ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.     

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.     

β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.     

Epitope expression on primate lymphocyte surface antigens

The cross-reactivity of peripheral blood mononuclear cells from 28 nonhuman primates was investigated with ten kinds of Leu series of monoclonal antibodies specific to human T-, natural killer/killer-, and B-cells. The chimpanzees possessed all ten epitopes examined but the orangutan lacked Leu4 and Leu7 epitopes and the gibbons lacked Leu4, Leu7, and Leu12 epitopes. In addition to the above epitopes, the Old World monkeys lacked Leu1 and Leu10 epitopes. The Leu3a/Leu2a cell ratios varied from 0 to 1.56 among the 12 macaque species and this enabled classification of these species into three groups. In the New World monkeys, Leu2a epitope was absent, whereas Leu11a epitope was detected in several species and Leu3a epitope was found only in the owl monkeys. The prosimians expressed only HLA-DR epitope.     

Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity

Spleen cells from rats which had been hyperimmunized with mouse lymphokine-activated killer (LAK) cells, were fused with the mouse myeloma cell line, P3 X 63 Ag8.653. Antibodies secreted by 1500 cultures were selected by their blocking effect on LAK cell-mediated cytotoxicity in the absence of complement. Two monoclonal antibodies (KBA4 and KBA6) greatly inhibited the cytotoxic activity of LAK cells, which were induced from mouse spleen cells by culture with recombinant human interleukin 2 (r-IL-2). These antibodies also blocked the cytotoxic activity of natural killer (NK) cells, but activated macrophages (A-M phi) were only slightly sensitive to them. However, no effect of the antibodies on the cytotoxic activity of cytotoxic T lymphocytes (CTL) was detected. These data suggest that the specific antigen, lymphokine-activated cell-associated (LAA) antigen, defined by these monoclonal antibodies may be associated with the recognition mechanisms of broad-reactive killer (BRK) cell-mediated cytotoxicity. The observation that low levels of LAA antigen are distributed in all lymphoid cells and that it was significantly enhanced by treatment of the cells with r-IL-2 suggests that the antigen may be involved in lymphocyte-activation mechanisms. We also found that the LAA antigen consists of two distinct polypeptides with Mr of 180,000 and 95,000 Da, which are similar to that of LFA 1 antigen. However, the biological characteristics of LAA antigen did not coincide with those of LFA 1. Therefore, KBA MAb may recognize a carbohydrate epitope distinct from that of LFA 1.     

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.     

The human lymphocyte function-associated (HLFA) antigen and a related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies

The structural and functional relations between the alpha- and beta-subunits of the human lymphocyte function-associated antigen (HLFA) and the human Mac-1 antigen (HMac-1) have been analyzed with the use of five monoclonal antibodies that react with these proteins. The specificities of these antibodies were examined by immunoprecipitation of proteins from 125I-labeled cells and purified HLFA and HMac-1 antigens. Three antibodies reacted with the Mr 95,000 common beta-subunit of the proteins, and also co-precipitated the Mr 175,000 HLFA alpha-subunit, the Mr 165,000 HMac-1 alpha-subunit, and a third polypeptide alpha-subunit of Mr 150,000. The other antibodies were specific to noncross-reactive epitopes present on the alpha-subunits of HLFA or HMac-1. These specificities were confirmed in sequential immunoprecipitation studies. Peptide mapping showed that the beta-subunits of HLFA and HMac-1 were identical, whereas the two alpha-subunits differed considerably. The HLFA alpha-subunit-specific monoclonal antibody inhibited phytohemagglutinin stimulation, the mixed lymphocytes reaction, cytolytic T lymphocyte-mediated killing, and tetanus toxoid stimulation, but did not affect natural killer cell-mediated killing or complement receptor type 3 function. The HMac-1 alpha-subunit-specific monoclonal antibody inhibited complement receptor type 3 function but had no effect on T cell or natural killer cell functions. Three monoclonal antibodies to the beta-subunit inhibited all functions tested, including T cell, natural killer cell, and complement receptor type 3 activities. The results suggest that the functions of the HLFA and HMac-1 molecules may be determined by the alpha-subunit, and that the common beta-subunit also bears functionally important epitopes.     

Reconstitution of killer cell inhibitory receptor-mediated signal transduction machinery in a cell-free model system.

Recognition of class I MHC molecules on target cells by killer cell inhibitory receptors (KIRs) blocks natural cytotoxicity and antibody-dependent cell cytotoxicity of NK cells and CD3/TCR dependent cytotoxicity of T cells. The inhibitory effect of KIR ligation requires phosphorylation of the cytoplasmic tail of KIR and subsequent recruitment of an SH2-containing protein tyrosine phosphatase, SHP-1. To better understand the molecular mechanism of the KIR-mediated inhibitory signal transduction, we developed an in vitro assay system using a purified His-tag fusion protein of KIR cytoplasmic tail (His-CytKIR) and Jurkat T cell lysates. We identified a target molecule of SHP-1 by comparing the phosphorylation of major cellular substrates following in vitro phosphorylation of Jurkat cell lysates in the presence and absence of the His-CytKIR in this cell-free model system. The His-CytKIR was tyrosine phosphorylated by Lck in vitro, and the phosphorylated His-CytKIR recruited SHP-1. Interestingly, we observed that among major substrates phosphorylated in vitro, PLC-gamma exhibited a dramatic decrease in phosphorylation when the His-CytKIR was mixed with Jurkat T cell lysates. However, PLC-gamma exhibited no decrease in phosphorylation when SHP-1 or Lck was depleted or deficient in this reaction mixture, suggesting that the SHP-1 recruited by the phosphorylated His-CytKIR directly mediate the dephosphorylation of PLC-gamma. The cell-free model system could be used to reveal the detailed molecular interactions in the KIR-mediated signal transduction.     

Cell-type-specific tyrosine phosphorylation of the herpes simplex virus tegument protein VP11/12 encoded by gene UL46

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play key roles in limiting herpesvirus infections; consequently, many herpesviruses, including herpes simplex virus (HSV), have evolved diverse strategies to evade and/or disarm these killer lymphocytes. Previous studies have shown that CTL and NK cells are functionally inactivated following contact with HSV-infected fibroblasts. During studies of the mechanisms involved, we discovered that HSV-inactivated NK-92 NK cells and Jurkat T cells contain a strikingly prominent, novel, ca. 90-kDa tyrosine-phosphorylated protein that we identified as the HSV tegument protein VP11/12. Inasmuch as VP11/12 produced in fibroblasts and epithelial cells is not obviously tyrosine phosphorylated, these data suggested that VP11/12 serves as the substrate of a cell-type-specific protein tyrosine kinase. Consistent with this hypothesis, VP11/12 was also tyrosine phosphorylated in B lymphocytes, and this modification was severely reduced in Jurkat T cells lacking the lymphocyte-specific Src family kinase Lck. These findings demonstrate that HSV tegument proteins can be differentially modified depending on the cell type infected. Our data also raise the possibility that VP11/12 may modulate one or more lymphocyte-specific signaling pathways or serve another lymphocyte-specific function. However, HSV type 1 mutants lacking the UL46 gene retained the ability to block signaling through the T-cell receptor in Jurkat cells and remained competent to functionally inactivate the NK-92 NK cell line, indicating that VP11/12 is not essential for lymphocyte inactivation. Further studies are therefore required to determine the biological function of tyrosine-phosphorylated VP11/12.     

Effect of anti-human pan-T monoclonal antibodies on lymphocyte proliferative and cytotoxic functions

Effects of anti-human pan-T-specific monoclonal antibodies of the Second International Workshop on Human Leucocyte Differentiation Antigens were investigated in a number of lymphocyte functional tests. Monoclonal antibodies blocking antibody-dependent cytotoxicity (ADCC), PWM-induced IL-2 release, or Con A- and PWM-induced lymphocyte proliferation were found among anti-CD2 and CD3 reagents. Inhibition of lectin-dependent cellular cytotoxicity (LDCC) was found as an exclusive effect of anti-CD2 (the sheep red cell receptor) antibodies. Several anti-CD2s blocked natural killer (NK) activity and/or PWM-induced interferon production. These two effects were exerted by antibodies against epitopes on resting T cells but not by those directed to activation epitopes. The inhibitory activity of individual antibodies in the LDCC and NK tests showed a good correlation. Also, PHA-mediated cytotoxicity (LDCC) and proliferation were in good correlation. Concerning anti-CD3 (T3) reagents, some effects were characteristic for the majority of the antibodies in this group. Namely, induction of proliferation, enhancement of IL-2-dependent cell division, IL-2 consumption by antibody-triggered cells, inhibition of mitogen-induced proliferation but not IL-2 and interferon production were observed. None of the CD3-specific reagents exerted all of these effects. In general, no correlation of the effects with immunoglobulin subclass or CD3 subcluster specificity could be found. Further epitope analysis and affinity data may be required to understand the basis of heterogeneity in functional effects of monoclonal antibodies to the CD3 molecule.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

High epitope expression levels increase competition between T cells

Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs) or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.     

Natural killer-like function of activated T lymphocytes: differential blocking effects of monoclonal antibodies specific for a 90-kDa clonotypic structure

A monoclonal antibody termed anti-NKTb has been generated following immunization of mice with cloned human cells (JT9) displaying natural killer (NK)-like activity. This antibody has the capacity to block cytotoxicity of the immunizing clone against several targets. In the present study, anti-NKTb was compared with a monoclonal antibody termed anti-NKTa that had previously been generated against JT9 cells and that had also been shown to block the NK-like function of these cells. The expression of a NKTb determinant, like that of NKTa, was found to be restricted to two NK active clones derived from the same individual, JT9 and JT10, both of which have the same mature T-cell phenotype (T3+, T8+, T11+). Comodulation, immunoprecipitation, and competitive binding experiments showed that both antibodies are directed to the same 90-kDa heterodimer associated with the T3 structure on the cell surface. However, cytotoxicity blocking studies suggested that NKTa and NKTb may represent functionally distinct epitopes of this 90-kDa molecule. Anti-NKTa uniformly blocked the cytotoxicity of both JT9 and JT10 cells when tested against 11 randomly selected target cell lines. In contrast, anti-NKTb totally blocked the cytotoxicity of these cloned cells against some targets (i.e., HPB-ALL, Nalm-1) but had very little effect when cytotoxicity was measured against other target cells (i.e., K562, U937, KG-1). This selective blocking effect, therefore, supports the notion that the heterodimer defined by the NKT antibodies is involved in the process of target cell recognition rather than in the cytolytic pathway of the cloned effector cells. Moreover, the unique functional effects of anti-NKTb suggest that additional levels of complexity exist in the specific recognition mechanisms of these clonal populations of NK active mature T lymphocytes.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Role of natural killer cells in resistance against friend retrovirus-induced leukemia

We have previously shown that immunization with a synthetic peptide that contains a single CD4(+) T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4(+) or CD8(+) T cells in the spleen, virus antigen-specific proliferative responses of CD4(+) T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8(+) effector cells. However, the same treatment markedly reduced the killing activity of CD4(-) CD8(-) effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.     

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.     

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.     

Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease.

We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.     

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.