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 (12). 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 theLyt-2 and4Lyt-3 loci have been constructed. They are: C.C58-Lyt-2^a, Lyt-3^a and C.AKR-Lyt-2^a, Lyt-3^a, congenic with Balb/cAn and bearingLyt-2 ^a andLyt-3 ^a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2^b, Lyt-3^b, congenic with AKR/J and bearing theLyt-2 ^b andLyt-3 ^b alleles of Balb/cJ.Abbreviations used in this paper DMSO dimethylsulfoxide - NP40 Nonidet P-40 detergent - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoreses - NP-NET buffer 0.15 M NaCl, 0.005 M EDTA, 0.05 M Tris, 0.02% sodium azide, pH 7.4, containing 0.5% or 0.05% NP40 as stated in text     

Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species.

Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.     

BALB/c antiarsonate idiotypes: Gene complementation necessary for expression

The expression of two idiotype (id) families (5AF6 and 3C6) associated with the BALB/c p-azophenylarsonate-specific antibody response was examined in 11 mouse strains. Eight strains produced some of one or the other of these two id families with the mean percent expression in the anti-Ar responses of id(+) strains ranging from 8 to 43% for the 5AF6 and from 2 to 10% for the 3C6 idiotype. Four strains of mice (C58, AKR, PL, and RF) thought to have Lyt-3.1-linked V_L repertoire differences from other mouse strains (Lyt-3.2) were tested for their capacity to contribute to 5AF6 and 3C6 id expression. The RE strain was capable of producing 5AF6 id and small amounts of 3C6 id. Tests of Lyt-3.1 congenic strains C.AKR (AKR Lyt-3.1 on a BALB/c background) and C.C58 (C58 Lyt-3.1 on a BALB/c background) showed that C.AKR could produce 5AF6 id while C.C58 could not. 3C6 id expression was present but depressed in C.C58 mice compared with the high 3C6 id expression in C.AKR. Breeding studies mating C.C58 (bearing the required Igh-C^a-linked V _H genes) to other 5AF6(–) strains showed that gene complementation could result in 5AF6 expression in F₁ offspring. 5AF6(–) strains capable of complementation included CBA/J, C57BL/6J, AKR/J, and PL/J. C58/J (from which C.C58 were derived) was the only tested strain that failed to complement for 5AF6 id expression. Additional matings between C58/J[5AF6(–)] and CBA/J[5AF6 (–)] showed F1 offspring could produce 5AF6 id, indicating that C58/J can contribute functional V _H genes necessary for 5AF6 id expression. Depressed expression of 5AF6 and 3C6 id was noted in mice where the C58/J-derived Lyt-3.1 genotype was present. The possibility that the depression of 5AF6 and 3C6 id expression derived from C58/J mice was due to regulatory influences rather than a lack of the V _L structural genes is discussed.Abbreviations used in this paper Ar p-azophenylarsonate - DNP dinitrophenol - H heavy - HIS hyperimmune suppressed - id idiotype - Ig immunoglobulin - IEF isoelectric focusing - k kappa - L light - PC phosphorylcholine - S.D. standard deviation     

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.     

Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2^a mice with thymocytes from either B6-Lyt-2^a, Lyt-3^a or B6-Lyt-2^a, Lyt-3^a, H-2^k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2^a)F_a mice and progeny of the backcross, (B6 × B6-Lyt-2^a)F₁ × B6-Lyt-2^a, were immunized with B6-Lyt-2^a, Lyt-3^a, H-2^k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2 ^a ) shows very poor penetrance in Lyt-2^a/Lyt-2^b mice.     

T100: a new murine cell surface glycoprotein detected by anti-Lyt-2.1 serum.

A cell surface glycoprotein (designated T100) of apparent m.w. 100,000 by SDS-PAGE under reducing and nonreducing conditions was precipitated from NP-40 extracts of surface radiolabeled thymocytes from a variety of inbred strains of mice by the standard noncongenic Lyt-2.1-typing serum. The inbred stain distribution, trypsin sensitivity on intact cells, and apparent m.w. of T100 suggest that it is different from Lyt-2.1. Inheritance and expression of T100 suggest that it is determined by an allele at a single locus, and testing of CXB recombinant inbred strains and B6.C minor histocompatibility congenic strains suggest that this locus is linked to H-25. Antiserum absorption experiments, two-stage cytotoxicity assays, and results of immunoprecipitations performed after prebinding antibody to radiolabeled thymocytes suggest that some T100 is accessible to antibody on the intact cell surface. However, for unknown reasons the number of cells required to absorb anti-T100 precipitating activity from antiserum was much higher than for removal of anti-Lyt-2.1 activity. A molecule with properties of T100 was also detected on lymph node cells and on the AKTB-1 lymphoma.     

Conditions of anti-Lyt-2-mediated inhibition of TcR/CD3-induced IFN-gamma secretion by a CTL clone

The relationship between the T cell receptor (TcR) for antigen (Ag) and the Lyt-2/3 molecule during T cell activation was studied using the T cell clone KB5.C20, which is dependent upon Lyt-2 for target cell killing. This cytolytic T cell clone can be activated to secrete IFN-gamma by stimulation with H-2Kb expressing cells or with monoclonal antibodies directed against a clonotypic structure of the TcR or against associated CD3 molecules. IFN-gamma production induced by H-2Kb can be inhibited by anti-Lyt-2mAb. In addition, TcR-mediated activation using the anticlonotypic mAb Désiré-1 in soluble form can be inhibited by anti-Lyt-2 mAb in soluble form either as a divalent IgG or as its monovalent Fab fragment. Anti-Lyt-2 mAb immobilized on plastic wells was also inhibitory. Stimulation induced by the anti-TcR mAb or by anti-CD3 mAb immobilized on plastic can be inhibited only with plastic immobilized and not with soluble anti-Lyt-2mAb, however. These results are discussed in terms of local interactions between TcR and Lyt-2 molecules.     

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.     

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.     

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.     

Xenogeneic monoclonal antibody to an Ly-6-linked murine cell surface antigen: differential reactivity with T cell subpopulations and bone marrow cells

The patterns of cellular and strain reactivity of a monoclonal antibody (6C3 MAb) derived from the fusion of SP2/0 cells with splenocytes from rats immunized against MRL/MpJ-lpr/lpr T cells were characterized by using flow cytofluorometry (FCF) analysis. This MAb was found to stain 70 to 90% of T cells of mice with the lpr/lpr genotype and 20 to 60% of T cells of congenic +/+ strains. Dual-parameter FCF analysis of Lyt-2 vs 6C3 expression revealed the existence of several Lyt-2- and Lyt-2+ T cell subsets, one of which (Lyt-2- bright 6C3+) was expanded in lpr/lpr-bearing mice. The 6C3 MAb stained only 2 to 5% normal thymocytes but reacted with 40 to 50% bone marrow (BM) cells. A strain survey demonstrated the expression of the 6C3 antigen on peripheral T cells (and BM cells) of all strains examined, with the exception of NOD, NZB/B1NJ, and ST/bJ. Interestingly, in the positive strains, two types of 6C3 staining patterns of T cells were observed: bimodal or trimodal. Study of BXH and CXB recombinant inbred (RI) strains demonstrated that the bimodal and trimodal 6C3 patterns are associated with the Ly-6.1 and Ly-6.2 phenotypes, respectively. Linkage of 6C3 expression with the Ly-6 locus was confirmed by using the congenic C3H.B6-Ly-6b strain. Moreover, the 6C3 staining of T cells in Ly-6.2 strains was reduced by preincubation with the H9/25 and SK-142-446 MAb, which are known to recognize Ly-6.2-associated antigens. Therefore, the 6C3 MAb appears to detect a frame-work determinant on an Ly-6-linked antigen that is absent from T cells of NOD, NZB, and ST/bJ mice. Analysis of (NZB x C58) NX8 RI strains demonstrated a correlation between the lack of 6C3 expression on T cells and unresponsiveness in autologous mixed lymphocyte reaction (a property of NZB/B1NJ mice). The 6C3 MAb should prove useful for further genetic and biochemical analysis of the Ly-6 locus and its product(s), and for the delineation of functional subsets of T cells and BM cells in normal and lpr/lpr-bearing mice.     

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.     

Effects of anti-Lyt-2 and anti-L3T4 monoclonal antibodies on the function of cytotoxic T lymphocyte/helper T lymphocyte hybrid T cell clones

CTL/HTL hybrid clones provide a unique system that allows detailed analysis of the role of Lyt-2, L3T4, and other structures involved in T cell functions. We have demonstrated previously that the fusion of cloned murine CTL and helper T lymphocytes with defined specificity generated hybrid cells that expressed both Lyt-2 and L3T4 as well as two TCR. Data obtained with these hybrid clones demonstrated that cytolysis is closely linked to the CTL TCR. We have analyzed the effects of anti-Lyt-2 and anti-L3T4 as well as anti-TCR mAb on cytolysis, proliferation, and lymphokine release by a number of hybrid clones. We found that anti-Lyt-2 and anti-L3T4 mAb were able to inhibit both proliferation and lymphokine release by the hybrid clones in response to stimulation of either the CTL or helper T lymphocyte parent TCR. In contrast, only anti-Lyt-2 and anti-CTL TCR mAb were able to block cytolysis of target cells bearing the Ag recognized by the CTL TCR. These results provide further evidence that cytolysis is closely linked to the CTL TCR and that Lyt-2 and L3T4 have more than a passive role as accessory molecules on the surface of T lymphocytes.     

A new mouse lymphoid alloantigen (Lgp100) recognized by a monoclonal rat antibody

A new genetically polymorphic cell surface antigen recognized by a monoclonal rat anti-mouse antibody is expressed on mouse lymphoid cells. Fluorescence analysis on the fluorescence-activated cell sorter (FACS) locates the antigen on thymocytes, lymph node cells, and both T and B cells in the spleen. It also appears on approximately 40% of cells in the bone marrow.Immune precipitations from surface iodinated spleen cells followed by 2-D gel electrophoresis demonstrate that the antigen is a glycoprotein of approximately 100,000 daltons. Since it is expressed in all lymphoid tissues and on both T and B cells, we designate it lymphoid glycoprotein 100 (Lgp100).Strains with Lgp100 include A/J, AKR/J, AKR/Cu, BALB/c, 129/J, CBA/N, C3H/HeJ, CBA/2J, and SJL/J. Strains with no detectable antigen include C57BL/6J, C57BL/10J, C57BR/cdJ, C57L/J, and C58/J. Intercrosses and backcrosses establish a pair of alleles, a positive and a negative one, at a single locus. Heterozygotes display about 50% as much antigen as homozygotes by quantitative membrane immunofluorescence on the FACS. Tests for Lgp100 in 35 recombinant inbred strains from three crosses (CXB, BXB, and BXH) locate this locus on chromosome 1, closely linked to theMls locus.     

Lyt-2+ suppressor T cells are resistant to anti-Lyt-2 antibody blocking

Lyt-2 molecules play a role in antigen recognition by cytotoxic T lymphocytes (CTL). In an attempt to determine whether Lyt-2 molecules play a similar role in suppressor T cell (Ts) functions, the effect of anti-Lyt-2 antibodies on Ts generation and effector activity was studied. Allospecific Ts were induced in allogeneic mixed lymphocyte cultures (MLC). Anti-Lyt-2 antibodies added to MLC in the absence of complement abolished CTL generation, but had no effect on concomitant induction of Ts. In a different experimental system, allospecific Ts were induced in cultures treated with pyrilamine, which blocks generation of CTL but allows differentiation of Ts. The addition of anti-Lyt-2 antibodies to pyrilamine-treated MLC resulted in unaffected induction of Ts. It was further demonstrated that the effector activity of Ts was as resistant to anti-Lyt-2 antibodies as their induction, in contrast to the cytolytic activity of CTL, which was inhibited by the same antibodies. Ts in the present experimental system were Lyt-2+ antigen-specific cells. It therefore appears that Lyt-2 molecules, although expressed on both CTL and Ts, are involved in CTL activity, but do not play an essential role in Ts function.     

Expression and functional significance of the J11d marker on mouse thymocytes

Subpopulations of thymocytes have been characterized phenotypically and functionally in relation to their expression of the marker defined by the monoclonal antibody J11d. Cortical-type L3T4+, Lyt-2+ thymocytes are all J11d+. Thymocytes that share the phenotype L3T4-, Lyt-2+ with peripheral Lyt-2+ T cells contain a J11d+ and a J11d- subset. These J11d- cells behave like peripheral Lyt-2+ T cells in two functional assays: they form clonal growth bursts in response to immobilized antibody against the T cell antigen receptor, and they act as precursors of alloreactive cytotoxic T cells. The J11d+ cells are inert in both of these assays. In contrast, L3T4+, Lyt-2- thymocytes do not contain a J11d+ subset.     

Serological characterization and partial purification of an Lyt-1 homolog in tunicate hemocytes.

A panel of alloantisera and monoclonal antibodies specific to murine Lyt-1 allotypic and framework determinants was used in indirect immunofluorescence and FACS analysis to investigate the occurrence of an Lyt-1 homolog in tunicate (protochordate) hemocytes. Binding assays and quantitative absorption experiments established the expression of Lyt-1 cross-reacting determinants on a distinct population of tunicate hemocytes. These determinants were expressed exclusively by cells with the morphological characteristics of hemoblasts and lymphocytes. In a rapid two-step purification procedure, Lyt-1 glycoproteins from tunicate hemocytes and C57B1/6 mouse thymocytes were solubilized and partially purified by affinity chromatography using a mAb anti-Lyt-1 frame-work determinant. In both cell types, antigenic activities were associated with a major 67-kDa component. Our findings suggest an early phylogenetic emergence of an Lyt-1 homolog at this level of evolution.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. IV. Comparative analysis of the blocking effects of anti-Lyt-2 and anti-H-2 antibodies on allogeneic and PHA-mediated killing

Blocking of cell-mediated lympholysis (CML) by anti-Lyt-2 antibodies was compared with that by anti-H-2 antibodies which most likely inhibit CML by blocking antigen recognition by cytotoxic T lymphocytes (CTLs). Both antibodies were shown to inhibit the early Mg2+-dependent process of killing. Moreover, the anti-H-2-sensitive event was found to be reversible by the antibody as was the case with the anti-Lyt-2-sensitive event, suggesting that the two antibodies block the same event taking place during the Mg2+-dependent stage. Both types of antibody were also shown to be capable of inhibiting the phytohemagglutinin (PHA)-mediated non-specific killing activity of CTLs. However, in the case of anti-Lyt-2 antibodies, available monoclonal antibodies failed to inhibit PHA-mediated killing whereas conventional antisera did. The results thus suggest multiplicity and heterogeneity of Lyt-2 determinants or the existence of multiple products of Lyt-2-linked genes. In addition, an anti-H-2 antiserum also exerted a specific inhibitory effect on PHA-mediated killing. Thus there appears to be a general requirement for involvement of the Lyt-2 molecules on CTLs and major histocompatibility complex (MHC) products on the target cells. The implications of these observations are discussed.     

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.     

Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.