TCR Recognition Research Articles

MHC class I molecules with Superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs.

CD8(+) CTLs are essential for effective immune defense against intracellular microbes and neoplasia. CTLs recognize short peptide fragments presented in association with MHC class I (MHCI) molecules on the surface of infected or dysregulated cells. Ag recognition involves the binding of both TCR and CD8 coreceptor to a single ligand (peptide MHCI [pMHCI]). The TCR/pMHCI interaction confers Ag specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to Ag. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. In this study, we show that increasing the strength of the pMHCI/CD8 interaction by approximately 15-fold results in nonspecific, cognate Ag-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with superenhanced affinity for CD8 activate CTLs in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL Ag specificity.

Stimulation of Naive CD8+ T Cells by a Variant Viral Epitope Induces Activation and Enhanced Apoptosis

Classically, naive T cells recognize a specific peptide-MHC complex resulting in their activation and differentiation. However, it is known that T cells also have the ability to interact productively with variant ligands, indicating a flexibility in TCR Ag recognition. These altered peptide ligands have been shown to trigger responses ranging from complete activation to full inhibition of T cell responses, and thus may play an important role in initiating or sustaining T cell-mediated immunity. We have found that influenza virus-specific CD8(+) TCR transgenic T cells differentially respond to a native (agonist) and variant viral epitope, differing in two amino acids that are thought to alter TCR recognition. In response to stimulation with the agonist epitope, these cells activate, proliferate, and differentiate into effector CTLs. Conversely, stimulation with the variant epitope results in activation, proliferation, and development of effector activity followed by rapid and extensive apoptotic cell death. Stimulation of the T cells with the altered ligand results in an inability to sustain the expression of the prosurvival molecules, Bcl-2 and Bcl-xL. These data suggest that the response to the agonist and variant epitopes may reflect TCR avidity-dependent differential signaling through the TCR, resulting either in activation-dependent T cell proliferative expansion and survival or in the accelerated death of acutely activated differentiating T cells. This process of CD8(+) T cell activation, proliferation, and differentiation followed by rapid cell death may represent a novel mechanism of altered peptide ligand-induced apoptosis programmed by initial Ag receptor engagement.

The recognition of γδ TCR to protein antigen does not depend on the hydrophobic I97 residue of CDR3δ

The crystal structure analysis demonstrated that the hydrophobic amino acid residue (isolecuine/leucine/valine) at conserved position 97 of Vdelta2 TCR plays an important role in recognizing the non-peptide antigen. But its importance to protein antigen remains unclear until now. In the present study, we focus on the role of hydrophobic amino acid residue at conserved position 97 of Vdelta2 TCR in complementarity determining region (CDR)3delta-mediated binding to protein antigen. We employed CDR3delta peptide and membrane-engineered gammadelta TCR as detecting molecules with mutated 97 hydrophobic amino acid residue in CDR3delta (nominated as OT10), a Vdelta2 CDR3 sequence derived from tumor infiltrating lymphocytes in ovarian epithelial carcinoma (OEC). Binding assays revealed that OT10 peptide and membrane-engineered gammadelta TCR (gammadelta TCR transfected cells with OT10 sequence) could bind specifically ovarian tumor cell line (SKOV3). The mutant analysis indicated that any amino acid substitution at position deltaI97 could abolish the response of the transfected cells to iso-butylamine, a known non-peptide antigen of gammadelta T cells. But amino acid substitution of isoleucine at position delta97 did not change the responsiveness of gammadelta TCR transfected cell to protein antigen. Our data suggested that a mechanism other than non-peptide antigen might mediate the recognition of Vdelta2gammadelta T cells for protein antigen. This finding may provide a possibility that gammadelta TCR recognize different ligands in diversity manners.

Tolerance Induction by Immature Dendritic Cells Is Mediated by Distinct MHC Dependent and Independent Mechanisms: A Novel Role for Perforin, Granzyme A and Toll Like Receptor 7.

Abstract 65The mechanism underlining tolerance induction by immature myeloid dendritic cells (IMDC) is poorly understood. To address this issue, we generated mouse IMDC from hematopoietic stem cells (HSC) via differentiation to early myeloid cells (10 days with a cytokine cocktail containing: TPO, Flt-3L, SCF, IL-3 and IL-6) and further differentiation to IMDC (10% GM-CSF for an additional 10 days). The phenotype of the cells obtained at the end of this procedure was CD11c+, CD11b+, B220−, CD80+, MHC class II + Low and CD86−.Addition of the IMDC to long term ( 3 days) mixed lymphocyte reaction (MLR) in which TCR transgenic (Tg) CD4+ or CD8+ alloreactive T cells are reacted against their cognate MHC antigens, led to a marked inhibition of proliferation (reaction ratio 1 IMDC: 2 T cells). This suppression was found to be mediated In short term MLR ( 5 hours) through killing of the effector cells.Killing of CD4+ alloreactive T cells was found to be MHC independent; namely, IMDC expressing MHC not recognized by the TCR transgene exhibited similar inhibition as that exhibited by IMDC expressing the cognate MHC (55±3% versus 59±4% killing upon addition of cognate MHC or non-cognate MHC IMDC, respectively).As previously shown, this non-specific killing is likely mediated by the NO system, as it could be completely abrogated by pre-treatment with N (G)-nitro-L- arginine methyl ester (L-NAME ) (5±4% and 4±3% killing upon addition of pre- treated IMDC bearing cognate MHC or non-cognate MHC, respectively).In contrast, killing of CD8+ alloreactive T cells by IMDC was only slightly reversed by L-NAME (74±3% killing with non-treated IMDC bearing cognate MHC, compared to 68±4% after pre treatment with L-NAME), and the remaining killing activity was found to be MHC dependent. Thus, in contrast to cognate IMDC, very low killing of alloreactive TCR transgenic CD8 T cells was exhibited by non-cognate IMDC in the presence of L-NAME (5±2% killing). When tested in a mouse model of T cell mediated bone marrow (BM) allograft rejection, only IMDC of donor, but not of third party origin, could prolong graft survival upon infusion of 3×106 IMDC (median survival of 24 versus 10 days respectively, p<0.05) suggesting that only the MHC dependent tolerance mechanism is operative in this graft rejection model.Surprisingly, the MHC dependent killing exhibited by IMDC in short term MLR was completely lost when using IMDC generated from perforin KO mice or when IMDC were pre-treated with Bapta-AM or Concanamycin A (6±4%, 5±3% and 3±3% killing, respectively). Thus, killing of the responding alloreactive CD8+ T cells by their cognate IMDC involves a perforin/granzyme mediated mechanism. Indeed, Electron Microscopy (EM) analysis revealed that IMDC were loaded with granules containing perforin and Granzyme A, mostly localized in proximity to the cell membrane. While these granules could barely be detected in early myeloid cells, formation of granules was markedly enhanced upon differentiating into IMDC.Further EM analysis revealed that recognition of the IMDC by alloreactive CD8+ T cells induced granules polarization towards the area of contact. Live video imaging (using perforin and granzyme A immunostaining) further supported the suggestion that this granule polarization leads to specific killing of the responding CD8+ T cell. Furthermore, pre-treatment of IMDC with a blocking peptide for Toll like receptor 7 (TLR7) but not for TLR1, TLR5, TLR6, TLR9, TLR10, or TLR11, completely abolished this granule degranulation, and the eventual killing of alloreactive CD8+ T cells (blocking TLR7 reduced the MLR inhibition from 68±4% to 8±3%, p<0.01). Thus, MHC dependent killing of alloreactive CD8+ T cells by IMDC is likely triggered by TCR recognition of the cognate MHC on the IMDC which, in turn, activate Toll receptor signaling leading to degranulation and granule mediated killing.In conclusion, IMDC can induce immune tolerance through two distinct mechanisms. The first, mediated by the NO system, is MHC independent and is limited to ablation of CD4+ T cell responses. The second, mainly targeting CD8+ T cells, is MHC dependent and is mediated by cytotoxic granules in the IMDC which polarize towards the contact area upon recognition by the T cells. This MHC- specific mechanism is clearly dependent on perforin and is specifically triggered by TLR7 ligation. The latter mechanism was predominant when testing IMDC in a mouse model for T cell mediated BM allograft rejection. Disclosures:No relevant conflicts of interest to declare.

Oxidative stress can alter the antigenicity of immunodominant peptides

APCs operate frequently under oxidative stress induced by aging, tissue damage, pathogens, or inflammatory responses. Phagocytic cells produce peroxides and free-radical species that facilitate pathogen clearance and can in the case of APCs, also lead to oxidative modifications of antigenic proteins and peptides. Little information is available presently about the consequences of such modifications on the immune response. To model oxidative modification of an immunodominant antigenic peptide, we oxidized the methionine residue of the human CMV pp65(495-503) (NLVPMVATV) peptide. Such modifications of an antigenic peptide can affect MHC binding or TCR recognition. Using binding and dissociation assays, we demonstrate that oxidative modification of the CMVpp65(495-503) peptide leads to a decreased binding of the pMHC complex to the TCR, whereas binding of the peptide to the MHC class I molecule is not impaired. Additionally, we show that CD8(+) T cells have a decreased proliferation and IFN-gamma production when stimulated with oxidized CMVpp65(495-503) peptide. Spectratyping the antigen-binding site of the TCR of responding T cells demonstrates that the CMVpp65(495-503) and the CMVoxpp65(495-503) peptides preferentially stimulate BV8 T cells. Sequencing of this dominant BV family reveals a highly conserved CDR3 amino acid motif, independent of the mode of stimulation, demonstrating the recruitment of the same T cell clonotypes. Our results suggest that oxidative modification of antigenic peptides may affect T cell responses severely by binding T cell clones with different affinity. This may lead to an altered immune response against infectious agents as well as against tumor or autoantigens under oxidative stress conditions.

A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism.

Structural Evaluation of Potent NKT Cell Agonists: Implications for Design of Novel Stimulatory Ligands

Natural killer T (NKT) cells are a subset of T cells that are activated by CD1d–glycolipid complexes through a semi-invariant αβ T cell receptor (NKT TCR). Upon activation, NKT cells secrete regulatory cytokines that are implicated in T helper cell responses. α-Galactosylceramide (α-GalCer) is a potent NKT cell agonist when presented by CD1d. Phenyl ring substitutions of the α-GalCer fatty acid moiety were recently found to be superior in eliciting regulatory cytokines. Crystal structures of four new mouse CD1d–lipid complexes (five structures), a new PBS-25 complex, and CD1d with an endogenous ligand, at 1.6–1.9 Å resolution, reveal that the α-GalCer phenyl analogues impart minor structural differences to the A′-pocket, while the sphingosine and galactose moieties, important for NKT TCR recognition, remain virtually unchanged. The observed differences in cytokine-release profiles appear to be associated with increased stability of the CD1d–glycolipid complexes rather than increased affinity for the NKT TCR. Furthermore, comparison of mouse CD1d–glycolipid complexes in different crystallographic space groups reveals considerable conformational variation, particularly above the F′-pocket, the primary site of interaction with the NKT TCR. We propose that modifications of the sphingosine moiety or other substitutions that decrease α-GalCer flexibility would stabilize the F′-pocket. Such compounds might then increase CD1d affinity for the NKT TCR and further enhance the stimulatory and regulatory properties of α-GalCer derivatives.

Efficient help for autoreactive B‐cell activation requires CD4+ T‐cell recognition of an agonist peptide at the effector stage

T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

Fatty Acyl Structures of Mycobacterium tuberculosis Sulfoglycolipid Govern T Cell Response

CD1b-restricted T lymphocytes recognize a large diversity of mycobacterial lipids, which differ in their hydrophilic heads and the structure of their acyl appendages. Both moieties participate in the antigenicity of lipid Ags, but the structural constraints governing binding to CD1b and generation of antigenic CD1b:lipid Ag complexes are still poorly understood. Here, we investigated the structural requirements conferring antigenicity to Mycobacterium tuberculosis sulfoglycolipid Ags using a combination of CD1b:lipid binding and T cell activation assays with both living dendritic cells and plate-bound recombinant soluble CD1b. Comparison of the antigenicity of a panel of synthetic analogs, sharing the same trehalose-sulfate polar head, but differing in the structure of their acyl tails, shows that the number of C-methyl substituents on the fatty acid, the configuration of the chiral centers, and the respective localization of the two different acyl chains on the sugar moiety govern TCR recognition and T lymphocyte activation. These studies have major implications for the design of sulfoglycolipid analogs with potential use as tuberculosis subunit vaccines.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

Casitas B-Lineage Lymphoma b Inhibits Antigen Recognition and Slows Cell Cycle Progression at Late Times during CD4+ T Cell Clonal Expansion

Optimal clonal expansion of CD4(+) T cells during the primary response to Ag requires prolonged TCR recognition of peptide Ag/MHC complexes. In this study, we investigated the capacity of Casitas B-lineage lymphoma b (Cbl-b) to counter-regulate late TCR signals necessary for continued cell division in vivo. During the first 24 h of a primary response to Ag, Cblb(-/-) 5C.C7 CD4(+) T cells demonstrated no alteration in CD69, CD25, and CD71 up-regulation or cell growth as compared with wild-type cells. Nevertheless, beyond 24 h, both the expression of CD71 and the rate of cell division were increased in the genetic absence of Cbl-b, leading to an augmented clonal expansion. This deregulation of late T cell proliferation in the absence of Cbl-b resulted in part from an inability of Cblb(-/-) T cells to desensitize Akt, PLCgamma-1, and ERK phosphorylation events downstream of the TCR/CD3 complex, in addition to their failure to undergo a growth arrest in the absence of Ag. These observations now suggest a novel role for Cbl-b in triggering the exit from cell cycle at the end of a CD4(+) T cell clonal expansion.

Quantitative Microarray Analysis of Intact Glycolipid−CD1d Interaction and Correlation with Cell-Based Cytokine Production

The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates T(H)1 and T(H)2 chemokine release. In this study, a variety of glycolipid ligands were attached to a microarray surface and their binding with dimeric CD1d was investigated. An alpha-galactosyl ceramide (alpha-GalCer) bearing a carbamate group at the 6'-OH position was tethered to the surface, and the dissociation constant on surface with CD1d was determined to reflect the multivalent interaction. Competition assays were then used to determine the dissociation constants (Ki) of new and intact glycolipids in solution. The 4-fluorophenyloctanoyl-modified alpha-GalCer (18) was found to bind most strongly with CD1d (Ki 0.21 microM), 2 orders of magnitude stronger than alpha-GalCer and more than three times more selective than alpha-GalCer for IFN-gamma release from NKT cells. Various alpha-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-gamma production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the T(H)1 pathway.

Characterization of HLA Class II/Peptide-TCR Interactions of the Immunodominant T Cell Epitope in Art v 1, the Major Mugwort Pollen Allergen

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 1(25-36) (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 1(25-36)-specific T cell responses. Further assessment of binding of Art v 1(25-36) to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 1(25-36) bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 1(25-36)-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 1(19-36) allowed the identification of Art v 1(25-36)-specific T cells by flow cytometry. In summary, the immunodominance of Art v 1(25-36) relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.

Molecular dynamics simulation study on the interaction of KRN 7000 and three analogues with human CD1d

Many analogues of KRN 7000, a synthetic glycolipid (α-galactosylceramide) exhibiting immuno-stimulatory activity and antitumor properties, were previously synthesized and tested in order to understand the reasons for the resulting biological activity and Th1/Th2 cytokine profile. Principles have been established for the interaction of such glycolipids with the human CD1d molecule but the exact mechanism by which different ligands with the same polar head elicit distinct biological responses remains unclear. Based on these experiments and on the available crystal structures, protein–ligand interactions are explored using molecular dynamics simulations. Hydrogen bond interactions are examined with regard to the polar group orientation. The influence of modulations on the dynamic behavior of the CD1d–glycolipid complex is addressed. Overall, our data support the mechanism by which the shortening of the α-GalCer sphingosine chain causes a significant twist of the CD1d α1 helix structure from residue Phe84 that affects the position of CD1d residues involved in the TCR recognition.

Autoimmunity in atherosclerosis: a protective response losing control?

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of oxidized lipoproteins, increased cell death and hypertrophic degeneration of the arterial intima. The disease process is associated with local formation of modified self antigens that are targeted by both innate and adaptive immune responses. Although it remains to be firmly established it is likely that these autoimmune responses initially have a beneficial effect facilitating the removal of potentially harmful rest products from oxidized LDL and dying cells. However, studies performed on hypercholesterolaemic mice deficient in different components of the immune system uniformly suggest that the net effect of immune activation is pro-atherogenic and that atherosclerosis, at least to some extent, should be regarded as an autoimmune disease. These observations point to the possibility of developing new treatments for atherosclerosis based on modulation of immune responses against plaque antigens, an approach presently tested clinically for several other chronic inflammatory diseases with autoimmune components. Pilot studies in animals have provided promising results for both parental and oral vaccines based on oxidized LDL antigens. The time when this concept is ready for clinical testing is rapidly approaching but it will be important not to underestimate the difficulties that will be encountered in transferring the promising results from experimental animals into humans.

Eradication of B Cell Malignancies by Anti-3rd Party CTLs: A Novel Role for MHC Class I.

Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

Recognition of allo-peptide is governed by novel anchor imposition and limited variations in TCR contact residues

Immune specificity of a T cell is determined by the TCR contact residues exposed on the antigenic peptide/MHC complex. Naturally processed, biallelic epitopes from H7 minor histocompatibility (mH) antigen vary in position 7 (p7) from aspartic acid (D) to a glutamic acid (E), which differ by an additional methylene (–CH 2) in the side chain. Here, we show that this variation generates a strong anti-H7a or anti-H7b cytotoxic T cell responses. Further, the H7 allelic peptides use p6 asparagine as their central anchor residue and amino acid variations in either the canonical p5 or the predicted p6 anchor positions in the antigenic epitope were detrimental for TCR recognition. In addition, introduction of any other amino acids, except asparagine, in the polymorphic p7 significantly abolished the ability of anti-H7b TCR recognition. This demonstrates that only an asparagine with an amine group as a side chain instead of a charged oxygen radical could effectively stimulate the anti-H7b specific T cells. Our findings provide evidence that mH antigen-specific TCRs are highly stringent in recognizing their cognate epitopes.

Raf signaling but not the ERK effector SAP-1 is required for regulatory T cell development.

Regulatory T cells (T(reg)) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and T(reg) development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, T(reg) development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4(+) cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3(+) and Foxp3(-) CD4(+) populations. In SAP-1-deficient CD4(+)CD25(+) T(reg) cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4(+)CD25(+) T(reg) cells to suppress CD4(+)CD25(-) cell proliferation in vitro. Finally the suppressive activity of CD4(+)CD25(+) T(reg) cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of T(reg) cells in the thymus are thus distinct from those required for "conventional" T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of T(reg) cells.

Structures of MART-1 26/27–35 Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26–35 decamer, although only the 27–35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26–35 and 27–35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1 26/27–35-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27–35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.

A Novel Loop Domain in Superantigens Extends their T Cell Receptor Recognition Site

Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the α3-β8 loop, a unique ∼ 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVβ5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG–TCR complexes, which results in the α3-β8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vβ domain specificity of SEK and other group V SAGs.