL-5 mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate and a selective antagonist for platelet activating factor (PAF) CV-6209 decreased the first peak of OVA-induced cutaneous eosinophilia in the mouse. These results indicate that CD4+ T cells, but not CD8+ T cells, cause the second peak of antigen-induced eosinophil recruitment of cutaneous LPR and that IL-5 mediates this eosinophil recruitment. In contrast, the first peak of antigen-induced eosinophil recruitment of cutaneous LPR is mediated by mast cells and PAF.

The role of leucyl-leucine methyl ester-sensitive cytotoxic cells in skin allograft rejection.

Treatment of murine spleen cells (SpC) with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) depletes L3T4(+) and Lyt2(+) cytotoxic T lymphocyte precursors and the capacity to generate lethal graft-versus-host disease in semiallogeneic class I + II MHC and multiple non-MHC-disparate recipient mice, whereas T helper cell function is preserved. In the present studies the role of Leu-Leu-OMe-sensitive CTL in skin graft rejection was examined. C57BL/6J (B6) mice were serially thymectomized, lethally irradiated, reconstituted with T cell-depleted bone marrow, and treated with intraperitoneal injections of anti-L3T4 and anti-Lyt2 monoclonal antibodies. These adult thymectomized, bone marrow-reconstituted, T cell-depleted (ATXBM, TCD) mice were unable to reject B6xDBA/2F1 (B6D2F1) skin grafts. When such ATXBM, TCD mice were reconstituted with 7 x 10(7) control B6 SpC, acute rejection of B6D2F1 skin was observed. When B6 donor SpC were Leu-Leu-OMe-treated prior to transfer to ATXBM, TCD mice, uniform rejection of B6D2F1 skin grafts was still observed, although a significant delay in the time to rejection was observed. More rigorous T cell depletion of ATXBM, TCD host mice by infusion of antithymocyte globulin did not prevent delayed rejection of B6D2F1 skin initiated by transfer of Leu-Leu-OMe-treated B6 SpC. Despite the lack of complete prevention of skin allograft rejection, Leu-Leu-OMe treatment of B6 donor cells prevented lethal GVHD even in thymectomized B6D2F1 recipients. Precursors of anti-B6D2F1-specific CTL were greatly reduced or undetectable in unreconstituted ATXBM, TCD mice or in irradiated B6D2F1 recipients of Leu-Leu-OMe-treated B6 SpC. By contrast, ATXBM, TCD recipients of Leu-Leu-OMe-treated B6 SpC were found to contain a population of anti-class I MHC-specific CTL precursors of host origin within 28 days of reconstitution. These findings have indicated a number of features of the cells involved in skin graft rejection. First, Leu-Leu-OMe-sensitive CTL play a major role in acute rejection of class I + II MHC and multiple non-MHC antigen-disparate skin grafts. Moreover, the thymus-independent expansion of host-derived CTL precursors in ATXBM, TCD mice reconstituted with syngeneic Leu-Leu-OMe-resistant T helper cells also appears to play a role in mediating rejection of allogeneic skin grafts.

Some approaches to improve the therapeutic effectiveness of adoptive chemoimmunotherapy with spleen cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor.

Spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells) were previously shown to be capable of bringing about the complete regression of a large (15 to 20 mm) s.c. MOPC-315 tumor in a substantial percentage of T-cell-deficient (athymic nude) mice that had been treated with low-dose L-PAM (adoptive chemo-immunotherapy; ACIT). Here we show that aggressive depletion of CD4+T-cells by treatment both of spleen-cell donors and of recipients with anti-L3T4 monoclonal antibody (MAb) greatly improved the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT. In the light of the apparent importance of cytotoxic T-lymphocytes (CTLs) for tumor eradication in low-dose L-PAM-treated MOPC-315-tumor bearers, it is interesting that treatment of L-PAM TuB spleen-cell donors with anti-L3T4 MAb was found to result in the generation of enhanced splenic anti-MOPC-315 cytotoxicity. Although most athymic nude mice in which the tumor had apparently completely regressed following ACIT remained tumor-free, approximately 1/3 of the mice relapsed. However, a substantial percentage of the relapsing mice were rescued by a low dose of L-PAM, which was not effective in causing tumor regression in athymic nude mice bearing a comparably large tumor if the mice had not been subjected previously to ACIT. Almost all athymic nude mice that had been "cured" of a large MOPC-315 tumor by ACIT but did not resist a subsequent MOPC-315 tumor challenge were rescued by low dose L-PAM. Thus, the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT may be improved by aggressive depletion of CD4+ T-cells, suggesting that a low dose of L-PAM, which leads to the acquisition of potent splenic-tumor-eradicating immunity in BALB/c mice bearing a large MOPC-315 tumor, does not eliminate completely (or possibly not at all) the inhibitory activity of CD4+ T-cells. In addition, athymic nude mice that are not endowed with fully protective tumor-eradicating immunity following ACIT still have a substantial residual anti-tumor immune potential that can be exploited to bring about eradication of a large tumor burden following low-dose L-PAM therapy.

Cell subsets responding to purified hepatocytes and evidence of indirect recognition of hepatocyte major histocompatibility complex class I antigen. I. The role of L3T4+ T cells in the development of allospecific cytotoxicity in hepatocyte-sponge matrix allografts.

The purpose of this study was to determine the role of L3T4+, Ly2- T cells in the development of allospecific cytolytic T cells in response to purified allogeneic MHC class I+, class II- hepatocytes in vivo in hepatocyte-sponge matrix allografts (HC-SMA). In previous studies we have shown that 99% pure murine hepatocytes stimulate the development of allospecific cytolytic T cells in vitro in mixed lymphocyte-hepatocyte culture (MLHC) and in vivo in HC-SMA. Furthermore, depletion of L3T4+, Ly2- T cells from responder splenocytes inhibits the development of allo-CTLs in response to purified hepatocytes in mixed lymphocyte-hepatocyte culture. Here, using an anti-L3T4 monoclonal antibody, we tested the effect of in vivo immunodepletion of L3T4+, Ly2- T cells on the subsequent development of allo-CTLs in HC-SMA. Sponge cells were harvested on day 4 and day 12 after grafting from control and treated groups and phenotypically analyzed by FACS and immunofluorescent labelling. Splenocytes from the same animals were similarly analyzed to assess for completeness of immunodepletion. Allospecific cytotoxicity was assessed on day 12 after grafting. We found that immunotherapy with anti-L3T4 mAb was effective in depleting L3T4+, Ly2- T cells from the spleen; however, a similar number of L3T4+ cells was isolated from the sponge between control and treated groups. Furthermore, the development of allo-CTLs in response to hepatocytes in HC-SMA was completely abrogated by both local and systemic immunotherapy with anti-L3T4 mAb. We conclude from these and previous data that host or responder L3T4+, Ly2- T cells and responder accessory cells in MLHC or host macrophages in HC-SMA may participate in "indirect" recognition of hepatocyte class I antigen both in vitro and in vivo.

Interleukin-1 enhances the development of type II collagen-induced arthritis only in susceptible and not in resistant mice

The development of type II collagen-induced arthritis (CIA) in DBA/1 mice is readily accelerated by treatments with interleukin-1β (IL-1β). In an attempt to further characterize this IL-1β-mediated enhancement of CIA, we first examined the effects of IL-1β treatments in other “CIA-susceptible” strains and “CIA-resitant” mice. It was observed that treatments with IL-1β also enhanced the onset of arthritis in two B10 recombinant CIA-susceptible strains, B10.T (6R) and B10.DA, and in the SJL mice which develop CIA with a relatively low and variable incidence. On the other hand, IL-1β failed to augment the expression of arthritic disease in several CIA-resistant strains. We also investigated the potentiating effects of IL-1β in mice that were depleted of L3T4 + T cells. It was found that the ability of IL-1β to accelerate the development of CIA was significantly reduced in DBA/1 mice pretreated with the monoclonal anti-L3T4 antibody. In further studies, we demonstrated that the induction of CIA upon transfer with collagen-primed spleen cells was also augmented by IL-1β, and this enhancing effect by IL-1β on the adoptive transfer of CIA was associated with a significant increase in the levels of serum anti-collagen antibodies. Moreover, IL-1β treatments did not potentiate the induction of CIA in mice that were transferred with either collagen-immune splenic cells that were depleted of L3T4 + T cells or only T cells obtained from collagen-immunized animals. However, IL-1β enhanced the development of arthritis in animals that had been transferred with two subpopulations of collagen-immune cells: (i) enriched T cells and (ii) splenic cells that were depleted of L3T4 + T cells. Thus, IL-1β potentialed the inflammatory responses in animals that were genetically predisposed to developing arthritis. In contrast, IL-1β was incapable of accelerating the development of arthritis in various mouse strains that were genetically resistant to CIA. The administration of IL-1β also failed to potentiate the development of CIA in L3T4-deficient mice or in animals transferred with collagen-primed spleen cells that were depleted of L3T4 + T cells. These results indicate that IL-1β readily accelerates the induction of arthritis when the disease is present, but that IL-1β is incapable of promoting the expression of the arthritis in the absence of underlying disease.

EVIDENCE FOR DIRECT AND INDIRECT PATHWAYS IN THE GENERATION OF THE ALLOIMMUNE RESPONSE AGAINST PANCREATIC ISLETS

The role of the direct and indirect pathways of alloantigen presentation in the generation of the alloimmune response was dissected using the murine mixed lymphocyte-islet coculture system (MLIC). Stimulator DBA/2J (H-2d) pancreatic islet populations consisted of whole islets (MHC class I+, II+) or FACS-purified beta cells (MHC class I+, II-). Responding C57Bl/6 (H-2b) splenocyte populations were either: (1) untreated; (2) depleted of helper T cells with anti-L3T4 monoclonal antibody plus complement; (3) depleted of cytotoxic T lymphocytes with anti-Lyt2 mAb plus complement; or (4) depleted of antigen-presenting cells by passage through a Sephadex G-10 column. Whole islets were capable of stimulating a significant C57Bl/6 anti-DBA cytotoxic T cell response if the responding population was untreated or treated with complement alone. Depletion of responding splenocytes with either anti-Lyt2 or anti-L3T4 mAb plus complement abrogated the generation of allospecific CTL. If the responding splenocyte population was depleted of APCs, the allo-CTL response against whole islets was decreased, but still significant. If, however, the stimulator population consisted of FACS-purified DBA 2J beta cells, APC-depleted C57Bl 6 splenocytes were incapable of generating any CTL response. Adding responder type (C57Bl/6) APCs back to the microwells restored the capacity for both whole islets and purified beta cells to stimulate a strong allo-CTL response. These data demonstrate that both indirect and direct pathways of alloantigen presentation function in the MLIC.

Cytotoxic T lymphocyte precursor cells specific for the major histocompatibility complex class I‐like antigen, Qa‐2, require CD4+ T cells to become primed in vivo and to differentiate into effector cells in vitro

Experiments were performed to determine whether CD4+ T cells are required for the generation of cytotoxic T lymphocytes (CTL) specific for the nonpolymorphic major histocompatibility complex (MHC) class I-like antigen, Qa-2. Splenic T cells from BALB/cBy (Qa-2b) mice that had been immunized with irradiated BALB/cJ (Qa-2a) splenocytes generated CTL following in vitro stimulation with BALB/cJ splenocytes. These CTL lysed all Qa-2+, but not Qa-2- targets, regardless of the H-2 haplotypes of target cells or their non-MHC backgrounds. This apparent MHC class I-unrestricted recognition of Qa-2 antigen was confirmed using Qa-2-specific CTL clones. The Qa-2-primed CTL precursor cells (CTLp) and CTL were found to be CD8+ T cells. Primed splenocytes depleted of CD4+ T cells prior to culture failed to generate CTL, but addition of lymphokines to the culture restored the CTL generation. Stimulation of primed splenic T cells with irradiated Qa-2+ T blast cells, instead of splenocytes or B blast cells, led to little to no CTL generation, suggesting that MHC class II molecules are involved in the presentation of Qa-2 antigen to CD4+ T cells. This was also supported by the results of experiments using Qa-2+, class II- thymoma cells of BALB/c origin. Stimulation of the thymoma-primed splenic T cells with the mitomycin C-treated thymoma cells resulted in no generation of anti-Qa-2 CTL, despite the fact that high levels of CTL specific for minor histocompatibility (H) antigens and H-2d were generated by immunizing the corresponding allogeneic hosts with the thymoma. However, the addition of lymphokines rendered thymoma-primed T cells capable of generating anti-Qa-2 CTL. Both CD4+ and CD8+ T cell populations, isolated from the BALB/cJ splenocyte-primed responder cells, proliferated in vitro in response to the Qa-2+ splenocytes, suggesting that Qa-2-reactive CD4+ T cells were present in the immunized mice. Depletion of CD4+ T cells from thymectomized BALB/cBy mice with anti-L3T4 monoclonal antibodies markedly reduced, but did not eliminate anti-Qa-2 CTL generation. In contrast, depletion of CD8+ T cells led to a complete abrogation of the CTL response. Addition of lymphokines to the culture of responder cells depleted of either T cell subset did not restore their reactivity. It is concluded that anti-Qa-2 CTLp need "help" from CD4+ T cells to become primed in vivo. Furthermore, primed CTLp also need "help" or lymphokines provided by CD4+ T cells to differentiate into effector CTL in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection.

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.

Importance in timing of cyclophosphamide on the enhancement of interleukin-2-induced cytolysis.

We investigated the in vivo effects of cyclophosphamide (CY) on interleukin-2(IL-2)-induced cytolytic function and spleen cell immunophenotype. Pretreatment of A/J mice with CY (25 mg/kg or 75 mg/kg) i.p. on days -10 and -15 followed by IL-2 (50,000 U i.p. on days 0 to +3) resulted in increased lysis of YAC-1 target cells compared to the group receiving IL-2 without previous CY therapy. In contrast, when CY was given on day -5, the cytotoxicity against YAC-1 was not enhanced. Phenotypic analysis of splenocytes obtained from mice treated with CY on day -10 or -15 revealed a relative decrease in L3T4- and Lyt2-positive T cells. In vivo depletion of natural killer (NK) cells by anti-asialoGM1, prior to IL-2 therapy, abrogated the enhancing effect of CY on cytolysis while in vivo elimination of T cells by anti-L3T4 and anti-Lyt2 monoclonal antibodies did not, indicating that in the absence of T cell antigenic challenge, the increased cytolytic function after CY administration is probably mediated through NK cells. These findings provide evidence that CY may be used more effectively in IL-2-based immunotherapy protocols, if consideration is given to timing of CY and IL-2 administration.

The adoptive transfer of T‐cell dependent immunity to Plasmodium chabaudi chabaudi in CBA/Ca mice is achieved only after superinfection of immune spleen cell donors

The transfer of spleen cells from CBA/Ca mice recovered from a P. c. chabaudi AS primary infection into irradiated syngeneic recipients conferred very poor protection. Neither elimination of Ly2 cells from immune spleen cells nor reinfection of the donors some days before transfer improved protection significantly. Significant protection was transferred with spleen cells from donors which had been infected 7 times prior to cell transfer. Transferred protection was reduced or eliminated by pretreatment of cells with anti-Thy-1 or anti-L3T4 monoclonal antibodies but not with anti-Ly2.

T helper cells in cytotoxic T lymphocyte development: Role of L3T4 +-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1 −2 +) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1 +2 − Th cells because: (i) cultures depleted by negative selection of Lyt-1 + T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1 +2 − T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4 + Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4 + cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.

Development of insulitis in NOD mice after early continued administration of anti-L3T4 monoclonal antibody

Development of insulitis in NOD mice after early continued administration of anti-L3T4 monoclonal antibody

Effect of Low-Temperature Culture and L3T4 Antibody on the Survival of Pancreatic Islets Xenograft (Fish to Mouse)

Catfish (Ictalurus punctatus) islets were transplanted into the renal subcapsular space of diabetic C57BL/6J mice. The xenograft mean survival time (MST) of islets cultured for 6 days at 24 degrees C was 4.9 +/- 0.5 days, while xenotransplantation of 1-hour-cultured islets did not restore normoglycemia in the diabetic mice. A modest but significant prolongation of the MST was obtained by administration of anti-L3T4 monoclonal antibody to the recipients (13.4 +/- 2.1 days). Anti-L3T4 in conjunction with low-temperature culture did not produce a further increase of the MST (9.1 +/- 2.7 days). These findings indicate that catfish islets may be a source of islet tissue for transplantation in diabetic recipients. However, procedures that demonstrated to be effective in prolonging the MST of xenografts transplanted across wide species barrier, such as low-temperature culture and anti-L3T4 treatment, failed to prolong markedly the xenograft survival. Further experimental work is needed to study the possibility of improving islets survival after xenotransplantation across distant species barrier (fish to mouse).

Analysis of the molecular requirements for T cell recognition and activation by using la-containing lipid vesicles and stopped-flow fluorometry

Using Ia antigen-containing lipid vesicles, we investigated by stopped-flow fluorometry the requirements for helper T cell recognition and activation. When azobenzenearsonate-L-tyrosine (ABA-L-Tyr)-specific, I-Ak-restricted helper T cell hybridomas 2-45-12 were mixed with ABA-L-Tyr and purified I-Ak molecules on lipid vesicles, an increase of intercellular calcium ion concentration ([Ca2+]i) in the T cells were detected within 1-2s. The average increases of [Ca2+]i were not much different when the lipid vesicles were composed of dimyristoylphosphatidylcholine or of egg phosphatidylcholine, but they were dependent on the density of I-Ak molecules on the liposomes. The increase of [Ca2+]i was inhibited in the presence of anti-I-Ak monoclonal antibody 10.2.16, but not by the addition of anti-L3T4 monoclonal antibody GK1.5. However, the addition of anti-L3T4 antibody during the first 3 h of cultivation completely inhibited the T cell activation [interleukin (IL-2) production]. In our experimental system, IL-2 production was observed either when L3T4-positive T cell hybridomas 2-45-12 were stimulated with ABA-L-Tyr and Ia molecules on the vesicles in the presence of phorbol 12-myristate 13-acetate, or when L3T4-negative T cell hybridomas 3H60.12 were incubated with ABA-L-Tyr and Ia molecules on the planar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of L3T4+ lymphocytes in protective immunity to systemic Candida albicans infection in mice

Protective immunity to lethal Candida albicans challenge in vivo and activation of splenic macrophages with highly candidacidal activity in vitro were detected in mice infected with low-virulence agerminative yeast cells of the variant strain PCA-2, at a time when a strong delayed-type hypersensitivity (DTH) reaction to C. albicans occurred in the footpads of PCA-2-treated mice. The DTH reaction was transferable with spleen cell populations from these animals, and enrichment of splenic lymphocytes in L3T4+ cells significantly increased the footpad swelling. The reactivity transferred by L3T4+ cells was a radiosensitive (2,500 rads in vitro) phenomenon that required collaboration with radioresistant, silica-sensitive syngeneic cells in the host and was inhibited by treatment of recipient mice with antibodies to the L3T4 antigen or murine gamma interferon. In vitro, the PCA-2-immune L3T4+ cells produced various lymphokine activities upon incubation with C. albicans, including gamma interferon and granulocyte-macrophage colony-stimulating factor. Anti-L3T4 monoclonal antibody treatment of PCA-2-infected mice significantly impaired their footpad reaction and resistance to C. albicans, as shown by increased recovery of yeast cells from the kidneys of anti-L3T4-treated mice. These results suggested that the mechanisms of anti-Candida resistance induced by PCA-2 may involve specific induction of a DTH response mediated by inflammatory L3T4+ T cells and lymphokine-activated phagocytic effectors. However, the survival rate of the PCA-2-immune mice challenged with C. albicans was not significantly modified by administration of the anti-L3T4 antibody, thus allowing for the conclusion that compensatory mechanisms lead to considerable anti-Candida resistance when the activity of L3T4+ cells is deficient.