Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4(+) T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20. DR3.Ab0 mice, immunized with bacillus Calmette-Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1-20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guérin but not to p1-20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.     

Processing of endogenously synthesized hen egg-white lysozyme retained in the endoplasmic reticulum or in secretory form gives rise to a similar but not identical set of epitopes recognized by class II-restricted T cells

To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.     

CD40-mediated stimulation contributes to lymphocyte proliferation, antibody production, eosinophilia, and mastocytosis during an in vivo type 2 response, but is not required for T cell IL-4 production

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.     

Somatically mutated B cell pool provides precursors for insulin antibodies

Antibodies to insulin are products of autoreactive B lymphocytes that escape inactivation or clonal deletion and are examples of "clonal ignorance." To understand the genetic origin of Abs from clonally ignorant B cells, the roles of somatic mutation and germ-line V(H) structures were examined for two murine IgG1 mAb that bind human and rodent insulin. Engineered mAb constructs that express germ-line or mutated V(H) genes show that somatic mutations introducing aspartic acid in or adjacent to CDRH2 play a key role in insulin binding. When either of the two anti-insulin V(H) regions is returned to its germ-line (unmutated) sequence, neither mAb binds insulin and the germ-line-encoded mAb are not polyreactive. Reconstruction of the somatic evolution of insulin binding in both mAbs shows that a single mutation in CDRH2 is sufficient to generate anti-insulin activity from a nonbinding precursor. When the role of somatic mutation in the binding of rodent insulin is examined, autoreactivity is associated with single mutations in both Abs. Together these findings indicate that, despite a low mutation frequency, IgG insulin Abs may not be derived directly from germ-line (unmutated) precursors. The requirement for somatic mutation as a prerequisite for measurable insulin binding suggests these Abs have their origin in a previously mutated B cell pool as a consequence of the individual's immune history. Low avidity interaction with endogenous insulin may play a role in selection of these B cells and contribute to the origin of clonal ignorance.     

Dendritic cells and resting B cells form clusters in vitro and in vivo: T cell independence, partial LFA-1 dependence, and regulation by cross-linking surface molecules

Initiation of an Ab response requires interaction between dendritic cells (DC), T cells, and B cells in a T cell area. We demonstrate that rat DC and B cells form T cell-independent clusters in vitro and in vivo. In vitro clusters form within 1 h and dissociate within 24 to 48 h. Clustering is restricted to resting B cells, is energy, cytoskeleton, and protein kinase C dependent, and is inhibited by anti-LFA-1 but not anti-ICAM-1 mAbs. Spleen and lymph node B cells cluster more strongly than those from lymph or blood, suggesting up-regulation of adhesiveness during transendothelial migration. Bone marrow B cells do not form clusters. DC from spleen and lymph nodes show the most clustering, lymph-borne DC are intermediate, and DC from lamina propria, Peyer's patches, and those grown from bone marrow form the fewest clusters. Clustering is stimulated by cross-linking MHC class II (whole mAb or F(ab')2) on DC or B cells or Thy-1 on DC, but not MHC class I, CD45, or CD44. Stimulation by mAb is energy, cytoskeletal, and protein kinase C dependent, but is not inhibited by anti-LFA-1 mAbs, suggesting involvement of other, unidentified adhesion molecules. We suggest that interactions between DC and B cells will occur regularly during B cell recirculation. Cross-linking of MHC class II-peptide molecules on DC by specific T cells would increase binding avidity, causing retention of Ag-specific B cells on DC long enough for the B cells to process Ag, thereby facilitating cognate interactions between T and B cells.     

Definition of the mitogenic factor (MF) as a novel streptococcal superantigen that is different from streptococcal pyrogenic exotoxins A, B, and C

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA-DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA-DR or HLA-DQ molecule. Recombinant MF selectively stimulated V beta 2, V beta 7, V beta 8, V beta 18 and V beta 21-bearing T cells, whereas rSPEA and rSPEC activated V beta 2 and V beta 6-bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR V beta in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR V beta required for rMF-mediated T cell activation are distinct from those required for rSPEA or rSPEC-mediated activation. Therefore, the MF is a novel streptococcal super-antigen which is different from SPEA, SPEB, and SPEC.     

Negative signaling by anti-HLA class I antibodies is dependent upon two triggering events

mAb with specificity for the alpha3 domain of HLA class I antigens, such as mAb TP25.99 and W6/32, are capable of inhibiting the proliferation of stimulated T cells in vitro by binding to their surface HLA class I antigens. The inhibitory potential of another HLA class I alpha3 domain-specific mAb, A1.4, was evaluated. In contrast to mAb TP25.99 and W6/32, which routinely inhibited superantigen (SEB) stimulation of T cells by >90%, mAb A1.4 at equivalent concentrations demonstrated only 20-50% inhibition. Univalent Fab fragments of all three mAb lacked inhibitory activity. Interestingly, however, by combining univalent W6/32 (or TP25.99) Fab fragments with intact, bivalent mAb A1.4 (at a non-inhibitory, sub-threshold concentration of 1 microg/ml), significant inhibition of SEB-driven T cell proliferation was obtained. Inhibition by the anti-HLA class I mAb W6/32 and TP25.99 was evident even when SEB was used in conjunction with paraformaldehyde-fixed HLA class I-, class II+ Daudi cells, suggesting that the inhibitory activity of these mAb results from direct HLA class I epitope engagement on the T cell. These findings suggest that effective antibody-mediated induction of the HLA class I inhibitory pathway within T cells is dependent upon two separable molecular triggers at the T cell surface. The first can be delivered by univalent mAb derivatives that engage one or more critical HLA class I epitope(s). The second requires intact mAb, though seems to be less selective as to the HLA class I specificity. This model may explain why some, but not all, anti-HLA class I mAb are inhibitory when used singly. Achieving synergies between a wider array of anti-HLA class I mAb and their derivatives may provide a path for more effectively tapping into the HLA class I inhibitory pathway in a therapeutic context.     

Limited diversity of human scFv fragments isolated by panning a synthetic phage-display scFv library with cultured human melanoma cells

To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.     

A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.     

The rate of dissociation between antibody and antigen determines the efficiency of antibody-mediated antigen presentation to T cells

We analyzed the role of Ab affinity on Ab-mediated Ag uptake and presentation to T cells. Hen egg white lysozyme (HEL) was captured by bifunctional hybrid proteins (Fv-MalE) in which the variable fragment (Fv) of the anti-HEL mAb D1.3 was covalently linked to the Escherichia coli MalE protein. These complexes were targeted via two anti-MalE mAbs to an APC expressing a receptor for the Ab constant region. The combination of Fv-MalE and anti-MalE mAbs increased, specifically, HEL presentation. With this experimental system, we evaluated the impact of six different mutations, affecting the Fv-MalE complementarity determining regions, on the increase of HEL presentation by the corresponding hybrids. These mutations increase the dissociation rate constant (k(off)), and, thus, the dissociation constant of the HEL/Fv-MalE interaction, up to 650-fold, as compared with the wt Fv-MalE. Increasing the k(off) from 7 x 10(-4) s(-1) up to 300 x 10(-4) s(-1) did not interfere with the enhancement of HEL presentation. A mutant with a k(off) of 600 x 10(-4) s(-1) had a reduced enhancement ability, and mutants with k(off) higher than 5700 x 10(-4) s(-1) did not enhance HEL presentation at all. These results show that affinity determines the efficiency of Ab-mediated Ag presentation to T cells. One consequence is that affinity maturation in specific B lymphocytes can drastically enhance their ability to collaborate with T cells in an MHC-restricted way. This may contribute to the selection of high affinity B cell clones.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. V. In vivo expression of individual antibody clones is dependent on Ig CH haplotypes and the categories of antigen

Antibodies (Ab) to the polysaccharide capsule of Haemophilus influenzae type b (Hib-PS) provide protection against Haemophilus influenzae type b disease in children, and Hib-PS vaccines with different immunologic properties are widely used clinically. The repertoire of human anti-Hib-PS Ab induced by these vaccines is relatively restricted and can be divided into two types by the structure of the light chain V region. Ab using A2-V kappa II gene product, which account for the majority of anti-Hib-PS Ab response in most patients, show little somatic mutations. In contrast, non-Ab using A2-V kappa II gene product use VL genes from the V kappa I, V kappa II, V kappa III, V kappa IV, and V lambda subgroups, are variably expressed among patients, and contain somatic mutations. To further study the expression of these two types of anti-Hib-PS Ab, we have produced KB13, a mAb specific for V kappa II subgroup, and used mAb specific for various other VL subgroups to develop immunoassays specific for anti-Hib-PS Ab of each VL subgroup. When Ig allotypes were studied for the effect on the Ab repertoire, A2-V kappa II (A2) Ab were found to be expressed less in patients expressing fb or zag CH haplotypes (p < 0.05). When the T cell-independent Hib-PS carbohydrate vaccine was compared to two T cell-dependent Hib-PS protein conjugate vaccines for their effect on Ab repertoire, Ab using V kappa III VL were found to be more often elicited with the conjugate vaccines than with the Hib-PS carbohydrate vaccine (p < 0.01). Thus, individual members of the anti-Hib-PS Ab repertoire differ not only in their V region structure but also in the control of their expression.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.     

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.     

An Epstein-Barr virus-associated superantigen

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.     

Early anti-nucleosome autoantibodies from a single MRL+/+ mouse: fine specificity, V gene structure and pathogenicity

In systemic lupus erythematosus, the nucleosome assumes a central role in the autoimmune response to self antigens. To gain insight into the etiology and pathogenesis of anti-nucleosome antibodies (Ab), we analyzed a panel of six IgG-secreting hybridomas derived from a single young MRL +/+ mouse at the onset of the autoimmune response. All monoclonal antibodies (mAb) bound exclusively the native nucleosome, and represented five different clonotypes that recognized diverse nucleosomal epitopes, typical of a polyclonal response. The VH-complementarity-determining region (CDR)3 regions exhibited unique stretches of charged amino acids with different polarity that may be important for the interaction with the nucleosome. These early anti-nucleosome mAb displayed striking structural differences with not only anti-DNA, but also with anti-nucleosome Ab, that appear later in disease. Two of the mAb deposited in kidney glomeruli after in vivo administration to RAG-1-deficient mice, suggesting that diverse B cell clones, possibly selected by the nucleosome itself, may play a role in the initiation of kidney damage.     

Intersite helper function of T cells specific for a protein epitope that is not recognized by antibodies

Humoral responses to a protein require T-B cell communication for B cell activation by T cells. Previous studies from this laboratory have mapped the T and B cell recognition sites (epitopes) on sperm-whale myoglobin (Mb) and several other proteins. It was found that, five of six regions on Mb recognized by T cells are also recognized by B cells (i.e. antibodies). There is, however, one region (E6) residing within Mb residues 61-77, that is recognized only by T cells and to which no antibody (Ab) responses are detectable. To investigate the function of this exclusive T cell epitope, we established, from E6-primed BALB/c mice, an E6-specific T cell line (T(e6)) which comprised Th2-type cells. These T cells provided help in vitro to B cells from Mb-primed BALB/c mice and activated them to produce anti-Mb Abs of the IgM (58.2%) and IgG (41.8%) isotypes. The helper activity of T(e6) cells was dependent on the concentration of the challenging Ag (intact Mb or peptide E6) in culture. Action of soluble factors released from E6-activated T(e6) cells on B(mb) cells led to low production of anti-Mb Abs, suggesting that activation of the B cells was more dependent on their contact with T cells. Mapping of the epitope recognition of the anti-Mb Abs produced in vitro by B(mb) cells on activation by T(e6) revealed that this activation was not general to all antigenic regions recognized by anti-Mb Abs in BALB/c mice. E6-specific T cells caused in vitro activation and differentiation of B(mb) cells into plasma cells that secreted anti-Mb Abs directed, in decreasing order, against the following Mb regions: E4 (107-120) > E3 (87 - 100) > E1 (10 - 22). Little or no Ab responses could be detected against peptides E2 (50 - 62), E5 (141 - 153) and E6 (61 - 77). With B cells of peptide-primed BALB/c mice, T(e6) cells activated strongly E4-, E3- or E1, and only very slightly E2- or E6-, primed B cells to secrete Abs against the correlate peptide, but failed completely to activate E5-primed B cells. The results show that a protein T cell epitope, to which no Abs are detectable, plays an active role in B cell responses against other epitopes within the same protein.     

Staphylococcal enterotoxin B-activated T cells can be redirected to inhibit multicycle virus replication

Cell-mediated immunity is a crucial part of recovery from virus infections. Adoptive transfer of T cells into infected animals is restricted by the need for Ag-specific and MHC-restricted T cells. One way to overcome these limitations is to use bifunctional Abs to redirect the T cells against virus-infected cells. We have demonstrated that bifunctional Abs can inhibit virus replication in the presence of activated T cells. To generate a large number of activated T cells in a short time, we tested the ability of the superantigen, staphylococcal enterotoxin B (SEB), to activate T cells. We demonstrate that SEB-activated T cells are effective killers when bridged to Fc receptor-bearing target cells using anti-CD3 Abs. SEB T cells can lyse virus-infected target cells in the presence of HHA6, a bifunctional Ab specific for the V beta 8 TCR product and the H1 hemagglutinin of influenza A/PR/8/34 virus. In addition, bifunctional Ab and SEB T cells can inhibit multicycle virus replication in vitro. In a conventional 4-h chromium release assay, SEB-activated CD8 T cells are efficient killers, whereas CD4 T cells are not. Yet both subpopulations have the ability to inhibit multicycle replication in vitro. Superantigens may represent a potent method for generation of effector cells for use in redirected immunotherapy protocols.     

Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand

Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag-nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag-specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC-mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells.     

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.     

Major histocompatibility complex-specific prolongation of murine skin and cardiac allograft survival after in vivo depletion of V beta+ T cells

The preferential usage of certain T cell receptor (TCR) V beta genes has been well established in several major histocompatibility complex (MHC)-restricted immune responses. However, V beta usage among allogeneic responses remains unclear. Because recent findings of ours and others indicate that V beta 8 predominates in certain Ld-restricted, peptide-specific responses, we examined the V beta 8 usage in allogeneic responses to Ld. To selectively recognize the Ld molecule, cells from BALB/c-H-2dm2 (dm2), the Ld-loss mutant mouse, were stimulated in vitro or in vivo with wild-type BALB/c cells. We report here that after the intraperitoneal administration of the anti-V beta 8 monoclonal antibody (mAb) F23.1, peripheral V beta 8 T cells were depleted from dm2 mice. This in vivo depletion abrogated the ability of dm2 splenocytes to mount a primary response to Ld molecules. This abrogation was specific, since the response of V beta 8-depleted dm2 cells to Kb/Db antigens was the same as that of control nondepleted dm2 cells. Furthermore, in vivo depletion of V beta 8 cells was found to cause a dramatic prolongation of Ld-disparate skin grafts (mean survival time [MST] 22.1 +/- 2.1 vs. 10.3 +/- 1.1 d for saline-treated controls, or 10.9 +/- 1.7 d for controls treated with mAb KJ23 to V beta 17). By contrast, V beta 8 depletion had no effect on recipients grafted with haplotype-mismatched skin or single Dk-locus-disparate skin. These findings demonstrate that V beta 8+ T cells predominate in allogeneic response to Ld but not other alloantigens. The effect of V beta 8 depletion was found to be even more dramatic on recipients grafted with Ld-disparate vascularized heart transplants (MST > 100 vs. 8.6 +/- 0.5 d for controls). In total, these findings establish the efficacy of using mAb to the V beta gene family to specifically and significantly enhance the survival of allografts. The implications of detecting V beta 8 usage in both alloreactive or MHC-restricted TCR responses to the same class I molecule are discussed.