HLA class II-restricted CD4+ T cell responses directed against influenza viral antigens postinfluenza vaccination

The memory T cell response is polyclonal, with the magnitude and specificity of the response controlled in part by the burst size of T cells expanded from effector/memory precursors. Sensitive assays using HLA class II multimers were used to detect low-frequency Ag-specific T cells directed against influenza viral Ags in subjects immunized with the influenza vaccine. Direct ex vivo tetramer staining of PBMC from five individuals identified frequencies of hemagglutinin (HA) 306-318 tetramer binding CD4(+) T cells in the peripheral blood ranging from 1 in 600 to 1 in 30,000 CD4(+) T cells. These frequencies were validated by counting CFSE(low), tetramer-positive T cells after in vitro expansion. Low frequency of T cells directed to other influenza epitopes, including DRA1*0101/DRB1*0401-restricted matrix protein 60-73, DRA1*0101/DRB1*0101-restricted matrix protein 18-29, DRA1*0101/DRB1*0701-restricted HA 232-244 and DRA1*0101/DRB1*0101-restricted nucleoprotein 206-217 were also determined. T cells which occurred at a frequency as low as 1 in 350,000 could be ascertained by in vitro expansion of precursors. Peripheral HA(306-318)-responsive T cells expanded 2- to 5-fold following influenza vaccination. Examination of phenotypic markers of the HA(306-318)-responsive T cells in the peripheral blood indicated that the majority were CD45RA(-), CD27(+), CD25(-), CD28(+), and CD62L(-), while T cell clones derived from this population were CD45RA(-), CD27(-), CD25(+), CD28(+), and CD62L(-).     

HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-gamma responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8. 7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.     

Biosynthetic intermediates of HLA class II antigens from B lymphoblastoid cell lines

Detergent extracts of B lymphoblastoid cell lines (B-LCL) were subjected to immunoaffinity chromatography and gel filtration to purify HLA class II antigens. Class II antigens purified from B-LCL cultured for 24 hr in 10 microM monensin, in which glycoproteins are trapped in transit through the Golgi apparatus, exist in a large macromolecular complex composed of the alpha- and beta-subunits of class II molecules associated with the invariant (I) chain and a sulfated macromolecule that appears to be a proteoglycan. Gel filtration experiments on Sephacryl S-300 reveal that the complex has a Stokes radius corresponding to a globular protein of approximately 270,000 m.w. Analysis of radiolabeled preparations by two-dimensional gel electrophoresis suggests that the complex contains the alpha-, the beta-, and I chain subunits in a 1:1:1 ratio. Dissociation of the protein components followed by gel filtration of the proteoglycan indicates that the proteoglycan contributes approximately 180,000 m.w. to the complex. These results suggest that the complex contains one copy each of the alpha-, the beta-, and the I subunits associated with a proteoglycan molecule. This complex appears to represent a biosynthetic intermediate in the expression of class II molecules which is induced to accumulate intracellularly by monensin treatment of B-LCL.     

Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens

Substantial CD8(+) T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP(118-126)) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8(+) T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8(+) T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thus, neutrophils not only provide rapid innate defense against infection, but also contribute to shaping the specificity and breadth of the CD8(+) T cell response. In addition, cross-presentation of bacterial Ags from neutrophils may explain how CD8(+) T cell responses are generated against Ags from extracellular bacterial pathogens.     

Molecular models of the interaction between T4 antigen and HLA class II antigens or the LAV virus

HLA class II beta chains contain in their aminoterminal polymorphic domain a highly conserved tetrapeptide (RFDS) also present in protein F encoded by the LAV virus. Homology between this tetrapeptide and the fibronectin cell-attachment site (RGDS) has suggested a role in cell adhesion processes. I propose here that such a structure, that I call adhesiotope, would allow stabilization of intermolecular contacts between molecules present at the surface of interacting cells or viruses. Analysis of a three dimensional model of the T4 antigen suggests that the tetrapeptide RADS is located at the surface of the aminoterminal, immunoglobulin-like domain. A model is proposed in which interaction between the adhesiotopes present in class II antigens (RFDS) and T4 (RADS) is the molecular basis of conjugate formation between antigen presenting cells and T helper lymphocytes. The LAV virus, having the RFDS adhesiotope on its surface, would mimic class II antigens in their interaction with T4 and infect selectively T4 positive cells, resulting in the acquired immune deficiency syndrome.     

Structure and polymorphism of the HLA class II SB light chain genes

The HLA Class II region contains at least three groups of loci, DR, DC and SB, which play an important role in the immune response. The antigens encoded at these loci are heterodimers composed of an alpha and a beta chain. The sequence of a complete Class II beta cDNA clone whose sequence agrees closely with the limited N-terminal protein sequence available for the SB beta chain is reported. In addition the structure and coding sequence of genomic SB beta clones of two different SB haplotypes has been obtained and allows definition of some polymorphic regions. The SB beta gene appears to undergo alternate splicing at its 3' end, resulting in expression of two different intracytoplasmic regions. Partial sequencing of a second non-allelic SB beta-like gene, SX beta, indicates that it is a pseudogene.     

Soluble HLA class I and HLA class II antigens in the blood of HIV infected patients

The levels of HLA, class I, antigen and HLA-DR antigen in the blood sera of HIV-infected persons were determined by the enzyme immunoassay. The levels of soluble antigens HLR-DR and of HLA, class I, in serum samples containing only antibodies to HIV were elevated, respectively, 1.8- and 1.3-fold in comparison with the norm. The level of soluble HLA-DR antigen in samples containing the markers of other infections (HBsAg, antibodies to hepatitis C virus, anti-IgM antibodies to cytomegalovirus) was significantly higher in comparison with samples containing only antibodies to HIV and serum samples from healthy donors (p < 0.05). The concentration of soluble HLA antigen, class I, in the samples containing antibodies to HIV and markers of other infections was also elevated, but the statistically authentic increase in comparison with the normal level was observed only in the presence of the markers of HIV infection, hepatitis C and cytomegalovirus infection.     

T cell alloresponses against HLA-DQ and -DR products involve multiple epitopes on the CD4 molecule. Distinct mechanisms contribute to the inhibition of HLA class II-dependent and -independent T cell responses by antibodies to CD4.

The involvement of the human CD4 molecule in T cell alloresponses to transfected HLA-DQ and -DR Ag was investigated with antibodies to defined CD4 epitopes. Anti-CD4 reagents inhibited T cell proliferation in a dose-dependent manner, and affected responses to HLA-DQ and -DR products equally well. As previously observed for conventional alloresponses, saturating concentrations of CD4 antibodies were required (and sufficient) for substantial blocking of T cell responses to transfected HLA-DQ and -DR products, and antibodies to a wide range of CD4 epitopes were inhibitory. In contrast to these results on class II-dependent T cell proliferation, MHC-independent T cell activation (via CD3 antibodies) was largely resistant to inhibition with the same dose range of CD4 mAb (provided that CD3 and CD4 reagents could not compete for the same class of FcR). This observation validates our conclusion that despite the substantial sequence differences between HLA-DQ and -DR heterodimers, the participation of CD4 epitopes in T cell responses to these molecules is conserved.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.     

Interaction of CD4 with HLA class II antigens and HIV gp120

We have developed a cellular adhesion assay in which B lymphocytes expressing HLA class II antigens form rosettes with COS cells expressing high levels of cell surface CD4 upon transient transfection with a CDM8-CD4 plasmid construct. The assay is specific, quantitative, and overcomes the difficulties encountered with a previously described system using an SV40 viral vector. Rosette formation was inhibited by a series of CD4- and HLA-DR-specific antibodies, as well as by human immunodeficiency virus (HIV) gp 120, and a synthetic peptide derived from part of its binding site for CD4 (amino acid residues 414-434), but not by a variety of other effectors, including several soluble CD4 derivatives. The comparison of this pattern of inhibition with those observed in other systems further emphasizes the great similarity, but incomplete identity, in the CD4 binding sites for HLA class II antigens and HIV gp120, and supports a model in which CD4 is considered as an allosteric servomodulator of T-cell adhesion and function which probably is induced to interact with HLA class II antigens when associated with the Tcr/CD3 complex.     

Autoreactive T cell clones of MHC class II specificities are produced during responses against foreign antigens in man

Although the existence of autoreactive T cells has been widely reported in mice and in guinea pigs, a similar phenomenon is poorly documented in man. Here we report the study of three human autoreactive T cell clones isolated during immunization of HLA-DRw13 donors either against influenza A/Texas virus or against allogeneic cells. These clones are specific for autologous HLA-class II specificities either common to all HLA-DRw13 molecules or restricted to the HLA-DR products specific for the DW19 subtype of HLA-DRw13. They are also cytotoxic and they have the same specificity when tested for lytic activity or in proliferation assays. Furthermore, they are also able to help autologous B cells to polyclonally produce Ig. The possible implication of such clones in regulatory mechanisms involving HLA-class II molecules is discussed.     

T cell recognition of an engineered MHC class I molecule: implications for peptide-independent alloreactivity

Previously, we described H-2K(bW9) (K(bW9)), an engineered variant of the murine MHC class I molecule H-2K(b) (K(b)), devoid of the central anchor ("C") pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of K(b) and K(bW9) are largely nonoverlapping in vivo. On the basis of these observations, we used K(bW9) and K(b) to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of K(bW9) and K(b). Six of six K(b)-specific mAbs, directed against different parts of the molecule, recognized K(bW9) well, albeit at different levels than K(b). Furthermore, K(bW9) readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, K(bW9) mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between K(b) and K(bW9). Interestingly, when K(bW9) was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against K(b) and was specific for peptide-independent MHC epitopes. By contrast, K(b)-induced alloreactive CTLs recognized K(b) in a peptide-specific manner, did not cross-react on K(bW9), and were present at much higher frequencies than those induced by K(bW9). Thus, induction of rare peptide-independent CTLs depended on unique structural features of K(bW9), likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.     

Serological and biological cross-reactivity of class II antigens between mice and humans in antigen-specific T-cell proliferative responses

Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from B10.S(9R) which possess I-As and I-Ek molecules serologically cross-reacting with human Class II antigens. On the contrary, B10.S(9R) T cells primed to PPD were stimulated by PPD-pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.     

Delineation of three subsets of class I human T antigens (HTA) on Molt-4 cells: serologic and regulatory relationship to HLA class I antigens

Three subsets of class I human T antigens (HTA) were serologically identified on the surface of the Molt-4 T lymphoma cell line. The HTA 1 subset is defined by NAI/34, D47, or 10H3.9 cross-reactive m.Ab. and by BL6 m.Ab. The HTA 2 and HTA 3 subsets are defined by M241 and 4A7.6 m.Ab., respectively. We obtained no evidence of any additional HTA subset. The different HTA antigens share only few epitopes with human leukocyte antigens (HLA-A, -B, and -C). Interestingly, these epitopes all belong to the same cluster defined on HLA class I molecules, but differ from one HTA subset to another. These results would therefore suggest that HTA and HLA class I antigens display a limited structural homology, but have a conserved epitopic area whose detailed structure differs for each HTA subset. Furthermore, the cell surface expression of each HTA class I molecule type is differently enhanced by natural interferon (IFN)-alpha or -gamma. This result additionally supports the serologic delineation of HTA subsets, and suggests that the corresponding genes in Molt-4 cells, are subjected to distinct regulations.     

Capping of the surface OKT3 binding molecule prevents the T-cell proliferative response to antigens: evidence that this molecule conveys the activation signal

The human T-lymphocyte receptor for antigen appears to have been localized to a cluster of associated surface glycoprotein molecules, among which is the OKT3 binding molecule. We have tested the hypothesis that selective removal of the OKT3 binding molecule eliminates the cellular immune response to antigens by clearing the surface of the molecule that conveys the activation signal. Enriched T cells were obtained from donors immune to purified protein derivative (PPD), streptokinase-streptodornase (SK-SD), or keyhole limpet hemocyanin (KLH). T-3 molecules were cleared from the cell surface by capping with OKT3 and F(ab')2 goat anti-mouse IgG. Regeneration of surface molecules was prevented by culturing the T-3 capped cells with OKT3 625 ng/ml. The capacity of capped T cells to proliferate in culture with antibody in response to antigens, alloantigens, and the mitogens, PHA and ionophore A23187, was compared to uncapped cells pretreated with media and to capped cells permitted to regenerate the OKT3 binding molecule. T-3 capped cells cultured in the presence of antibody failed to proliferate to antigens or alloantigens. However, T-3 capped cells cocultured with antibody also did not significantly proliferate to PHA, but did respond to A23187. In contrast, both media-treated T cells and cells which had regenerated the OKT3 binding molecule proliferated to mitogens, antigens, or alloantigens. The requirement for the OKT3 binding molecule was determined by utilizing T-1, T-4, and T-8 capped cells. T-1, T-4, or T-8 capped cells cultured in the presence of OKT1, OKT4, or OKT8 proliferated in response to antigens, alloantigens, and mitogens. These results demonstrate that in the absence of the OKT3 binding molecule, antigens, alloantigens, and PHA failed to induce a significant cellular proliferative response. In the absence of this molecule, PHA cannot bind to its carbohydrate moiety, and therefore cannot activate T cells to proliferate. These data support the concept that the molecule binding OKT3 conveys the transmembrane signal resulting in cellular activation.     

Association of HLA class I antigens and HLA class II alleles with vitiligo in a Turkish population

As is the case with many other autoimmune diseases, there is an association between vitiligo and HLA complex. HLA subtypes vary with racial/ethnic background. The purpose of this study was to determine which HLA class I antigens and HLA class II alleles are associated with Turkish vitiligo patients. Forty-one patients with vitiligo and 61 healthy control subjects were typed for HLA class II alleles. Thirty-three out of 41 patients with vitiligo and 100 healthy transplant donors were typed for HLA class I antigens. HLA DNA typing was performed by polymerase chain reaction/sequence specific primer method for class II. HLA typing for class I was performed by serological method. The frequency of HLA DRB1*03 was 0.6340 in patients compared to 0.2950 in controls (P = 0.0014). The frequency of HLA DRB1*04 was found to be 0.6830 in patients compared to 0.2950 in controls (P = 0.00026). The allele HLA DRB1*07 was present in 0.390 of patients compared to 0.0820 of the controls (P = 0.0004). A preventive antigen for the manifestation of vitiligo has not been identified in this study. Our findings suggest that DRB1*03, DRB1*04 and DRB1*07 alleles are genetic markers for general susceptibility to vitiligo in a Turkish population.     

Control of T cell responses to staphylococcal enterotoxins by stimulator cell MHC class II polymorphism

The bacterial toxic mitogens or superantigens are a family of related proteins that elicit potent T cell proliferative responses. These responses require APC that express MHC class II proteins, but they are not MHC restricted and they do not depend on a processing step, presumably because these mitogens bind directly to MHC class II molecules. These mitogens stimulate T cells by interacting in an unknown way with the portion of the TCR encoded by certain V beta gene segments. In this paper, we explore the importance of MHC class II polymorphism in T cell responses to staphylococcal enterotoxins. We find that certain MHC molecules present SEB to V beta 8-bearing T cells far better than others. These data suggest that one route of host defence against bacterial toxic mitogens may be to alter MHC class II molecules so that stimulation is inhibited.