Identification of T cell ligands in a library of peptides covalently attached to HLA-DR4

While T cells have been clearly implicated in a number of disease processes including autoimmunity, graft rejection, and atypical immune responses, the precise Ags recognized by the pathogenic T cells have often been difficult to identify. This has particularly been true for MHC class II-restricted CD4+ T cells. Although such cells can be demonstrated to have undergone clonal expansion at sites of pathology, they are frequently difficult to establish as stable T cell clones. Furthermore, in general, larger peptides in higher concentrations are required to stimulate CD4+ T cells than CD8+ T cells, which makes some of the techniques developed to identify CD8+ T cell Ags impractical. To circumvent some of these problems, we developed a model system consisting of two parts. The first part involves the construction of an indicator T cell hybridoma expressing a chimeric TCR comprised of murine constant regions and human variable regions specific for influenza hemagglutinin 307-319 presented by DR4. The second part consists of a library of fibroblasts each expressing multiple peptides as amino terminal covalent extensions of the beta-chain of HLA-DR4 (DRA1*0101, DRB1*0401). Using this model system, we screened approximately 100, 000 peptides and identified three novel peptides stimulatory for the HA1.7 TCR. While there is some convergence at residues known to be important for T cell recognition, all three peptides differ markedly from each other and bear little resemblance to wild-type hemagglutinin 307-319.     

Fine specificity of murine class II-restricted T cell clones for synthetic peptides of influenza virus hemagglutinin. Heterogeneity of antigen interaction with the T cell and the Ia molecule

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.     

Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor.

Autoimmune T cell lines specific for muscle nicotinic acetylcholine receptor (AChR) were propagated from the blood of three myasthenia gravis patients by the use of a pool of synthetic peptides (delta-pool) corresponding to the complete sequence of the delta-subunit of human muscle AChR. Propagation of AChR-specific T cell lines was attempted unsuccessfully from four other myasthenia gravis patients and from four healthy controls. The lines had CD3+, CD4+, CD8- phenotype, strongly recognized the delta-pool, and cross-reacted vigorously with non-denatured AChR purified from mammalian muscle. They did not cross-react detectably with pools of similar overlapping synthetic peptides corresponding to the complete sequences of the alpha- and gamma-subunits of human muscle AChR. The sequence segments of the delta-subunit that contain T epitopes were identified by investigating the response of the three CD4+ T cell lines to the individual synthetic peptides forming the delta-pool. Each line had an individual pattern of peptide recognition. Although no immunodominant region, recognized in association with different DR haplotypes, could be identified, the sequence segments most strongly recognized by the CD4+ T cell lines were clustered within residues 121-290. One of the peptides more strongly recognized by the T cells corresponded to a sequence segment with high predicted propensity to form an amphipathic alpha-helix, a structural motif proposed to be typical of T epitopes.     

Inhibition of CD4+ T cell activation and adhesion by peptides derived from the gp160.

It has been previously demonstrated that the HIV envelope glycoprotein gp160 can inhibit the activation of T cells triggered by phytohemagglutinin, anti-CD3 antibody and Ag, caused in part by the modulation of the expression of CD4. In this study, we show that gp160 is also able to inhibit the Ag-independent adhesion of CD4+ T cells to B cells as anti-CD4 antibodies do. In addition, synthetic peptides (14 to 21 mer) derived from the gp160 sequence and analogous to the putative binding site of gp160 to CD4 (residues 418-460), and also covering residues 460 to 474 inhibit the capacity of both CD4+ T cell proliferation induced by tuberculin and anti-CD3 antibody and adhesion. This was not associated with inhibition of Ca2+ flux in T cell activation. These inhibitory activities are specific because a) CD4+ T cells but not CD8+ T cells are susceptible to their effects, and b) soluble CD4 neutralizes the inhibitory activities. Peptides are, however, about 100- to 1000-fold less potent inhibitors than the native gp160. In addition, they do not induce CD4 modulation. It is thought therefore that at least part of the gp160 inhibitory activity is not secondary to CD4 modulation but may rely either upon steric hindrance of CD4-MHC class II interaction, of CD4/CD3 TCR complex interaction, or upon negative signaling through binding to CD4. The latter hypothesis is suggested by the inhibition by gp160, gp160-derived peptides, and anti-CD4 antibodies of the Ag-independent adhesion of CD4+ T cells. This adhesion process has been previously shown to be mediated by the LFA-1 and CD2 molecules and not by the TCR/CD3 complex and by CD4. Together, these results support the role of part of the 418-460 region of gp160 as a binding site to CD4, and suggest that binding of part of this region to CD4 can alter T cell proliferation and adhesion. It is proposed that these effects are mainly mediated by negative signaling through CD4.     

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.     

Overlapping epitopes that are recognized by CD8+ HLA class I-restricted and CD4+ class II-restricted cytotoxic T lymphocytes are contained within an influenza nucleoprotein peptide.

Viral epitopes that are recognized by both HLA class I-restricted and class II-restricted T cells have been defined for a type A influenza virus nucleoprotein (NP) peptide. CD8+ and CD4+ CTL lines have been generated against a synthetic peptide encompassing residues 335 to 349 of NP that are restricted by HLA-B37 and HLA-DQw5, respectively. Both of these CTL populations were capable of specifically lysing influenza A virus-infected targets, indicating that a naturally processed NP peptide(s) was being mimicked by the NP (335-349) peptide. Amino acid residues that are critical for recognition of this NP determinant in the context of HLA-B37 and HLA-DQw5 were investigated by the use of panels of truncated and alanine-substituted NP peptides. The results demonstrate that: 1) truncations in the amino- or carboxy-terminal ends differentially affect CD8+ and CD4+ CTL recognition; 2) the NP (335-349) sequence contains two octapeptide epitopes that share a core of six amino acid residues (NP 338-343); and 3) alanine substitutions at five of these residues abrogated recognition by at least one of the CD8+ and CD4+ CTL lines. Thus, these class I- and class II-restricted CTL lines recognize similar but distinct epitopes, and different structural features of the NP peptide are required for presentation by HLA-B37 and HLA-DQw5. Comparison of the amino acid sequences of the NP peptide presented by HLA-B37 and HLA-DQw5 with other peptides known to be presented by both class I and class II molecules revealed a common motif among these peptides.     

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.     

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.     

Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2

An influenza B virus nucleoprotein (BNP) peptide, residues 82-94, defined by limited sequence homology with an HLA-A2-restricted peptide from influenza A matrix protein, was recognized by HLA-A2-restricted CTL. Reciprocal inhibition of T cell recognition by the two peptides suggest that the BNP peptide may have lower avidity for HLA-A2 molecules than the matrix peptide. The interaction between this peptide and HLA-A2 was explored by studying the CTL recognition of BNP 82-94 presented by mutant HLA-A2 molecules. Mutations at residues 9, 99, 70, 74, 152 and 156 were found to abolish T cell recognition of the BNP peptide. These results were compared with results previously obtained with the influenza A matrix peptide and suggest that the two peptides bind differently in the peptide binding site.     

Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.     

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

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

Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus.

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in beta cell lysis and induction of diabetes, we have used a transgenic mouse model in which beta cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet beta cells by either pathway.     

Calcium and T lymphocyte activation

A prolonged (at least 2-4 hr) elevation of [Ca2+]i accompanies early T cell activation by TCR/CD3-specific ligands. Ca2+ is generally thought to be an essential second messenger for early activation, but the precise molecular events contingent upon the Ca2+ signal remain to be determined. The Ca2+ signal can be separated into an early transient peak due to InsP3-released Ca2+ from intracellular stores, and a sustained plateau due to altered transmembrane Ca2+ flux. Patch clamp studies have identified an InsP3-activated, Ca2+ permeable channel in the plasma membrane of T lymphocytes that may be responsible for the sustained elevation of [Ca2+]i during continuous TCR/CD3 occupancy. The Ca2+ signal can be further resolved at the level of the single cell into a series of repetitive oscillations between peak and trough levels with a period of 16-20 s. The oscillations may be part of a frequency-encoded signaling system. Several nonlinear internal feedback controls may contribute to the periodic nature of the Ca2+ signal: PKC-mediated phosphorylation of the CD3 gamma subunit, which is a feedback inhibitor of TCR/CD3 function; amplification of Ca2+ release from endoplasmic reticulum by a highly cooperative step in the opening of Ca2+ channels by InsP3, and Ca2+-dependent feedback enhancement of PLC function; autoregulatory negative feedback on Ca2+ influx by Ca2+, both by a direct effect on the plasma membrane Ca2+ channel and by induction of membrane hyperpolarization secondary to Ca2+-activated K+ efflux. In addition, several other internal feedback controls on TCR/CD3 function, by CD4-induced tyrosine-specific phosphorylation of the CD3 zeta subunit, or on the Ca2+ signal, by extracellular Cl- or by GM1 gangliosides, are also postulated. The question of whether a G protein couples TCR/CD3 to PI hydrolysis and to Ca2+ mobilization is unresolved, although some indirect evidence for the involvement of GTP binding proteins in T cell activation has recently been obtained with cholera toxin. There is also preliminary evidence that TCR/CD3 may structurally conform to G protein coupled receptors, i.e., having a core structure of seven alpha helical transmembrane spanning segments, a ligand recognition site, loci for regulatory phosphorylation, and a putative nucleotide binding site.     

A dominant Th epitope in influenza nucleoprotein. Analysis of the fine specificity and functional repertoire of T cells recognizing a single determinant.

The sequence 260-283 of the nucleoprotein (NP) of influenza A virus is an epitope recognized by virus-immune lymph node cells from CBA (H-2k), B6 (H-2b), and B10.S (H-2s) mice. Further analysis shows that there are at least two Th epitopes within this sequence: the one close to the N-terminal (p260-273) is recognized by T cells from CBA and B6 mice while that close to the carboxyl-terminal (p270-283) is a dominant Th determinant in B10.S mice. The fine specificity of the recognition of this epitope by NP-specific T cell clones is also studied. When B10.S mice were infected intranasally or i.v. with live influenza virus, or immunized by different ways with various Ag preparations, P270-283 persistently emerged as a dominant T cell epitope. Immunization of B10.S mice with peptide p270-283 induces T cells with different in vivo functions including class II-restricted cytotoxicity, cognate help for Ag-specific antibody synthesis and delayed type hypersensitivity. This may have important implications for the understanding of the differentiation and classification of subsets of CD4+ T cells. The corresponding sequence of the NP of an equine influenza virus, A/Eq/Prague/56, which has a substitution (leucine to proline) at position 283, was not recognized by the lymph node cells from mice primed with either A/Okuda or A/Eq/Prague. However, the peptide, p270-283(E), representing this sequence induced T cell responses to both human and equine viruses. The data are discussed with respect to the development of viral vaccines.     

Selective modulation of CD4+ T cells from lupus patients by a promiscuous, protective peptide analog

A peptide encompassing residues 131-151 of the spliceosomal U1-70K protein and its analog phosphorylated at Ser140 were synthesized as potential candidates for the treatment of patients with lupus. Studies in the MRL/lpr and (NZB x NZW)F1 lupus models have demonstrated that these sequences contain a CD4+ T cell epitope but administration of the phosphorylated peptide only ameliorates the clinical manifestations of treated MRL/lpr mice. Binding assays with soluble HLA class II molecules and molecular modeling experiments indicate that both peptides behave as promiscuous epitopes and bind to a large panel of human DR molecules. In contrast to normal T cells and T cells from non-lupus autoimmune patients, we found that PBMCs from 40% of lupus patients selected randomly and CFSE-labeled CD4+ T cells proliferate in response to peptide 131-151. Remarkably, however, we observed that phosphorylation of Ser140 prevents CD4+ T cells proliferation but not secretion of regulatory cytokines, suggesting a striking immunomodulatory effect of phosphorylated analog on lupus CD4+ T cells that was unique to patients. The analog might act as an activator of regulatory T cells or as a partial agonist of TCR.