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Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling. published

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the α-chain connecting peptide motif (α-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing α-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of α-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or α-CPM-deficient forms of the T1 TCR. This Kd-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide252–260 IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that α-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 Å resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

Mature B and T lymphocytes, as well as natural killer (NK) cells, express on their surfaces receptors that recognize antigen and elicit effector functions. B and T cell receptors are encoded by genes formed by rearrangement, but receptors utilized by NK cells are not. Keywords: lymphocyte; receptor; immunology; antigen

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252–260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-β-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR. published

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

Cytotoxic T cells present at the periphery or at tumor sites recognize tumor epitopes presented by melanoma cells but fail to induce tumor rejection in melanoma patients. Inhibitory receptors including PD-1 and Tim-3 expressed by spontaneous CD8+ T cells directed against the cancer-germline antigen NY-ESO-1 play a role in promoting tumor-induced T cell dysfunction. In the present study, we show that BTLA is upregulated on spontaneous NY-ESO-1-specific CD8+ T cells and that BTLA+PD-1+Tim-3− CD8+ T cells represent the largest subset of NY-ESO-1-specific CD8+ T cells in melanoma patients. BTLA+PD-1+Tim-3− CD8+ T cells are partially dysfunctional, producing less IFN-γ than BTLA−PD-1+Tim-3− and BTLA−PD-1−Tim-3− but more IFN-γ, TNF and IL-2 than highly dysfunctional BTLA+PD-1+Tim-3+ NY-ESO-1-specific CD8+ T cells. Unlike PD-1, BTLA expression by NY-ESO-1-specific CD8+ T cells does not increase with higher T cell dysfunction or upon cognate antigen stimulation, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Finally, BTLA pathway blockade adds to PD-1 and Tim-3 blockades, enhancing NY-ESO-1-specific CD8+ T cell expansion, proliferation and cytokine production. Collectively, our findings support the targeting of BTLA, PD-1 and Tim-3 pathways to reverse tumor-induced T cell dysfunction in patients with advanced melanoma. This work was supported by the National Institutes of Health/National Cancer Institute grants R01CA90360 and R01CA157467 (to H.M. Zarour), an award from the Melanoma Skin Spore grant NCI P50CA121973 (to H.M. Zarour) and a grant from the Cancer Research Institute (to H.M. Zarour). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5407. doi:1538-7445.AM2012-5407

A novel procedure is presented describing the induction of antigen-specific cytolytic T lymphocytes (CTL) in vivo, that uses as immunogen syngeneic Concanavalin A stimulated spleen cells expressing H-2Kd (Kd) molecules photocrosslinked with a photoreactive peptide derivative. The Kd restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was conjugated with photoreactive iodo-4-azidosalicylic acid (IASA) at the NH2-terminus and with 4-azidobenzoic acid (ABA) at the TCR contact residue Lys259 to make IASA-YIPSAEK(ABA)I. Selective photoactivation of the IASA group allowed specific photoaffinity labeling of cell-associated Kd molecules. Optimal peptide derivative binding to Kd molecules of concanavalin A stimulated spleen cells was obtained upon 4-6 h incubation at 26 degrees C in the presence of human beta 2 microglobulin. Photocrosslinking prevented the rapid dissociation of cell-associated Kd-peptide derivative complexes at 37 degrees C. The photoaffinity labeled cells were injected i.p. into syngeneic recipients. After 10 days, the peritoneal exudate lymphocytes were harvested and in vitro stimulated with peptide derivative pulsed P815 mastocytoma cells. The resulting bulk cultures displayed high cytolytic activity that was specific for IASA-YIPSAEK(ABA)I and YIPSAEK(ABA)I. In contrast, peritoneal exudate lymphocytes from mice inoculated with concanavalin A blasts that were pulsed, but not photocrosslinked, with IASA-YIPSAEK(ABA)I expressed only marginal levels of IASA-YIPSAEK(ABA)I-specific cytolytic activity. This immunization strategy, using neither adjuvants nor potentially hazardous transfected/transformed cells, is safe and should be universally applicable.

The paradoxical coexistence of spontaneous tumor antigen-specific immune T cell responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction and exhaustion. We have previously observed in patients with advanced melanoma that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1-specific CD8+ T cells that upregulate PD-1 expression and that a fraction of PD-1+ NY-ESO-1-specific CD8+ T cells upregulates Tim-3 expression. We have found that Tim-3+PD-1+ NY-ESO-1-specific CD8+ T cells represent highly dysfunctional T cells in terms of cytokine production and proliferation. Importantly, Tim-3/Tim-3L blockade together with PD-1/PD-L1 blockade enhanced cytokine production and proliferation of NY-ESO-1-specific CD8+ T cells upon prolonged antigen stimulation. Whether the upregulation of PD-1 and Tim-3 is associated with tumor antigen-specific CD4+ T cell dysfunction/exhaustion in patients with advanced melanoma remains unknown. Here, our findings show that in contrast to EBV-specific, CMV-specific and total CD4+ T cells, PD-1 and Tim-3 expression is upregulated by NY-ESO-1-specific CD4+ T cells isolated from peripheral blood lymphocytes of patients with advanced melanoma. Interestingly, PD-1/PD-L1 blockade synergized with Tim-3/Tim-3L pathway blockade to restore the numbers of cytokine-producing and proliferating NY-ESO-1-specific CD4+ T cells. Collectively, our findings support the use of Tim-3/Tim-3L blockade in association with PD-1/PD-L1 blockade to reverse tumor-induced CD8+ and CD4+ T cell exhaustion/dysfunction in patients with advanced melanoma. Our findings have significant implication in terms of novel immunotherapeutic strategies aiming at promoting T cell-mediated tumor regression in patients with advanced cancers. This work was supported by NIH/NCI Grants CA90360 and CA112198 (HMZ) and Cancer Research Institute grant (HMZ). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3657. doi:10.1158/1538-7445.AM2011-3657

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

A panel of 15 single alanine substitutions on the floor of the peptide binding groove of the murine class I histocompatibility molecule H-2Kd has been analyzed. All but two mutant molecules were expressed on the cell surface, and were tested for peptide binding and presentation to specific cytotoxic T lymphocytes. Eleven out of 13 mutant molecules appeared to be functionally altered. Five of the substituted residues were involved in the presentation of all peptides tested. Three participated in the presentation of certain peptides but not others. Three other residues participated in epitope formation through indirect interactions. Only two mutations had no detectable effect.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

T LYMPHOCYTES recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histo-compatibility (MHC) molecules1,2. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules3–6 purified solubilized class I molecules either bind relatively poorly7 or show degenerate specificity8–11. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

Photoaffinity labeling was used to evaluate optimal conditions for purification of I-A k histocompatibility molecules in functionally active form. We assessed the biological activity of I-A k primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [125I]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-A k on B cell hybridoma membranes and their detergent solubilisates, at the alpha chain. Following extensive detergent dialysis, the intensity of this labeling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-A k from various monoclonal antibodies and by determining the denaturation of I-A k under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-A k purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus I-A k was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-A k on cell membranes, purified I-A k in detergent showed extensive labeling of the beta chain. The overall labeling intensity and the extent of beta chain labeling substantially changed upon addition of certain lysophosphatides.

We have studied the interaction of six photoreactive conjugates of the immunogenic hen egg-white lysozyme peptides HEL(46-61) or HEL(49-61) with the murine histocompatibility class II molecules I-Ak, I-Ad, I-Ek, and I-Ed. All compounds tested selectively labeled the alpha chain of the class II molecules. This was true when testing class II molecules on cell membranes or solubilized in detergents. The COOH-terminal conjugate of HEL(49-61) with (4-azidobenzoyl)cystine preferentially labeled I-Ak. However, addition of hydroxyl or iodine substituents to the photoreactive moiety increased the labeling efficiency and resulted in labeling of the other class II molecules. The data suggest that the photoreactive groups enhanced the binding affinities of these peptides to class II molecules, reflected by the increased labeling efficiencies. Conversely, introduction of an iodine substitution into the tyrosine residue of HEL(46-61) or HEL(49-61) strongly decreased the photoaffinity labeling, possibly due to steric interference with ligand binding to class II molecules. Judicious use of photoaffinity probes that conserve binding specificity of the peptide should be useful for mapping the antigen-binding site of a class II molecule.

The conjugation of two lipophilic p-oxymethylbenzyl-3β-cholestanylsuccinate groups to fully benzylpenicilloylated eicosa-L-lysine [BP20Lys20(OSuco)2] greatly potentiated its ability to suppress anti-BPO IgE antibody formation in vivo in mice. This enhanced tolerogenicity was due to the ability of BPO20Lys20(OSuco)2 to induce T suppressor cells (Luescher, I. F. et al., Eur. J. Immunol. 1984.14:68). In the present study the role of adherent cells in the induction of T suppressor cells by BPO20Lys20(OSuco)2 was investigated. Low numbers of macrophages pulsed in vitro with BPO20Lys20(OSuco)2, but not with the hydrophilic analog BP020Lys20-OH, suppressed anti-BPO IgE antibody formation upon transfer into syngeneic recipients. This suppression was antigen and IgE specific, and was manifested in naive as well as in previously immunized mice. By cell transfer experiments it was found that the suppression was sensitive to cyclophosphamide. The IgE suppression was not due to carry-over of BPO20Lys20 (OSuco)2 by the transferred macrophages since the amount of cell-associated antigen was approximately 500-fold too low to account for the observed suppressions. The capability of BPO20Lys20 (OSuco)2 to elicit anaphylaxis in passively sensitized rats was significantly lower in comparison to the hydrophilic analog BPO21Lys20OH. Adherent cells pulsed with BPO20Lys20 (OSuco)2, although strongly suppressing IgE antibody formation, failed to elicit any detectable anaphylaxis. The role of adherent cells in the induction of T cell-mediated suppression of IgE antibody formation by lipid-modified antigens is discussed and novel therapeutical concepts to achieve desensitization of drug allergic individuals are suggested.

A rapid analytical procedure is described to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins specifically adsorbed on antibody-coupled nitrocellulose membrane filters. Purified antibodies were adsorbed on nitrocellulose filters and were immobilized by treatment with dilute glutaraldehyde. Efficient adsorption of specific antigen occurred upon filtration of the antigen solution (e. g. cell supernatant or cell lysate) within a few minutes. Theim-munoadsorbed proteins were released from the membrane filters in the presence of urea, Nonidet P-40 detergent and electrophoresis sample buffer and were directly analyzed by SDS-PAGE. All adsorptions and washing steps were performed by filtration utilizing a filtration manifold. This allowed the processing of a large number of samples in a short time. Furthermore, and in contrast to the Western blotting method, antigen-antibody recognition preceeded gel electrophoresis, which greatly reduced potential failures in antigen recognition. Similarly, SDS-PAGE of proteins non-specifically adsorbed on nitrocellulose filters was also possible. Dilute protein solutions, like cell supernatants, were filtered through nitrocellulose membrane filters. Hereby proteins, but not low molecular weight compounds such as inorganic salts, amino acids or detergents, were rapidly and quantitatively adsorbed. The adsorbed proteins were released from the filters and subjected to SDS-PAGE as in the previous method.

This is a summary of some of the research taking place in our laboratory. The paper represents a condensed version of a recent review paper (Unanue et al ., 1989).

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the mol. basis of this sensitive recognition process, the authors studied dimeric pMHC complexes contg. linkers of different lengths. Although dimers contg. short (10-30-.ANG.) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers contg. long linkers (>=80 .ANG.) did not. Based on this and on fluorescence resonance energy transfer expts., the authors describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their const. domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, the authors demonstrated that pMHC dimers contg. one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition. [on SciFinder (R)]

CD8+ cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2Kd molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules. published

Biologically active human T-cell receptor (TCR) was expressed as a secreted recombinant protein in 293 cells. Correct α/s chain pairing was facilitated by complementary charged peptides (leucine zipper) at the C terminus. Two plasmids, coding for the α and s chains were constructed and prepared using commercially available purification kits. After optimizing transfection conditions for secretion of heterodimeric TCR in 12 well plates, transfections were performed in spinner flasks. The transfection efficiency was determined with a co-transfected s-gal expression vector nd correlated with TCR levels as determined by ELISA. Product was isolated by affinity chromatography. Biological activity, i.e. ligand binding was tested. Transiently produced recombinant soluble TCR showed similar affinity to the ligand as receptors purified from cell membranes.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Meeting abstracts Tumor antigen-specific CD4+ T cells generally orchestrate and regulate innate and adaptive immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4+ T cells is restricted at local tumor sites where antigen-presenting cells are