HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes

The specificity of four cytotoxic T lymphocyte (CTL) clones which recognize class II major histocompatibility complex (MHC) antigens was analyzed. All clones recognized antigens associated with the serologically defined HLA-DRw6 specificity. The activity of two of these clones, JR-2-2 and JR-2-10, could be inhibited by a monoclonal antibody Q 5/13 specific for a monomorphic determinant present on HLA-DR. In contrast, the activity of the two other CTL clones, JR-2-19 and JR-2-26, was not blocked by Q 5/13, but by a new monoclonal reagent, SPV-L3. This latter monoclonal antibody precipitated a two-chain structure of 28 kDa and 33 kDa and reacts with a monomorphic determinant. The molecular weight of the polypeptides precipitated with SPV-L3 was slightly less than those precipitated with a HLA-DR-specific monoclonal reagent. In addition two-dimensional gel electrophoresis showed that the antigen precipitated by SPV-L3 differed in charge from those precipitated with the anti-HLA-DR antibody. These results indicate that SPV-L3 recognizes a class II MHC product different from HLA-DR. This observation was confirmed by partial amino acid sequence analysis of the two chains which revealed that the molecule precipitated by SPV-L3 is homologous to HLA-DC/DS molecules. Therefore this report provides the first evidence that human cytotoxic T cells can recognize HLA-DC/DS antigens.     

Dual overlapping peptides recognized by insulin peptide B:9-23 T cell receptor AV13S3 T cell clones of the NOD mouse

T cells isolated from islets of non-obese diabetic (NOD) mice are enriched for insulin-reactive cells. The great majority of these T cells recognize insulin B chain peptide (B:9-23). B:9-23 reactive T cell clones are diabetogenic and show a dramatic TCR alpha -chain restriction (predominant AV13S3). We have studied the reactivity of five different B:9-23 reactive T cell clones to truncated peptides and alanine substituted analogues of B:9-23. Amongst these AV13S3 T cell clones, one reacted with peptide B:9-16 and four with B:13-23. The two peptides have in common only four amino acids (B:13-16; EALY). Having defined minimal peptide epitopes, we evaluated a mutant insulin sequence (B:13 glutamine) which retains metabolic activity. As predicted, this single amino acid change abrogated T cell reactivity. In addition, we have created a modified I-A(g7)gene with the B:9-23 peptide covalently linked to I-A(g7). Antigen presenting cells transfected with this construct were excellent presenting cells for all clones studied. The definition of dual peptide motifs and creation of bioactive covalent I-A(g7)-B:9-23 should facilitate studies of the pathogenic significance and antigen recognition by B:9-23 reactive diabetogenic T cells.     

Specific inhibition of class II MHC gene expression by anti-sense RNA

We have established an anti-sense RNA system which is capable of regulating expression of the class II (Ia) molecule coded for by the major histocompatibility complex in cultured mouse cells. Various areas of the I-A beta chain gene were subcloned in an anti-sense orientation to the 3' of the dihydrofolate reductase (DHFR) cDNA under the control of the human metallothionein IIa gene promoter. These anti-sense DNA constructs were transfected into M12.4 cells, a BALB/c B lymphoma cell line which expresses both I-A and I-E molecules on the cell surface. I-A expression of selected clones transfected with anti-sense DNA encompassing the 5' untranslated region (UT) (100 or 310 bp) including the translation start site or the poly(A) addition signalling sequence in the 3' UT (250 bp) of the I-A beta chain gene were specifically reduced to less than 5% of the control M12.4 cell surface I-A expression. These clones had normal levels of I-E expression. However, transfection of the anti-sense DNA to the beta 1 domain (510 bp) including the splicing donor and acceptor sequences did not affect the expression of I-A molecules. The same antisense DNA constructs (100 bp of the 5' UT or 250 bp of the 3' UT) without the DHFR cDNA (710 bp) did not down-regulate the expression of I-A molecules, indicating that either the physical length of the anti-sense RNA or specific DHFR cDNA sequences are also important.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen

Injection of native type II collagen (CII) to susceptible strains of mice (H-2^q) induces a rheumatoid arthritis-like disease. To study the role of CD8⁺ T cells in the collagen-induced arthritis (CIA),we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H.Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CH-pulsed macrophages and recognize different antigenic epitopes in association with K^q molecules. When these T cell clones were irradiated and inoculated into (C3H.Q × AKR)F₁ mice 21 days prior to priming with native CII/ complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.     

The MT4 Allodeterminant Is Borne on an HLA-DS Molecule on DR5 Cells

Human HLA-DR molecules have been shown to be structurally homologous to the murine I-E subregion molecules by amino acid sequence analysis. Recent studies have demonstrated the isolation of an I-A subregion-homologous molecule (HLA-DS) from human B-cell lines with the rabbit antiserum RbO3, made against a marmoset I-A-like Ia molecule. Previous work from our laboratory has demonstrated that the DR5 homozygous lymphoblastoid cell line Swei expresses at least two different Ia alpha chains and four different Ia beta chains, which associate to form four distinct human Ia molecules, alpha 1 beta 2, alpha 1 beta 3, alpha 2 beta 1, and alpha 2 beta 4, and that the alpha 2 beta 1 molecule bears the allodeterminants MB3 and MT4. To determine whether the MT4-bearing alpha 2 beta 1 molecule was an HLA-DS molecule, the alpha 2 beta 1 molecule reactive with an anti-MT4 alloserum was compared with the Ia molecule reactive with the rabbit xenoantiserum RbO3 by two-dimensional gel electrophoresis and sequential immunoprecipitation. The alpha 2 chain and the RbO3-reactive alpha chain yielded essentially similar spot patterns. The beta 1-chain spot pattern was a subset of the RbO3-reactive beta-chain spot pattern. Sequential immunoprecipitation indicated that RbO3 removed all molecules reactive with MGH88B. These results indicate that on DR5 cells the allosera-reactive alpha 2 beta 1 molecule, which bears MT4, is an HLA-DS molecule.     

Mechanisms by which HLA-class II molecules protect human B lymphoid tumour cells against NK- and LAK-mediated cytolysis.

We have previously shown that mutant B lymphoblastoid cell lines, totally deficient in expression of human leucocyte antigen (HLA)-class II molecules, but with normal HLA-class I expression, develop enhanced susceptibility to natural killer (NK) and lymphokine-activated killer (LAK) cell lysis. The current investigations were aimed at examining the role of HLA-DR and native peptides occupying the antigen-presenting grooves of HLA-class II molecules in protecting mutants of the same B-lymphoid lines against LAK-mediated lysis. No augmentation in LAK lysis was observed despite using two mutant B-cell lines (9.22.3 and 3.1.0) that lacked HLA-DR. Both these lines expressed HLA-DP and HLA-DQ. However, when using other B-cell lines with point mutations in certain regions of the HLA-DR alpha-chain (78, 80 and 96) significantly increased their susceptibility to LAK lysis despite normal expression of HLA-DR and the other class I and II molecules. Of particular interest was the finding that absence of native peptides in antigen-presenting grooves of all the HLA-class II molecules did not render the mutant B cell (9.5.3) susceptible to LAK lysis. These observations support the concept that there are different NK or LAK clones. Certain LAK clones recognize ''self'' major histocompatibility complex (MHC) antigens (but not the native peptides in their antigen-presenting grooves). Presence of ''self'' MHC antigens inhibits such clones. Conversely, other NK or LAK clones recognize ''non-self'' in the context of MHC antigens. Hence, point mutations at certain specific sites on the MHC molecules or foreign peptides in the antigen-presenting grooves enhances the susceptibility of these cells to LAK clones recognizing ''non-self''.     

Analysis of the fine distribution of thymic epithelial microenvironmental molecules by immuno-electron microscopy

Normal T cell development depends upon an interaction between progenitor cells and their microenvironment. Previously raised monoclonal antibodies to subtypes of human thymic epithelium were used with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which these Mabs bind. Mab MR6, thought to recognize the human IL-4 receptor, shows strong surface labeling of cortical epithelial cells and thymic nurse cells, and very weak staining of thymocytes, medullary macrophages and interdigitating cells. Mab 1st 8.18 labels the surface of Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by these two antibodies are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast Mabs MR10 and 19 recognize Intracellular molecules within subcapsular, perivascular and some medullary epithelium. These molecules may represent soluble material awaiting secretion from the cell; alternatively, they may be internal structural proteins.     

Strong lymphoproliferative suppressive function of PLT clones specific for SB-like antigens

From a total of 37 different priming combinations between donors matched for HLA-A,B, and/or Dw/DR, but mismatched for SB, antigens, T cell clones strongly restimulated with concordance for SB specificities were isolated from only two. Most of the alloproliferative (PLT) clones obtained were restimulated by determinants not correlated with any currently known HLA product. Nonetheless, their stimulation was inhibited by a monoclonal antibody TU 39, which preferentially blocks stimulation by SB-, rather than by Dw/DR-associated determinants. Despite having an OKT4+, OKT-, Leu8- phenotype, and secreting Interleukin-2 after contact with stimulatory cells, these clones strongly suppressed proliferative responses of cloned PLT reagents as well as unprimed lymphocytes in mixed leukocyte cultures. They may thus represent a novel type of immunoregulatory T cell, stimulated by SB-related antigens, which despite their "helper/inducer" phenotype are able directly to suppress lymphoproliferative responses.     

The importance of cross-reactions between species: mouse allo-anti-Ia monoclonal antibodies as a powerful tool to define human Ia subsets

Three mouse allo-anti-Ia monoclonal antibodies recognizing epitopes specific for either I-E gene products (H81.98.21 and H40.315.7 Mabs) or shared between I-E and I-A gene products (H82.246.3 Mab) were used to analyze the human Ia molecules against which they cross-react. The results indicated that in the Ia pool of the human B cell line Raji the three Mabs were able to recognize at least three distinct subsets differing each other both in their alpha as well as in their beta subunits. The comparison with the previously defined human Ia subsets NG1, NGe, and DC1 suggested the following similarities: H81.98.21 specific Ia molecules similar to NG1; H82.246.3 specific Ia molecules similar to NG2, and including probably NG1; H40.315.7 specific molecules similar to DC1 in their alpha subunits and probably heterogeneous in their beta subunits with characteristics of the DC1, NG1, and NG2 subunits altogether. The heterogeneity of the human Ia molecules and the possible similarities with the mouse Ia system were discussed.     

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Interleukin-2 production in response to phytohemagglutinin is not necessarily dependent upon the T3-mediated pathway of T-cell activation

The present study was designed to investigate whether heterogeneity exists in the requirement for T3 molecules in the mechanism of T-cell activation and interleukin-2 (IL-2) release by IL-2-producing T-cell clones. Clones were derived from peripheral blood by a culture system which allows clonal expansion of essentially all T lymphocytes or from mixed lymphocyte culture (MLC) activated T-cell population. IL-2-producing clones were selected and cell aliquots treated with anti-T3 monoclonal antibodies (mAb) to induce modulation of T3 surface molecules. Although stimulation of modulated clones with different anti-T3 mAb did not lead to IL-2 production, 4/39 of these clones produced IL-2 after stimulation with phytohemagglutinin (PHA). The ability of these clones to be activated by PHA could not be explained by incomplete T3 modulation. In addition, two alloreactive clones were isolated from MLC population in which modulation of T3 antigens abrogated the IL-2 production induced by either anti-T2 mAb or allogeneic stimulation but had no effect on PHA-induced IL-2 release. These data further support the concept that PHA may trigger some T cells via surface molecules that are independent from the T3-Ti cell receptor molecular complex.     

CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules

Summary: A multigene family of immunoglobulin superfamily (Ig-SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H-2 products is mediated by members of the murine Ly49 C-type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C-type lectin receptor complex in NK cell-mediated recognition of Bw6+ HLA-B, HLA A and HLA-C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glyco-proteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig-SF receptor system. By contrast. NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94-specific mAbs. Remarkably, a set of Ig-SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94-associated molecules belong to the NKG2 family of C-type lectins; the NKG2-A gene encodes for the p43 subunit. which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.     

Dextran sulfate specifically interacts with the human LFA-1 molecule (leucocyte function associated antigen-1).

We have investigated by flow cytometry the action of dextran sulfate (DxS) on the expression of the LFA-1 molecule in human lymphocytes. This work was undertaken because of the involvement of the LFA-1 molecule in HIV-1 induced syncytia and because of the role of DxS played in the inhibition of syncytia formation. Firstly we detected five distinct topographic regions (epitopes) on the LFA-1 molecule with a panel of 11 monoclonal antibodies (Mabs). Then we demonstrated that DxS interacts with some epitopes mainly present on the alpha chain of the LFA-1 molecule. This inhibition on the LFA-1 expressions by DxS occurs after 1-3 hr of incubation of either 4 or 37 degrees C with complete reappearance of LFA-1 within 1 hr of placing cells in fresh medium. In addition both 5 and 500 kDa have been found to have a similar influence on the inhibition of the LFA-1 expression, while non sulfated dextran have no effect. Other sulfated polyanion (SP) such as heparin and chondroitin sulfate have no effect on the LFA-1 expression. Further at 4 degrees C, DxS does not alter the expression of molecules recognized by Mab such as Leu3a (CD4), Leu2a (CD8), Leu4 (CD3) and Leu5b (CD2). However at 4 degrees C, DxS decreases the expression of CD45R molecule which is recognized by Mab Gap8.3. At 37 degrees C, we observe a decrease also in CD4 expression after DxS exposure. It has also been found that DxS decreases LFA-1 expression to the same extent regardless of the basal expression of LFA-1 in each selected cell subset (LFA-1 low, dim or bright). These results suggest that the inhibitory effect of DxS on the HIV-induced syncytium formation could be due partially to a specific steric hindrance of some LFA-1 determinants.     

Specificity of alloactivated human T lymphocyte clones in secondary proliferation, cell-mediated lympholysis and interleukin-2 release

Alloreactive cytolytic T cell clones were generated from mixed leucocyte reactions (MLRs) between unrelated individuals. Two clones exhibited proliferative responses specific for class I HLA antigens B21 and B17. They were also found to be cytolytic toward cells bearing these HLA-B antigens as measured in cell-mediated lympholysis (CML) assays. Four clones exhibited primed lymphocyte test (PLT) and CML activity specific for various class II HLA antigens, namely DR1, DR5, DQw3 and DRw53. For each of these six clones, the CML specificity was identical to the PLT specificity. Both class I and class II specific clones released interleukin-2 (IL-2) upon restimulation with irradiated cells carrying the relevant HLA specificity. This stimulator-induced IL-2 release showed the same specificity pattern as that observed in the PLT assay. Monoclonal antibody (mAb) inhibition studies on alloreactive T cell clones showed similar inhibition profiles of PLT, CML and IL-2 release assays. These findings suggest that cytolytic activity, secondary proliferation and IL-2 release by alloactivated T cells may be induced by the same HLA encoded determinant.     

B lymphocyte activation upon exclusive recognition of major histocompatibility antigens by T helper cells

This study has investigated whether exclusive recognition of I-A or I-E molecules on the B cell surface by T helper cells is sufficient to activate resting B cells. Lines and clones of long-term-cultured T helper cells with specificity for I-A or I-E antigens have been derived from mixed lymphocyte cultures between spleen cells from major histocompatibility complex (MHC)-congenic mouse strains. These cells were tested for helper activity and proved competent to induce resting B lymphocytes expressing the specific MHC antigens to polyclonal expansion and maturation to Ig secretion. B cell activation was shown to require direct recognition of I-A/E antigens by the helper cells on the responding B lymphocyte surface and it could not be achieved by soluble factors released by "third-party" helper cell activity ongoing in the same cultures. Since B lymphocyte activation occurs in the absence of antigen recognition by the responding B cells, these observations suggest that I-A and I-E molecules expressed on the B cell surface participate in the functional reception of T helper cell-derived induction signals.     

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.     

The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86

The response to tetanus toxoid (TT) was studied in immune donors that carry two alleles of DR5 that differ only at DRβ residue 86: DRB1*1101 (G86, abbreviated 1101) and DRB1*1104 (V86, abbreviated 1104). A large number of TT-specific T cell clones was isolated and the epitopes recognized in association with 1101 and 1104 were mapped. We found that two epitopes (p2 and p32) can be recognized in association with both 1101 and 1104 while three epitopes (p23, p27 and p30) are recognized in association with 1101, but never in association with 1104. The sets of naturally processed self peptides displayed by 1101 and 1104 were characterized using alloreactive T cell clones. We found that all 1104 alloreactive clones recognize both 1104 and 1101, while ?30% of the alloreactive 1101 clones fail to recognize 1104. Thus it is apparent that both naturally processed TTand self peptides displayed on 1104 molecules represent a fraction of those displayed on 1101 molecules. The mechanism responsible for this differential presentation was investigated by comparing the capacity of 1101 and 1104 antigen-presenting cells to present TTor synthetic peptides to specific T cells and by measuring the binding of these peptides to DR molecules. Three sets of results suggest that V86 acts as a constraint to the binding of naturally processed peptides: (i) all 1104-restricted or alloreactive T cell clones recognize TT- or allo-epitopes presented by 1101 as well, thus ruling out a major effect of V86 as a residue determining T cell restriction specificity; (ii) presentation of naturally processed peptides correlates in general with the capacity of long synthetic peptides to bind to 1101 or 1104 and (iii) while the naturally processed p30 epitope discriminates between 1101 and 1104, a short synthetic peptide binds equally well to and is comparably recognized in association with both 1101 and 1104. Taken together these results suggest that the natural polymorphism at residue 86 might be a molecular switch that finely tunes the complexity of the peptide collection presented on DR molecules.     

Allospecific T-cell lines with functional activities.

A T-lymphocyte line and sublines from it reactive with Class II MHC alloantigens have been examined for proliferative responses and helper activity in vitro and in vivo. Both the parent cell line and its derived sublines recognize alloantigenic determinants encoded for by the I-A subregion of the H-2 complex in a proliferative assay, and have regulatory activity for allogeneic and syngeneic B cells in two different helper systems. The in vitro selected cell populations are approximately twenty-fold more active in functional helper assays than populations selected in vivo by priming. Differential analysis of the patterns of proliferative responses of sublines against allogeneic I-A molecules on different backgrounds indicates that one type of selected cells reacts preferentially to MHC molecules presented on the background used for selection.