Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Mls-1a-induced peripheral tolerance to host minor histocompatibility antigens in radiation bone marrow chimeras. Modification of T cell repertoire associated with active suppression and permanent presentation of host antigens.

Minor histocompatibility Ag (mHAg) can be responsible for the development of graft vs host reaction (GVHR) after bone marrow transplantation. In a mouse model, B10.D2 donor immunization against Mls-1a prevents lethal GVHR developed by CD4+ T cells against DBA mHAg in irradiated (DBA/2 x B10.D2)F1 hosts. Such F1 hosts become 100% chimeric and show long term survival (LS mice). The cellular mechanisms underlying the tolerance in LS mice was investigated. It was found that a state of tolerance can be induced in thymectomized F1 hosts. Although spleen cells from LS mice are able to initiate lethal GVHR in third-party H-2k-incompatible hosts, no GVHR is observed in secondary hosts incompatible for specific DBA/2 mHAg. Mixed lymphocyte experiments in vitro confirm that T cells from LS mice are unresponsive toward specific DBA/2 mHAg, although they are able to proliferate in response to H-2 or Mls-1a Ag. The responsiveness to Mls-1a correlates with the presence of V beta 6+ cells in LS mice, probably derived from mature T cells present in the donor inoculum. The tolerance in LS mice is not due to the lack of DBA/2 mHAg presentation; instead, permanent presentation of Ag (Ag I and Ag II) previously described as being responsible for lethal GVHR is consistently observed. A significant protection against GVHR is obtained by transferring normal B10.D2 cells together with spleen cells from LS mice, clearly indicating the contribution of active suppression in the state of tolerance; this is further confirmed by in vitro results obtained in limiting dilution assays. It is concluded that tolerance in chimeric LS mice 1) is due to a peripheral (thymus-independent) mechanism; 2) is specific for mHAg; 3) correlates with unresponsiveness of the repertoire to host mHAg, without alteration of the repertoire for H-2 and Mls-1a Ag; and 4) is associated with an active suppression and with a permanent presentation of at least two mHAg responsible for GVHR mortality.     

Analysis of T hybridomas prepared from a T cell clone with three specificities. Recognition of self + X and allo-H-2 determinants segregates from recognition of Mlsa determinants

To see k information on T cell recognition of Mlsa determinants, hybridomas were prepared from a well-characterized F23.2+ (V beta 8.2+) T cell clone specific for three different ligands, i.e., 1) Mlsa determinants, 2) fowl gamma-globulin (F gamma G) plus self-H-2 (H-2d), and 3) allo-H-2, e.g., H-2p, determinants. Fusion of the clone to the BW5147 thymoma line produced a triple-reactive T hybridoma which generated two types of spontaneous variants. One type of variant (type I) lost Mlsa reactivity but retained reactivity to both F gamma G/H-2d and allo-H-2p. These variants, which were generated at high frequency, stained strongly with a mAb, A1.57, with idiotypic specificity for the TCR molecules of the parental clone. The second type of variant (type II) reacted to Mlsa determinants but showed no reactivity to F gamma G/H-2d or to allo-H-2p. These variants failed to express the A1.57 idiotypic determinants of the parent clone, but were F23.2+ (V beta 8.2+); nonequilibrium pH gradient electrophoresis analysis suggested that these hybrids expressed a mixed TCR heterodimer composed of the parental clone beta-chain and the BW5147 alpha-chain. Three aspects of the data are very difficult to accommodate with the view that Mlsa, F gamma G, and allo-H-2 determinants are all recognized via a common TCR molecule: 1) the independent (and frequent) segregation of Mlsa reactivity from F gamma G/H-2d and allo-H-2p reactivity, 2) the retention of Mlsa reactivity by the type II variants despite loss of the parental clone alpha-chain, and 3) the loss of Mlsa reactivity by the type I variants despite high expression of the A1.57+ TcR molecules derived from the parental clone. The data support a model in which Mlsa determinants are recognized by a separate T cell structure, which we envisage as a monomorphic accessory molecule unrelated to the TCR. Since the type II hybridoma variants invariably retained quantitatively normal TcR expression, the triggering phase of anti-Mlsa responses appears to be TCR dependent. The model we favor is that anti-Mlsa/Mlsa interaction increases TCR binding with Ia epitopes to above the threshold required for cell triggering. A key feature of this model is that Mlsa and Ia determinants are recognized as separate structures rather than as a complex.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

T cell responses to Mls determinants are restricted by cross-reactive MHC determinants

The studies presented here investigated the relationship between T cell recognition of MHC-encoded products and non-MHC-linked Mls determinants. The first aspect addressed whether Mls-reactive T cells recognize Mls-encoded products alone or in association with MHC-encoded determinants. Initial studies used Mlsa-specific T cell clones that were generated by repeated stimulation of C57BL/6 or B10.A(5R) spleen cells with DBA/2 lymphoid cells. These clones recognized Mlsa on cells expressing MHC products of the H-2b, H-2d, and H-2k haplotypes, but not the H-2q haplotype. Thus, these cloned T cells were found to recognize Mlsa products in association with public but demonstrably polymorphic H-2 determinants. The question of whether T cell clones that were specific for self-H-2 determinants (autoreactive) or soluble antigen plus syngeneic H-2 (antigen-specific) could also be stimulated by Mlsa determinants was also addressed. A substantial proportion of the antigen-specific or autoreactive T cell clones tested were stimulated by Mlsa determinants. Furthermore, stimulation of these clones by Mlsa was H-2 restricted. The pattern of H-2-restricted recognition of Mlsa by these clones was not distinguishable from that observed in the Mlsa-specific T cell clones, nor was it influenced by the primary specificity or H-2 restriction pattern of a given clone. Although these findings provide a means of explaining the observation that Mls-reactive T cells exist at extremely high precursor frequencies, they also raise questions regarding the nature of the receptor structures which are used by a single T cell in the recognition of two or more apparently distinct stimuli.     

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.     

Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes

V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.     

Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage.

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.     

Activation requirements of cloned inducer T cells. III. Need for two stimulator cells in the response of a cloned line to Mls determinants

To gain insight into the nature of Mls determinants, we examined the stimulator cells responsible for the activation of inducer T cell clones by Mls determinants. Two types of clones responding to Mls determinants were identified. One type responded to purified B cells, but not to splenic adherent cells (SAC), from mice bearing Mls stimulatory determinants. The other type of Mls-reactive T cell clone, including the representative clone Ly1-N5, demonstrated a vigorous response to unfractionated spleen cells, but showed little or no response to B cells alone or to SAC alone from mice bearing the Mlsa or Mlsd stimulatory determinant. The response of these clones to Mls determinants required stimulation by two cell types. The failure of clone Ly1-N5 to respond to Mlsa-bearing B cells was reversed by the addition of SAC taken from mice bearing the Mlsa allele. In addition, SAC from mice bearing the nonstimulatory Mlsb allele could synergize with B cells from Mlsa-bearing animals. B cells were required to provide the Mlsa determinant, because the combination of Mlsa-bearing SAC and Mlsb-bearing B cells did not activate the clone. The response of clone Ly1-N5 to Mls is restricted by Ia determinants (shared by H-2b, H-2d, and H-2k haplotypes but not by the H-2q haplotype). The permissive H-2 alleles can be present either on the stimulator B cell or on the SAC. The optimal response of the clone was obtained by using B cells bearing Mlsa and the permissive Ia epitopes. However, a significant response of the clone to B cells bearing Mlsa but an inappropriate Ia (Iaq) was also seen in the presence of SAC bearing the nonstimulatory Mlsb allele but the permissive Ia epitopes.     

Effect of Mlsa on antigen presentation to class II-restricted T cells

The nature of the gene products encoded or regulated by the minor lymphocyte-stimulating (Mls) loci remains enigmatic despite extensive experimental evaluation. This work tested the hypothesis that the Mlsa genotype, when compared to the Mlsb genotype, facilitates Ag presentation to class II-restricted T cells. Titrated numbers of H-2-identical, Mls-disparate APC were used to stimulate proliferation of autoreactive, alloreactive, or Ag-specific class II-restricted T cell clones or lines. Apparent preferential presentation by Mlsa vs Mlsb APC obtained from H-2-identical strains was seen infrequently, and when observed, analysis with the use of APC from recombinant inbred lines revealed that preferential presentation did not correlate with the Mls genotype of the APC. These studies show that the Mlsa genotype does not influence overall Ag presentation to class II-restricted T cells.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

Characterization of streptococcal antigen-specific CD8+, MHC class I-restricted, T cell clones that down-regulate in vitro antibody synthesis.

T cell clones were generated from the peripheral blood of rhesus monkeys that had been immunized with a soluble Mr 185,000 Ag (SAI/II) derived from Streptococcus mutans. The clones were CD3+ CD8+ CD4- alpha beta TCR+ and were specifically stimulated to proliferate by SAI/II. The proliferative responses of the cloned cells were class I restricted, as demonstrated by reconstitution of the cloned T cells with APC matched at various MHC class I and II loci, as well as by inhibition with anti-class I and not anti-class II mAb. The function of the CD8+ cloned cells was examined in vitro for their effect on antibody synthesis by Ag-stimulated CD4+ cells and B cells from immunized animals. Indeed, four of the five clones suppressed SAI/II-specific IgG antibody synthesis when activated with SAI/II and the appropriate MHC-matched APC. Although activation of the suppressor clones was Ag specific, the effector function of the suppression of antibody synthesis was Ag nonspecific. The latter was probably mediated by lymphokines and, indeed, the culture supernatant generated by stimulating the cloned CD8+ cells with anti-CD3 mAb suppressed both the specific and nonspecific antibody synthesis. Cytotoxicity studies showed that all five CD8+ clones showed a low level of lectin-dependent cytotoxicity. However, because four of the five clones expressed significant suppression of antibody synthesis, the suppressor activity was unlikely to be a function of the weak cytotoxicity. The results suggest that immunization of rhesus monkeys with a soluble streptococcal Ag induced CD8+ alpha beta TCR+ T cell clones that show SAI/II-specific, MHC class I-restricted proliferative responses and nonspecific down-regulatory function of in vitro antibody synthesis.     

A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.