Degeneracy of T cell receptor recognition of an influenza virus hemagglutinin epitope restricted by HLA-DQ and -DR class II molecules

The T cell receptor (TcR) recognizes antigens in the form of short peptide fragments bound to major histocompatibility (MHC) molecules. TcR have an immunoglobulin (Ig)-like structure and, in an analogous manner to antigen recognition by Ig, the third complementarity determining regions (CDR3) of the TcR are believed to provide the primary contact with the peptide lying in the MHC groove. CDR1 and CDR2 are thought to contact the presenting MHC molecule. We have analyzed seven human CD4⁺ T cell clones that recognize a conserved peptide epitope (residues 255–270) within the influenza virus hemagglutinin (H3) HA1 subunit. Two T cell clones recognized the peptide in the context of HLA-DRB1*1001 and HLA-DQB1* 0602/DQA1*0102, respectively, and shared V_α, V_β and J_β gene segments. Only the junctional regions encoding the CDR3 regions of the two TcR chains were different. This suggests that the CDR3 regions of these TcR interact with the MHC class II molecule. Six of the T cell clones were restricted by the HLA-DRB1*1001. Two of these T cell clones expressed Vβ9.1 and three expressed Vβ13 gene segments; the remaining clone expressed Vβ7.2, a close homologue of V_β9.1. A diverse selection of V_α and J gene segments contributed to the junctional heterogeneity of the TcR, indicating a diversity of sequence combinations recognizing the epitope. Nevertheless, five out of six T cell clones bore a motif in the V_α CDR3 loop consisting of adjacent acidic and polar amino acid residues, eight residues from the carboxyl end of each CDR3.     

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: Selective in vivo expansion of T cells bearing defined receptors

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2⁺ and Vβ5.6⁺ clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2⁺ and Vβ5.6⁺ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.     

Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27

The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383–391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR Vα14 gene segment (but not those using TcR Vα12) were also sensitive to P1 substitutions, suggesting that the TcR α chain of these clones lies over the N terminus of bound peptide, and that the “footprint” of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are “flag” residues with TcR-accessible side chains.     

Redistribution of critical major histocompatibility complex and T cell receptor-binding functions of residues in an antigenic sequence after biterminal substitution

Residues critical for establishing a trimolecular interaction with a major histocompatibility complex (MHC)-encoded receptor and a T cell antigen receptor (TcR) were determined for an antigenic nonapeptide. The N-terminal residue proved to be involved in binding of the peptide to both receptors and the C-terminal residue was essential for MHC binding. While substitution of either of these critical terminal residues by alanine resulted in an almost complete loss of peptide antigenicity, simultaneous substitution of both created a new functional ligand for the same MHC molecule and the same TcR. Notably, in the biterminally substituted peptide, the core residues took on new roles in the trimolecular interaction in that a residue critical in the authentic nonapeptide for TcR binding became critical for MHC binding and former spacer residues became essential to various degrees for the interaction with either receptor or both. Thus, apparently, the loss of the terminal residues' contribution was at least partially compensated by a redistribution of the roles among the remaining residues. These results reflect a cooperative contribution of all residues of an antigenic peptide to its binding to both receptors and thus challenge a static definition of agretope and epitope as MHC and TcR binding sites.     

The influence of invariant chain on the positive selection of single T cell receptor specificities

The appearance of peptide-loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4⁺ T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain-deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single-positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of E^k. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti-MCC 88-103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene-encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptide in vitro. The most likely interpretation of these findings is that selection of most CD4⁺ T cells depends on MHC class II complexes loaded with peptide in an Ii-dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii-independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4⁺ T cells, for which there exists little prior evidence.     

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4⁺ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2⁺, CD4⁺, CD8^?, T cell receptor (TcR) αβ⁺ and produced interleukin-2 in response to CII peptides presented by I-A^q molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical Vα and Jα (VαBMB, Jα20) gene segments and two distinct Vβ (Vβ1 and Vβ4) associated with the Jβ2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both α and β chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4⁺ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.     

Characterization of an epitope of the human cytomegalovirus protein IE1 recognized by a CD4+ T cell clone

CD4⁺ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)-peptide-T cell receptor (TcR) interactions in the CD4⁺ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti-cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86–491 encoded by exon 4 of IE1 (GST-e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatographymass spectroscopy (HPLC-MS). We identified the 14-residue epitope 162-DKREMWMACIKELH-175 recognized by an HLA-DR8-restricted clone, BeA3. Synthetic elongated, truncated and di-Ala-substituted peptides of the 18-mer IE1 158-IVPEDKREMWMACIKELH-175 sequene were used to analyze the amino acid motifs involved in binding to HLA-DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW → AA), or decreased (RE → AA and MA → AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW → AA substitution was associated with poor HLA-DR8 binding. Decreased T cell function (RE → AA and MA → AA) was associated with good HLA-DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV → AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158–175). CI → AA substitution induced strong potentiation of HLA-DR8 binding, proliferation and interferon-γ and interleukin-4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4⁺ T cell response according to the peptide residues involved in the HLA-DR8-peptide-TcR interaction.     

Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma

We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the Vβ2 germ-line segment which paired with several Vα segments and that the putative CDR3 of both α and β chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 α^?β^?, by expressing the human α and β variable regions joined to the mouse α and β constant regions, respectively. The chimeric TcR, expressing the same Vβ germ-line segment (Vβ2), two expressing Vα21.1, twoVα17.1 and one Vα8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large differences in their capacity to react with the ligand. Second, interchanging the α and β chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line Vα17.1 could functionally replace Vα21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Vα replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.     

T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function

A new anti-p58 monoclonal antibody (mAb), termed CH-L, has been used to characterize a minor subset of T lymphocytes co-expressing p58 and CD3 molecules. In two-color immunofluorescence analysis, CH-L⁺CD3⁺ cells represented 0.5 to 6% of the peripheral blood lymphocytes (in 20 healthy donors). Clonal analysis showed that most CD3⁺CH-L⁺ T cell clones expressed the CD8⁺4^? T cell receptor (TcR) α/β⁺ phenotype, while only a few were CD8^?4⁺ TcR α/β⁺, CD8^?4^? TcR α/β⁺ or CD8^?4^? TcR γ/δ⁺. Western blot analysis indicated that the CH-L mAb identifies the same 56-58-kDa diffuse band in both T and natural killer cell (NK) clones. A minority of T cell clones also expressed other NK-related markers such as CD16, CD56 and CD94 and two clones also reacted with the anti-p58 mAb EB6. Interestingly, most clones displayed cytolytic activity in an anti-CD3 mAb-triggered redirected killing assay against the Fcδ receptor⁺ P815 target cells and NK-like activity against K562 and Raji cells. In contrast, the IGROV-1 ovarian carcinoma cell line was resistant to cytolysis by all of these clones. Since p58 molecules have previously been shown to exert regulatory functions on NK-mediated lysis, we investigated whether anti-p58 mAb could also influence cytotoxicity mediated by CD3⁺p58⁺ T lymphocytes. Lysis of P815 target cells, triggered by anti-CD3 mAb, could be inhibited by anti-p58 mAb in 8 out of 12 cytolytic clones tested, while 4 clones were not inhibited. In addition, anti-p58 mAb enhanced the cytolytic activity of 3 clones against IGROV-1 and of 4 other clones against Raji target cells. Taken together, these data indicate that p58⁺ T cells express heterogeneous phenotypes and different forms of TcR and, in most instances, display cytolytic functions. Perhaps more importantly, the p58 molecule appears to modulate the cytolytic activity triggered via the CD3/TcR complex.     

T cell development in a major histocompatibility complex class II-deficient patient

In this report we show that the major histocompatibility complex (MHC) class II-negative thymus of a bare lymphocyte syndrome (BLS) patient contains a reduced CD4⁺ CD8^? T cell population when compared to thymocytes derived from a MHC class II-expressing thymus. Of these CD4⁺ CD8^? BLS thymocytes, approximately only one third co-expressed the CD3 antigen, moreover at a lower expression level when compared to control thymocytes. This suggests a partial maturation of the CD4⁺ CD8^? T cells in the absence of MHC class II expression. Among the BLS thymocytes, CD4⁺ CD8⁺ thymocytes could easily be detected. Noteworthy, the number of CD4^? CD8⁺ thymocytes was significantly increased. CD4⁺ CD8^? T cells could also be found among the BLS peripheral blood mononuclear cells, albeit at reduced numbers. Despite the absence of peripheral MHC class II expression, the majority of these CD4⁺ CD8^? T cells co-expressed the CD45RO marker. In the BLS patient, thymocytes as well as peripheral CD4⁺ CD8^? T cells were not restricted in the use of the available T cell receptor (TcR) V gene family pool. However, the lack of detectable levels of thymic and peripheral MHC class II antigen expression in the BLS patient had altered the CD4^?skewing patterns of TcR V gene families which were present in normal individuals. In conclusion, the lack of MHC class II expression in the BLS patient does not completely inhibit the CD4+ CD8^? T cell development.     

An example of immunodominance: engagement of synonymous TCR by invariant CDR3 beta

The structural basis of the T cell response against immunodominant tetanus toxin (TT)-derived peptides was investigated using TT-specific T cell clones raised from a DRB1*0301 homozygous donor. Three peptides forming T cell epitopes were identified, including one, TT(1272-1284), that stimulated four different TT-specific T cell clones. TCR sequence analysis revealed that these synonymous TCR shared only arginine at the third position of the CDR3 beta loop. This prominent residue may form a salt bridge with a corresponding aspartate at the relative position 8 (P8) of the antigenic peptide TT(1272-1284) as suggested from amino acid replacement analysis. A similar scenario was observed for a second TT epitope, TT(279-296), and its corresponding TCR. These examples show that immunodominance may result from a single strong amino acid interaction between TCR CDR3 beta loops here in contact with the C-terminus of the antigenic peptide. Such a dominant interaction could compensate for weaker contacts between other residues of the TCR and the antigenic peptide, and would allow the recognition of a single peptide-MHC complex by a broader synonymous TCR repertoire and could thus contribute to its immunodominance.     

In situ analyses of in ovo graft-vs.-host reaction induced by thymic nurse cell lymphocytes

In both mammalian and avian systems, thymic nurse cells (TNC) have been shown to harbor a heterogeneous population of T lymphocytes (TNC-L) some of which exhibit a postselectional phenotype. By tranplanting micromanipulated single chicken TNC onto the chorionallantoic membrane (CAM) of major histocompatibility complex (MHC)-disparate embryos, an experimental system which allows for the detection of lymphocytes with graft-vs.-host (GVH) reactivity, we demonstrate here that TNC enclose lymphocytes that can develop into both CD4⁺ single-positive (sp) and CD8⁺ sp, T cell receptor (TcR)αβ⁺, or TcRγδ⁺ cells. This finding was additionally confirmed by serial transfer of primary expanded alloreactive T cells onto the CAM of secondary hosts. All donor TNC-L expressed MHC class II molecules and the interleukin-2 receptor a chain in primary and secondary GVH reactions. Furthermore, we observed selective accumulation of CD8⁺ and TcRγδ⁺ host lymphocytes in the CAM upon the induction of a local GVH reaction, most probably as a consequence of the pathological alteration of the epithelium.     

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2K^b-bound octapeptides. CTL recognition of such determinants is always sequencedependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on K^b-associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.     

Early degenerate selection of thymocytes by class I major histocompatibility complex

Ontogeny of T cells is accomplished in the thymus by a process of positive selection, in which interaction of the T cell receptor (TcR) expressed on CD4⁺8⁺ thymocytes with self major histocompatibility complex (MHC), expressed on cortical epithelial cells, determines the progress along the maturation pathway and confers self restriction to T cells. Conversely, cells behaving as self reactive by interaction with bone marrow-derived antigen-presenting cells are negatively selected by apoptosis. We show here that the presence of a class I-restricted soluble TcR (sTcR) in the fetal thymic microenvironment, early in T cell ontogeny, determines an enhanced negative selection of a sizeable number of CD4⁺8⁺ thymocytes, which have been previously subjected to a positive-selection event. We hypothesize that the generation of the mature thymic T cell repertoire stems from an interaction of TcR, under a critical affinity threshold, with a self peptide-MHC complex which is common to a great number of TcR specificities using the same restriction element. A shift in this affinity threshold, caused by sTcR, results in the generation of cells acting in a self-reactive manner, which are then deleted. In extended fetal thymus organ culture in the presence of sTcR, we have also observed the appearance of mature CD8⁺ T cells, which once adoptively transferred to syngeneic nude mice are expanded in the periphery, consistent with an enhanced avidity of these cells for self MHC.     

Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus

A CD4⁺ T cell epitope of the influenza virus matrix protein corresponding to the C terminus (QAYQKRMGVQMQRFK) was inserted into the VP7 gene of bluetongue virus (BTV). The chimeric protein was expressed by a dual recombinant Autographa californica polyhedrosis virus (AcNPV), which encodes the two inner capsid proteins VP3 and VP7 of BTV. When Spodoptera frugiperda cells (Sf9 cells) were infected with this recombinant BTV, core-like particles (CLPs) were formed as demonstrated by electron microscopy. To study the immunogenicity of a foreign epitope deprived of its natural flanking sequences in vitro, purified CLPs expressing the T cell epitope were used to stimulate two different MHC class II-restricted CD4⁺ human T cell clones. One of these T cell clones, ALF 3.7 was specific for the inserted epitope, whereas the other T cell clone ALF 4.4 recognized shorter derivates of the given epitope. CLPs with the inserted epitope were presented as efficiently as purified influenza virus matrix protein to the clone ALF 3.7, whereas clone ALF 4.4 showed no proliferative response. Received: 17 February 1998     

Increasing inflationary T‐cell responses following transient depletion of MCMV‐specific memory T cells

Murine CMV (MCMV) infection induces effector CD8⁺ T cells that continue to increase in frequency after acute infection (“inflation”) and are stably maintained at a high frequency, with up to 20% of the CD8⁺ T‐cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8⁺ T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8⁺ T cells. Treatment of MCMV‐infected mice with MHC‐Class I‐saporin tetramers led to partial (80–90%) depletion of epitope‐specific CD8⁺ T cells—rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8⁺ T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the “inflated” CD8⁺ T cell responses during persistent infection.     

Restricted alpha/beta receptor gene usage of idiotype-specific major histocompatibility complex-restricted T cells: selection for CDR3-related sequences.

We have sequenced the T cell receptor (TcR) V alpha and V beta genes of seven independent BALB/c CD4+ T cell clones specific for the immunoglobulin lambda 2 light chain produced by the MOPC 315 myeloma (lambda 2(315)). All the clones recognize a peptide of residues 91-101 of lambda 2(315) and are restricted by the major histocompatibility complex (MHC) molecule I-E(d). The results indicate that in BALB/c mice, this anti-idiotypic response uses a very limited number of TcR. The four clones which cross-react between Phe94 and Tyr94 peptide analogues use very similar receptors (V alpha 3, J alpha 1, V beta 6, J beta 1.1). The V alpha 3 gene used by all of these clones is identical and has not been previously described. Although the four clones differ in nucleotide sequence in the V/J borders, two had identical receptors at the amino acid level. One of the cross-reactive clones exhibits a heteroclitic response to the Tyr94 peptide variant resulting from a single amino acid exchange in the V/J junction of the alpha chain. The three remaining clones which recognize only the Phe94 and not the Tyr94 peptide have somewhat more diverse TcR, however, two of these three clones use V beta 6. One of these non-crossreacting clones is alloreactive, the specificity of which can be attributed to differences in the N-D-J sequences. Taken together these data indicate that this T cell response to an immunoglobulin idiotope is very restricted in terms of the TcR used. These data in conjunction with recently published results indicate that, although there can be strong preference for individual V alpha or V beta gene segments, certain V alpha/V beta combinations are preferentially selected for interacting with a given peptide/MHC combination, and that the CDR3-related regions are crucial for antigen fine specificity and alloreactivity.