CD28 costimulation regulates FOXP3 in a RelA/NF-κB-dependent mechanism.

The molecular mechanisms whereby CD28 alone or associated with TCR can regulate FOXP3 expression are not understood, although the importance of CD28 as a pivotal regulator of CD4(+) CD25(+) FOXP3(+) T cells is well recognized. We previously demonstrated that unique CD28-induced, NF-κB-dependent signals were sufficient to activate FOXP3 transcription in human CD4(+) CD25(-) T cells; however, the exact mechanisms are currently unknown. In this study, we have identified novel κB-binding sites on FOXP3 gene and demonstrated that CD28 signals mediated FOXP3 trans activation by nuclear translocation of RelA/NF-κB and not of c-Rel. The occupancy of FOXP3 κB-binding sites by RelA dimers that correlated with histone acetylation and recruitment of Pol II were required both to initiate FOXP3 transcription and to control the promoter occupancy by NFAT. Interestingly, knockdown of RelA in CD4(+) CD25(-) T cells stimulated through TCR and CD28 significantly affected FOXP3 expression, confirming that also the transcriptional activation of FOXP3 gene by TCR in the presence of CD28-costimulatory signals is RelA-dependent. In conclusion, these data suggest a new mechanism by which FOXP3 is activated and supports the critical role of CD28 in the regulation of peripheral tolerance.

Differential expression of H-2Dd and H-2Ld histocompatibility antigens.

To determine why the H-2Dd antigen is expressed on the surface of transfected cells at a rate several-fold higher than an analogously transfected H-2Ld molecule, we analyzed both previously described and new H-2 hybrid genes. These genes were constructed by exchanging domains between H-2 genes. Quantitative radioimmunoassay indicates that the region of the H-2Dd molecule responsible for its enhanced expression resides in the polymorphic N domain, the first domain of the mature class I molecule.

Use of DNA-mediated gene transfer to analyze the role of H-2Ld in controlling the specificity of anti-vesicular stomatitis virus cytotoxic T cells.

Mouse thymidine kinase (tk-) C3H L (H-2k) cells transformed by the technique of DNA-mediated gene transfer with the herpes simplex virus tk gene together with the BALB/c H-2Ld gene express H-2Ld molecules indistinguishable from their counterparts on spleen cells. An established cloned cell line (8-5) was used to assess the function of the H-2Ld antigen in determining the specificity of alloreactive as well as anti-vesicular stomatitis virus (VSV) cytotoxic T cells (CTL). Both anti-H-2d and anti-H-2Ld CTL displayed a cytotoxic effect against 8-5 cells but not a control cell line transformed with the tk gene only (tk+ cells). Further evidence that 8-5 cells express H-2Ld was provided by the finding that monoclonal anti-H-2Ld but not H-2Dd antibodies blocked target cell lysis by the effector cells. Both BALB/c (H-2d) and DBA/2 (H-2d) animals generated anti-VSV CTL that lysed infected 8-5 but not tk+ cells. To further establish that H-2Ld controlled the specificity of the effector cells, a monoclonal antibody directed against H-2Ld was shown to inhibit lysis of infected 8-5 target cells. To determine whether other H-2d-encoded gene products could serve as restricting antigens for anti-VSV CTL in BALB/c animals, unlabeled VSV infected 8-5 cells were tested for their ability to block lysis of 51chromium-labeled P815 (H-2d)-infected target cells. The 8-5-VSV inhibitor cells inhibited lysis to a slightly lesser extent than unlabeled P815-VSV cells, indicating that H-2Ld plays a major if not exclusive role in restricting anti-VSV CTL in H-2d animals.

Role of L3T4 in antigen-driven activation of a class I-specific T cell hybridoma.

The expression of L3T4/Lyt-2 on murine T cells has led to the association of these surface markers with recognition of either class II or class I major histo-compatibility complex (MHC) antigens. It has been suggested that these T cell surface antigens interact with nonpolymorphic determinants on MHC antigens. We have examined the role of L3T4 in the recognition of H-2Dd by the T cell hybridoma, 3DT52.5. Mouse L cells transfected with either the H-2Dd gene, or with both the alpha and beta genes of I-Ak and the H-2Dd gene have been used to assess the role of an L3T4/la interaction at varying doses of H-2Dd. A role of L3T4 in activation of 3DT52.5 becomes evident only at limiting doses of antigen. It appears that an L3T4/la interaction can influence T cell function during suboptimal stimulation, implying that the L3T4/la interaction serves to raise the functional affinity of interaction between the T cell and the antigen-bearing cell.

Expression and T cell recognition of hybrid antigens with amino-terminal domains encoded by Qa-2 region of major histocompatibility complex and carboxyl termini of transplantation antigens.

Coding potential of the Q6 gene from the Qa-2a region of BALB/c Crgl mice was analyzed by a combination of hybrid class I gene construction and DNA-mediated gene transfer. Recombinant genes were created by exon shuffling of the 5' coding region of the Q6 gene and the 3' coding region of a gene encoding a transplantation antigen (Kd, Dd, or Ld), or the inverse. Some of these hybrid class I genes were expressed in the transfected mouse fibroblasts (L cells). The hybrid class I molecules encoded by the 5' end of the Q6 gene and the 3' end of the Ld gene precipitated as 45,000 mol wt molecules associated with beta 2-microglobulin. The expression of the hybrid proteins indicates that 926 basepairs of the 5' flanking region upstream of the structural Q6 gene contain a promoter that functions as a transcription initiation site in L cells. The 3' portion of the Q6 gene appears to be responsible for the lack of cell surface expression of the intact Q6 and the hybrid Ld/Q6 genes in mouse fibroblasts. Accordingly, this portion of the Q6 class I gene may play a regulatory role in tissue-specific expression. Serological analyses of hybrid Q6 proteins suggested that Q6 may be a structural gene for CR (H-2 crossreactive) antigen found normally on subpopulations of lymphocytes. If this identification is correct, Q6 gene will define a new category of class I genes encoding approximately 40,000 mol wt molecules and carrying a characteristic truncated cytoplasmic tail. Analysis of L cells transfected with Q6 hybrid genes demonstrated also that the cytotoxic T cells specific for Qa-2a region-coded antigens recognize the amino-terminal alpha 1-alpha 2 domain of Q6 fusion products. This recognition can be blocked by anti-Qa-2a alloantiserum and monoclonal antibodies reactive with the alpha 3-beta 2-microglobulin portion of the Q6 hybrids. We propose that the structural requirements for the anti-Qa-2a cytotoxic T lymphocyte-specific epitopes on target molecules are the same as for anti-H-2-alloreactive cytotoxic T lymphocyte determinants on transplantation antigens and that the mechanism of target recognition is similar in both cases. This interpretation is consistent with the following structural similarities found in both categories of class I molecules: (a) Kd and Q6 alpha 1-alpha 2 domains share serologically defined epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells.

Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.

Humoral immunostimulation. V. Selection of variant cell lines.

A permanent L-cell variant cell line (LC1) was isolated by the growth of the parent L-cell line (L) in the presence of a cytostimulatory dose (1:200) of rabbit anti-L-cell antiserum (AL) for 9 mo. LC1 differed from L in many aspects: (a) it was larger (1,533 mm3 vs. 1,284 mm3), (b) it grew faster (1.5- to 2-fold), (c) it grew in aggregated fashion, (d) its growth was no longer stimulated by AL, (e) it was almost completely resistant to high concentrations of AL in the presence of complement (C), (f) its original membrane antigens (immunogenic for AL) were redistributed in sparse and patchy clumps as noted by fluorescence microscopy, (g) it contained about 65% of the total original 125I-AL membrane-binding sites (1.4 X 10(7)/cell vs. 2.2 X 10(7)/cell), (h) its AL-binding sites displayed a lower average affinity constant (K = 0.9 X 10(5) M-1 vs. 2.8 X 10(5) M-1), (i) it contained a smaller proportion of high affinity (K greater than 10(6) M-1) binding sites (13% vs 21%), and (j) LC1 was fully immunogenic in that it was readily killed by homologous antiserum (ALC1) and C, whereas L was not similarly affected by ALC1 indicating that LC1 contained new membrane antigens not present on L. Another variant (LC2) was produced by growth of LC1 in a 10-fold higher dose (1:20) of AL (cytotoxic for L) for 1 mo. LC2 was even more resistant to AL in the presence of C, contained 0.84 X 10(7) AL-binding sites/cell with an average affinity constant of 1 X 10(5) M-1 (unchanged from LC1), and was less susceptible than LC1 to lysis in the presence of ALC1 and C. These findings confirm and extend our previous in vitro and in vivo observations dealing with the direct stimulation effects of antibody on tumor cell metabolism and suggest that immunostimulation may be a mechanism of tumor escape from immune control in vivo possibly by immunoselection and antigenic modulation as proposed by other investigators.

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.

Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.

Dissection of serological and cytolytic T lymphocyte epitopes on murine major histocompatibility antigens by a recombinant H-2 gene separating the first two external domains.

A novel H-2 gene in which the first external (N) domain of the H-2Ld antigen was replaced with that of the H-2Dd antigen was constructed and introduced into L cells. A transformant expressing the products of the hybrid gene was studied for binding to monoclonal antibodies specific for H-2Ld and H-2Dd antigens. It was found that serological determinants are distributed both in the N (Dd) and Cl (Ld) domains. Determinants recognized by allospecific cytotoxic T lymphocytes (CTLs) and virus-specific CTLs also mapped to the N and Cl domains. Determinants recognized by vesicular stomatitis virus (VSV)-specific effect cells, however, were not present on the recombinant molecule. These results show that a recombinant gene of two H-2 antigens in which the first external domain has been reshuffled can express a functional H-2 antigen that can then be used to map serological and CTL determinants to specific domains.

Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens.

Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.

Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.

Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.

The T cell receptor repertoire influences V beta element usage in response to myoglobin.

T cell clones recognizing the sperm whale myoglobin (SpWMb) epitope 110-121 in association with H-2d major histocompatibility complex class II molecules display a very limited heterogeneity of T cell receptor (TCR) V beta usage in DBA/2 mice. All clones previously tested used the same V beta 8.2 gene segment and very restricted junctional regions. To investigate the significance of this observation in vivo, we immunized DBA/2 mice with the intact SpW Mb protein or peptide 110-121. Only the V beta 8+ T cells showed any significant response to the 110-121 epitope. The response to peptide 110-121 was then analyzed in mice which, either as a consequence of antibody depletion or through genetic deletion of TCR V beta genes, lacked V beta 8+ peripheral T cells. DBA/2 mice depleted of V beta 8+ T cells by antibody treatment responded poorly to the 110-121 peptide, and only at high antigen concentrations. In contrast, DBA/2V beta a mice (homozygous for a deletion of multiple V beta gene segments including the V beta 8 family) made a response at least as great as that made by DBA/2 mice, even though the DBA/2V beta a mice had a very restricted TCR V beta repertoire compared with DBA/2 mice. Mechanisms which might determine differences in the 110-121 specific response of DBA/2, DBA/2V beta a and F23.1-treated DBA/2 mice are discussed.

Humoral immunostimulation. IV. Role of complement.

When L cells were treated with anti-L-cell antibody in medium containing heat-inactivated fetal calf serum, nucleoside uptake and cell growth were stimulated. The response was markedly increased when fresh, unheated sera from calves, guinea pigs, humans, mice, or rabbits were also present. The factors in unheated serum responsible for the enhancement of immunostimulation were studied. Using low concentrations of sera deficient in various complement (C) components and low concentrations of antibody no augmentation of immunostimulation was seen with Clr-deficient human serum, C2-deficient human serum, C2,4-deficient human serum, C4-deficient guinea pig serum, C3-C9-depleted guinea pig serum (by administration of cobra venom factor to animals), but stimulation was observed with C5-deficient human serum, C5-deficient mouse serum, and C6-deficient rabbit serum. When the concentration of anti-serum was raised, however, augmentation was observed with C4-deficient guinea pig serum. Thus, at low concentrations of antiserum enhancement appeared to occur through the classical C pathway, whereas at high concentrations of antibody either the classical or alternate C pathways appeared to be involved. Stimulation was specifically restored by purified C2 in C2-deficient serum and by C3 in C3-C9-deficient serum. Under the usual reaction conditions consumption of guinea pig C component C4 could be demonstrated which provided direct evidence for activation of the classical C pathway under conditions leading to immunostimulation. By immunofluorescence, cells treated with antibody and normal human serum had human C3 deposited at the cell surface. Taken together these observations suggest that C activated through C3 by either the classical or alternate pathways has the potential to enhance nucleoside incorporation into DNA and cell growth of cells exposed to limiting amounts of antibody. Although the mechanism of stimulation is unknown, it is likely to involve a direct effect of C3 at the level of the cell membrane.

Major histocompatibility complex-restricted antigen receptor on T cells. VIII. Role of the LFA-1 molecule.

We show that the LFA-1 molecule on T cells does not play a role in the stimulation of T cell hybridomas by certain targets, namely antigen presented by L cell derivatives or polyvalent anti-receptor antibody. These results suggest that LFA-1 may act by binding to ligands that are not present on all cells. We hope this result will help us and others to establish the true role of LFA-1 in T cell responses.

Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients.

We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.

The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain.

The murine pre-B cell-specific genes VpreB and lambda 5, as well as the murine gene for mu heavy chain, were introduced into Ltk- fibroblast cells which normally do not express these genes. Stable transfectants carrying these genes produced the corresponding proteins of 15.5, 21.5, and 75 kD. They secreted the three proteins as a triple complex that could be immunoprecipitated by mu heavy chain-specific antibodies, consisting of one VpreB, one lambda 5, and one mu heavy chain. The mu heavy chain and lambda 5 were disulfide-bonded with each other, while the VpreB protein was noncovalently associated. These experiments proved that the VpreB, lambda 5 and mu H chain proteins can form a heavy/light chain-like heterocomplex.

Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells.

The TL region of the major histocompatibility complex of the mouse contains dozens of tandemly arranged class I genes, including those encoding the thymus leukemia (TL) antigens. TL antigens have been thought to be expressed only on the surface of some T lineage cells, namely immature thymocytes of some mouse strains (TL+ strains), some leukemia cells, and activated T cells. While the function of TL antigens is unknown, recent studies have implicated the products of at least some TL region class I genes as molecules that present antigens to gamma/delta T cells. Since some gamma/delta T cells are known to be specifically associated with certain epithelial tissues, we have investigated the expression of some TL region class I genes in a variety of epithelium-containing tissues. Our results show that the TL antigen gene of C57BL/6 mice, T3b, and the TL antigen genes of BALB/c mice, T3d (previously T3c) and T18d (previously T13c), are highly expressed in the epithelium of the small intestine. In the case of T3b, we further show, using a T3 product-specific antibody, that its product is expressed on the surface of the columnar epithelial cells. In addition, we demonstrated that two other TL region class I genes of C57BL/6 origin, T9b and T21b, are also expressed nearly exclusively in intestinal epithelial cells. These results are consistent with the hypothesis that the products of these TL region class I genes are recognized by gamma/delta T cell receptors of intestinal intraepithelial lymphocytes, a subset of gamma/delta T cells that is localized in the intestinal epithelium and has a restricted V gamma repertoire. Finally, our study indicates that the relative levels of expression of the two homologous TL antigen genes, T3d and T18d, differ widely between the thymus and the intestine.

The role of L3T4 in recognition of Ia by a cytotoxic, H-2Dd-specific T cell hybridoma.

The expression of T4/T8 surface markers on human T cells and of L3T4/Lyt-2 on murine T cells has lead to the association of these surface markers with recognition of either class II or class I major histocompatibility complex (MHC) antigens. It has been suggested that these T cell surface antigens interact with MHC antigens. We have examined the role of L3T4 in the recognition of Dd by the T cell hybridoma, 3DT52.5. This T cell hybridoma was found to be specific for the N/Cl domain of Dd. The recognition of a class I antigen by an Lyt-2-, L3T4+ T cell hybridoma allowed the separate evaluation of interactions between L3T4/Ia and the T cell antigen receptor, Dd. Recognition by this hybridoma resulted in the production of interleukin 2 (IL-2) and cytolytic activity. Antibody blocking experiments have demonstrated that L3T4 was involved in triggering the effector function of 3DT52.5 only on Ia+ stimulator or target cells. We have demonstrated that an L3T4+, Dd-specific T cell hybridoma, 3DT52.5, uses the L3T4 molecule to directly interact with nonpolymorphic Ia determinants.

Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes.

To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.

Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells.

A gene coding for the major histocompatibility antigen HLA-A2 was transferred into human HLA-A2 negative M1 cells and murine L cells. Following transfection, these cells expressed molecules at the cell surface that are biochemically indistinguishable from HLA-A2 antigens on the human cell line JY from which the HLA-A2 gene was isolated. The M1A2 cells were recognized and lysed by a cytolytic T-cell clone specific for HLA-A2. The transfected L cells which express HLA-A2 in association with human beta 2-microglobulin were not lysed by this T-cell clone. The specific cytolysis of M1A2 cells could be inhibited by monoclonal antibodies to HLA-A2, and monoclonal antibodies to T3, T8, and LFA-1 on cytotoxic T lymphocytes. These results suggest that killing by allospecific T cells requires HLA-A2 antigens as well as other species-specific structures on the target cell surface.