key: cord-0010084-ak1umcec
authors: nan
title: Lymphocyte activation
date: 2004-02-19
journal: J Cell Biochem
DOI: 10.1002/jcb.240560809
sha: 15d12c3555269e228f8fdeaedc064ec760646ba5
doc_id: 10084
cord_uid: ak1umcec

nan

The longterm persistence of memory cells within the immune system seems to be largely brought about by intermittent restimulation of these cells. Our experiments and those of others have illustrated the central role that antigen depots play in the continuance of this stimulation. For non-replicating antigens (ie. not viral) the most important sites of storage are on the surface of follicular dendritic cells (FDC), specialized cells that can keep antigen-antibody complexes on their surface for upto 1 year and possibly longer. Because the antigen stored on FDC is undegraded, presentation of this antigen to a memory T cell will require an intermediary APC (FDC are incapable of processing antigen themselves). As the only cells that can efficiently obtain antigen from FDC are antigen-specific B cells we have predicted that the longterm persistence of a memory T cell response will require the presence of antigen-specific memory B cells. As a corollary to this hypothesis it is logical to conclude that in the absence of a n antibody response, and the subsequent failure of FDC antigen localization, the maintenance of T cell memory will be impaired. We have tested this hypothesis in a variety of systems (including SCID mice reconstituted with T cells but not B cells and in normal mice immunized with peptide-pulsed APC to elicit T cell responses in the absence of antibody production). The results so far suggest that indeed the lack of antigen-specific B cells or antibody impairs the efficient development of a longterm memory T cell response. The interaction between a memory T cell and a memory B cell seems likely to be mutually beneficial as the transfer of memory B cells into nude mouse recipients, with antigen-antibody complexes but without T cells, leads only to a relatively short term survival of the memory B cells. We are currently investigating the role of the CD40-CD40 ligand interaction (in addition to lymphokines) in the memory survival process. Data from experiments involving the in vivo treatment of mice with soluble CD40-Fc, indicating a central role of CD40-CD40 ligand interaction in memory B cell generation will also be discussed.

The Kb mutant phenotypes are characterized by their inability to present certain peptide epitopes, e.g. ovalbumin257.2~ (OVA) to C n s . In order to determine whether the mutant phenotype is a result of lack of peptide binding we have determined the dissociation constants for complexes between OVA peptide and Kb mutant molecules Kbml or Kbms. The KD values determined did not differ more than a factor 5 from those measured for Kb, indicating that lack of binding of peptide is not the reason for the lack of presentation of this peptide. We are currently investigating wheter it is the structure of the Kbm' or Kh8-OVA peptide complexes that results in the lack of recognition by T-cells. Bulk C n s from C57BV6, K h l , and Kbms mice were stimulated with cells (either R or Drosophila melanogaster) transfected with Kbml and KhE and pulsed with OVA. The C n s generated were found able to kill OVA-pulsed Khl and Khs-nansfected 'I2 cells as well as Kbtransfected T2 cells. However, limiting dilution assay revealed that Kb-mutant mice had a 20-fold lower frequency of OVA-specific CTL precursors. Furthermore, we found that CTLs from Kb mutant mice were more dependent on CD8 for killing, especially if the targets were not syngeneic. These results suggest that the lack of recognition of certain peptides presented by Kb mutant molecules is a result of conformational differences. Under controlled conditions, major histmompatability complex class I and class I1 proteins can fold in vivo and exist on the surface of cells without their normal peptide ligands. Class I molecules can fold in virro, but the folding reaction seems to absolutely require peptides which are specific ligands of the class I protein. Class I1 a and p subunits produced in E . coli can also fold in vino in the presence of a peptide, but the issue of a requirement for a peptide in the class I1 folding reaction has not been resolved. We now show that heterodimers can be isolated from class I1 I-Ek folding reactions which do not include peptides, and that these molecules will subsequently bind specific peptide ligands. The pH dependence of peptide binding for the "empty", in vitro folded I-Ek heterodimers is apparently identical to that of soluble "empty" I-Ek produced in a lipid-linked form in CHO cells. We conclude that at least some class I1 proteins contain all of the information in their primary sequences necessary for folding. 

Drosophila cells were cotransfected with these cDNAs, a ml32m-pRMHa construct and a neo resistant plasmid. Stable transfectants were selected with G418 and cells that expressed soluble T13G M3 and CDl.I-L12m complexes were obtained. The soluble CDl heterodimers were used to screen a random peptide phage display library (see Miller e t al. abstract) to identify peptides that bind the non-classical class I CDl heterodimer. The sequences of the clones that bind to CDI describe a peptide motif which is heterogeneous in length at the Nterminus and contains three anchor residues in a seven aminoacids long stretch. Using codon-based deletion mutagenesis on selected peptide phage display clones we have determined the importance of the N-terminal positions to C D l -peptide binding. Surface plasmon resonance studies allowed direct measurement of peptide binding in solution and the determination of affinity constants. Alanine scanning mutagenesis on synthetic peptides will be done to verify the relative contribution of the putative anchor residues to the binding to CD1.

V 108 Mary Lynne Hedley, Robert G.Urban, and Jack L.

Strominger, Harvard University, Department of Biochemistry and Molecular Biology, Cambridge, MA 0 2 1 3 8 Class II heterodimers assemble in the endoplasmic reticulum (ER) with the help of at least one other protein, invariant chain (li). li is thought to prevent the class I 1 pocket from acquiring peptide in the ER and furthermore, directs class I 1 to the endosomal compartment of the cell. Here, li is released and peptides generated from endocytosed proteins bind to class II molecules. We have developed an in vitro system to study heterodimer formation and peptide binding. cONAs encoding invariant chain, DR alpha and DRl beta chains were independently cloned into RNA expression vectors. Minigenes encoding the DR alpha signal sequence followed by sequences coding for one of three peptides were generated by overlapping PCR. Two of the peptides encoded by the minigenes are known to bind DRl, whereas the third peptide does not bind. mRNA was transcribed from these constructs and was added to a rabbit reticulocyte lysate translation mix containing canine pancreatic microsomal membranes. Translation of each product was assayed by SDS-PAGE. Co-translation of alpha and beta chain mRNA followed by immunoprecipitation with LB3.1, a monoclonal antibody specific for alphdbeta heterodimers which does not react with single chains, demonstrated that DR alpha and beta assemble in the microsomal membranes in the absence of li. Co-translation of peptide minigene, DR alpha and beta mNRAs followed by immunoprecipitation suggest that certain peptides can interact with class II molecules in the absence of invariant chain.

heterodimedli complex and was used to determine if OR1 assembled with Ii in vitro. Co-translation of all three mRNAs (alpha, beta and invariant) followed by immunoprecipitation with DA6 .147 demonstrated that the OR alphdbeta complex assembles with li in vitro. Data from additional experiments using the in vitro system suggests that at least some peptides can compete with li for binding to alphdbeta complexes in the ER. restricted T cells has been demonstrated by us, to react with MHCassociated, haptenated peptides largely independent of the carrier peptides' amino acid composition (J. Immunol. 149 (1992) 2569 and 151 (1993) 678). It was then argued that chemically trinitrophenylated cells might present an unusually repetitive pattern of TNP-epitopes to such T cells, and that this might relate to the hapten's pronounced allergenic properties. However, allergic responses such as contact sensitivity are widely discussed as being mediated primarily by T cells of the CD4+, class I1 MHC-restricted compartment. We have therefore produced TNP-specific T cell lines and hybridomas of the CD4+ phenotype, restricted to I-Ab, LAd or I-Ed encoded class I1 molecules. Our cells were selected for antigenic reactivity to TNBS modified irradiated syngenic spleen cells. 25 % of our hybridomas and 50 % of our T cell lines were reactive to syngenic spleen cells modified with TNP-protein digests. We also screened several synthetic peptides. known from the literature to bind to I-Ab, I-Ad or I-Ed in which lysine residues were e-N-alkylated by TNP. The same peptides without TNP were not antigenic for our T cells and the recognition could be inhibited by TNP-or MHC class 11-specific antibodies. Some of our T cells crossreactively reacted with TNP-BSA and one of our synthetic TNPpeptides. We computer-compared the sequence of this peptide with the sequence of BSA. The three most similar TNP-BSA-peptides revealed maximally 31 % identity (46 % similarity) to the synthetic peptide, but were not antigenic for our crossreactive T cells. The antigenic peptide within the TNP-BSA sequence must, therefore, be of still lower homology to our antigenic TNP-peptide than the three synthesized sequences. As for class I, also class I1 MHC-restricted TCR, thus apparently may interact with TNP-epitopes in a carrier-independent fashion. We take this to further support our hypothesis that the strong antigenic, and possibly the allergenic properties of haptens such as TNP may be influenced by the repetitive type of hapten-determinants on different carrier peptides resulting from chemical cell modification. In the human, HLA-DR molecules can be clustered into groups based on shared common serologic determinants. Membars of a group (microvariants) differ only subtty from one another in B chain amino acid sequence. One intriguing and complicated group of molecules bear both D R l l and DR13 serologic determinants. To study the effect of microvariation on function, 17 DR11-and DR13expressing wild type and mutant murine L cell transfectants were generated. Mutant DR B chain cDNAs were created by altering amino acids in and around the antigen binding groove. The substitutions utilized amino acids found at the same position in other microvariants in the same group. Cells expressing DRBl*llOl/ *1303/*1304 bound HA 307319 peptide at a higher level than cells expressing other wild type molecules. When DR(u,B1*1304) was mutated to alter position 57 or 58, HA binding was decreased 5 fold. Changing positions 32 and 37 in DR(o,Bl*1304) to residues found in DR(u,B1*1301) reduced HA binding to the level observed for DR(u,B1*1301). Interestingly. changing these positions in the reverse direction ("1301 to *13C4) also decreased HA binding. These results suggest that single amino acid residues in the groove can affect binding; however, peptide binding is also influenced by the milieu of the antigen binding groove of each DR molecule. The effect of microvariation on allorecognition (and sew peptide binding) was assessed using alloprolierative T cell clones (TLC) (R:DRB1*1101, S:DRB1*1102 and vice versa). Some of these TLC recognize L cells expressing DRB1*1102 as well as cells expressing DR13 allelii products. The patterns of recognition suggest that recognition involves the shared u helical regions of these DR molecules as well as a peptide component. These observations demonstrate the power of apparently subtle evolutionary changes in creating this diverse repertoire of antigen binding molecules.

Clone AH11112-2 is an HLA-A2.1 -restricted, xenoreactive CTL clone isolated from a C57BU6 (H-Zb) mouse. These CTL efficiently lyse EL4/A2.1 transfectants pulsed with the peptide ALWGFFPVL, corresponding to the naturally occurring epitope identified by mass spectrometric analysis (as AXWFFPVX) of peptides eluted from purified A2.1 molecules. Molecular modelling of the A2,VALWGFFPVL complex suggested that peptide residues P4 and P6 are appreciably solvent exposed, and might form impollant TCR contacts. To address the predictions of this model. we examined the ability of variously substituted derivatives of ALWGFFPVL to sensitize target cells for lysis by clone AH11112-2. Unexpectedly. single substitutions at most positions only minimally affected target lysis. while substitutions Ala-P3 and Ala-P5. occurring at positions expected to be nearly or completely buried in the MHC groove, each gave rise to reduced cell lysis while retaining high affinity (lC5,,-10nM) binding to A2.1. However, Phe-P3 and Tyr-P5 substitutions gave results comparable to the parent peptide. Guided by these initial results. we constructed a limited synthetic peptide library comprised of 1296 different peptides having 6-fold degeneracy at positions P3-P6. in order to examine an expanded array of potential epitopes. The HPLC-fractionated library was tested for its ability to sensitize target cells for lysis by clone AH11112-2. This analysis revealed two prominent. late-eluting peaks of activity. one of which corresponds to the retention time of ALWGFFPVL. as welt as lesser peaks at earlier retention times. We are currently charaderizing these peaks. Our observations thus far indicate that TCR recognition of the class llpeptide epitope shows a non-rigid dependence on peptide sequence, and are consistant with recent observations of others who have suggested that TCR recognition may be sensitive to peptide-induced MHC conformation. However, it is equally plausible that residue substitutions alter the conformation of the MHC-bound peptide, consequently altering T cell recognition. Additionally, our results suggest the utility of synthetic. combinatorial peptide libraries as a means to identify immunologically active and potentially useful peptides for a given T celVtarget system in the absence of information regarding the exact sequence of the naturally occurring epitope. Random peptide phage display libraries were screened with empty soluble class I molecules (purified from Drosophila melanogaster cells which were cotransfected with p 2-microglobulin) following enrichment of candidate phage by panning. Sequencing of positive clones allowed rapid delineation of peptide motifs displayed by these phage. The peptide motifs described are consistent with data obtained from: crystal structures of class I peptide complexes, peptides eluted from cellular class I molecules, and class I-restricted peptide antigen studies. In addition to providing "anchor residue" information, this method provides important binding data concerning the contribution of amino acids which lie proximal to these anchor residue positions. Screening of murine H-2Kb-specific clones with empty class I molecules representing alternative alleles (H-2Kbm1, H-ZKbmB), yields information concerning the requirements for both shared and allele-specific discrimination in peptide binding. Surface plasmon resonance studies allowed direct solution competition measurements of various peptide motif analogs, and were utilized to quantify binding. Murine HMT, CD1 molecules, and several H-2Kb alleles are among the class I molecules successfully screened using this technique. The N-terminal peptide, Acl-11, of myelin basic protein (MBP) can induce experimental autoimmune encephalomyelitis (EAE) in PL/J and (PL/JxSJL/J)FI mice, but cannot in SJL/J mice, since Acl-1 1 binds to LA", but not to I-As. We have studied the interaction of Acl-I1 and I-A" as a model system for therapeutic intervention of the autoimmune response seen in EAE. In this study we have identified a major interaction between the arginine at position five in Acl-11 and glutamic acid at position 74 in the beta chain of I-A". We have also identified two residues that differ between I-A" and I-AS that are critical for peptide binding to I-AU. These two residues, positions 26 and 28, lie on the bottom of the peptide binding groove on the beta chain. Mutants that contain an AaU chain paired with an ADS chain which has only these two residues changed to those of APU bind, Acl-I1[4A], a high binding analog of Acl-11, with much greater affinity than does the wild type I-AU. Furthermore, when position 70 in the beta chain, which lies on the helix, is changed to an arginine, as ia found in I-A", binding is reduced to wild type levels. Arginine at position 70 in the beta chain, however, is required for recognition by four of five Acl-I I , I-AUspecific T cell hybridomas that we have tested. The requirement of arginine for the four T cells tested correlates with the presence of a negatively charged residue in the CDRZ region of the T cell receptors, suggesting that the CDR3 regions of the T cell receptors may recognize this residue as well as the peptide. Residues 26, 28, 70, and 74 of the beta chain of I-A" lie in close proximity and may form a pocket that allows Acl-I1 to bind to I-A". These results have provided us with a better understanding of how Acl-I1 sits in the groove of I-AU and will help us to design immunotherapeutic molecules that can specifically interfere with the interactions we have identified between the peptide and MHC. Class I molecules bind peptitles based on the presence of ;inchor residues, whose position and identity depend on the individual class I molecule. We have recentl) detemiiiied peptide-binding motifa for I-ILA-A1, -A3, -BX, -B14 and -BwJI, and have used these motif? to identify new antigenic peptides. I n order to incre:ise the predictive power of tl.ese motifs, u e have developed a ni:ithemsticiil model th;tt w e c:ill the independent binding of sidec1i;iins (IBS) hypothesis to interpret peptide-binding d;it;t. According to tlie 1BS hypothesis, ench amino acid within tlie peptide cmtributes to binding in a f;ishicin that is independent of 11ie rest of tlie peptide. To test the IDS hypothesis, a11 equation is writien out for c:icIi peptide for which binding data is ;Ivnil;tble, in which the for tiiasociatioii is set equal to the product of coc.l'iicietit~ that rspresent the quantirutive importance of each reaitlue. The coefficients are detemiined by solving the set of simii1t;ineous equations coiisi	ing of all of the avsihble binding d:it:i. 13:it:i oti nonbiti~litig 2cptidc.s :ire :ilso useful because they pl:ice :in uppcr limit oii the vdiies of cert:iiii coefficients. When thebe c~i l c~~l~i t i o~~s :ire pcrl'ortiied on peptidebinding data for HLA-A?, it is found th:u tlic. IBS hypothesis :iccouiits for tlie be1i:ivior of tlie v:ist m:ijority of thi: peptides, indicating that signific.uit sidrcliain-sidecli;iiii ititer;ictiotis are unusual. Msny antigenic peptidcs bind tightly to HLA-A2 compared to the pi-edictal binding affinily of all other peptides from the same protein, indic:iting antigenic peptides :ire often among the best possible binding prptides. Occasional antigenic peptides are of rather weak relative affinity, suggesting that these peptides may be processd unusu:illy efficiently, or :~ltern:itively, that certain T cells :ire originally :ictivated by ;I higher affinity peptide, followed by cros\-re:ictive recogiiitioli. University Hospital, Leiden, The Netherlands. Two naturally occuring DR3 molecules dffer by 4 8-chain amino acid residues (positions 26, 28, 47 and 86) whose side chains point into the class II antigen binding groove. Previous analysis has shown that positions 26, 47 and 86 influence recognition by alloproliferative T cell clone suggesting that the functional differences between the DR3 microvariants are mediated primarily through the influence of the variant positions on peptide binding. Murine fibroblast cell lines expressing DR(u,B1*0301), DR(u,B1*0302) and 11 mutant DR3 molecules (created by exchanging the amino acid residues found at the variant DR3 positions) were examined for their ability to bind peptide antigens. The binding of HA 307-319 (DR(u,81*0302)specific) was affected only when position 28 was mutated in conjunction with position 26 or 86 suggesting a cooperative interaction between these positions for HA binding to DR(u,,91*0302). Two peptides (HSP3-13 and SWM 132-151) that are DR(u.81*0301)specific utilize different Combinations of variant DR3 amino acid residues for binding. Changing position 28 or 86 alone significantly reduced HSP 3-13 binding to DR(u,B1*0301) which suggests that both positions are equally important for binding HSP. All of the mutant molecules examined, including single residue mutant 0302 molecules, were able to bind SWM to some degree indicating that all four variant DR3 positions interact with SWM; thus, only by changing all four residues (0301 to 0302) is binding completely abrogated. These data suggest that different peptides utilize different combinations of DR contact residues for binding and may explain why not all peptides isolated from an individual DR molecule contain a similar binding motif. Finally, it is clear that subtle evolutionary changes which created such DR microvariants result in dramatic differences in the peptide repetoires that bind to these molecules. Gp160 and gp120 specific CD4+ T lymphocyte lines were developed and evaluated from I I HIV seropositive volunteers enrolled in a double blinded phase I1 gp160 vaccine trial. We were able to generate gpl60 specific T cell lines in 8/11 and gp120 specific T cell lines in 11/11 volunteers after 3 cycles of stimulation. Each lines was started from PBMC collected 150 days from the beginning of the trial. We challenged the specific T cell lines with a panel of peptides covering constant and variable regions of gp160. We arbitrarily characterized the response as broad or narrow according to the number of peptide pools the antigen-specific T cell lines were able to recognize. All but one gp160 specific T cell lines were able to recognize V3 peptide pool. Moreover 3/7 narrow responders showed the presence of p24 in their culture supernatants demonstrating active viral replication and 014 broad responder had detectable levels of p24. Despite the lines being dev-eloped with IIIB antigens, all of the lines which were tested were able to recognize gpl20(MN). Taken together these preliminary findings, observed in this unique cohort of patients, provide new insight into the character of gp120 peptide recognition by T lymphocytes. This information not only contributes to a better understanding of the functional capabilities of the cellular immune response, but will in the future, help determine the capacity of vaccines to modify this response. MHC class I molecules bind short peptides for cytotoxic T cell scrutiny at the cell surface The peptide binds to a groove formed by the a 1 4 domains of class 1 molecules. Peptide binding occurs in the endoplasmic reticulum (ER) where class I molecules also associate with b2-microglobulin (P2m) through their a3 domains. Although p2m is necessary for efficient transport of the complex to the cell surface, whether it is required for efficient peptide-class I binding is still in question Moreover, the events that occur to form the trimolecular complex (i e , class LP2m-peptide) are not yet hlly understood, since work with purified molecules has been hampered by technical ditficulties, such as obtaining preparations of class 1 molecules free of peptides and developing an experimental system that reilects results obtained with intact cells or cell lysates We have used purified, full-length, class I Db molecules isolated from the cell line M A -S (RMA-S-Db) to obtain efficient, specific binding of peptides in the presence or absence of p2m. Peptide binding was determined by a1 domain refolding, as measured with conformationspecific monoclonal antibodies (mAb) in an ELSA assay system In a second set of experiments, Db molecules isolated from RMA cells @MA-Db) and subjected to heat (H-Db) or acid (A-Db) treatment experienced unfolding of both a1 and a3 domains. A-Db regained the al domain conformation upon addition of specific peptides, but, surprisingly, reappearance of the native conformation of the a3 domain also occurred only upon addition of Db-restricted peptides. While H-Db retained the ability to undergo peptide-induced a1 refolding, the native conformation of the a3 domain could not be rescued. These data indicate that first, pZm is not necessary for peptide binding; second, acquisition ofthe native conformation of the al domain of class I MHC is dependent on specific peptides, but not on the structure of the a3 domain, and, third, specific peptide binding affects not only the tertiary structure of the a1 domain but also affects folding of the more distal a3 domain. 1993 ) reflects the conformation adopted by the heterodiier after having bound peptide. However, it is clear from a number of analyses that the conformation of the peptidefree, or "empty" class I1 heterodimer differs from that of the "full" heterodimer. Since it is the "empty"

conformation of the class II molecule that must initially bind peptide, we have been interested in determining how this conformation differs from that of the fully folded, peptideassodated class II molecule. Using a broadly DW specific antibody, TAL14.1, we have found that the determinant recognised is dependent on the conformation of the B chain but is also present in monomeric DRP chains except that of the DRB5' 0101 chain. The failure to bind to the monomeric DRB5"OlOl chain is not simply as a result of the loss of the epitope, since TALl4.1 does bind to the peptide assodated DRB5'0101 ap heterodimer, but results from the conformational lability of this molecule compared to other DRB chaiis. We have determined that the instability in the DRB5' 0101 chain is a consequence of ionic interactions between aspartate residues, two of which are unique to the DRB5W101 sequence. By mutating these residues we have increased the stability of the p chain and restored binding of TAL14.1. From the positions of the aspartate residues in the structure of the class II molecule we can conclude that at least the first three p strands and the short (pl) a helix are formed and must interact, suggesting that they are in an orientation similar to that in the native conformation.

PEPTIDES, Robert G. Urban, Roman M. Chicz, and Jack L.

Strominger. Harvard University, Department of Biochemistry and Molecular Biology, Cambridge, MA 02138

The predominant peptides bourid to MHC class I1 molecules expressed on human B cells are derived from a relatively limited number of self proteins. We have performed a set of experiments to determine which, if aiiy of these bound self-peptides are uniquely released at pH 4.0 and thus may be released during MHC class I1 recycling in vivo or more readily exchanged than others during pH binding experiments in v i m .

To avoid the complications arising from the presence of detergent, intact immunoaffinity purified HLA-DR1 molecules were digested with papain to remove the cytoplasmic tail and transmembrane domain. These highly purified water soluble HLA-DRI molecules were exposed to differing pHs in the presence or absence of high-affinity synthetic peptide. The resulting bound peptides were then acid extracted, and separated by reversed-phase HPLC. Using a combination of mass spectrometry, amino acid analysis, and ultraviolet spectroscopy the ratio of prebound self-peptides to newly bound synthetic peptide was determined. The results unequivocally demonstrate that most of the predominant self-peptides bound to HLA-DRI are not appreciably released during extended exposure to acidic pH. However, some, but not all, forms of invariant chain derived peptides are substantially reduced after extended acidic exposure. These findings provide new insight into the processes of peptide exchange in vitro and imply that the prebound self-peptide repertoire would provide a formidable obstacle to productive recycling of human MHC class I1 molecules in vivo. MHC class &restricted T cell recognition of systematically shortened synthetic peptides derived from a 13-residue T cell epitope was investigated. All 65 possible analogues with a size between 3 and 12 residues were prepared with uncharged termini and with free termini. When the peptides were tested for recognition by a panel of HLA-DR2 restricted T cell clones, a dynamic interaction of MHC and TCR contact residues was deduced. Sometimes, the addition of a C-terminal amino acid residue was found to compensate for the truncation of an important N-terminal residue, and vice versa. Occasionally, deletion of additional terminal residues from a peptide that was non-stimulatory for a T cell clone resulted in a peptide that was recognized again by the clone. To the charged analogues, very few positive responses were recorded. Finally, non-overlapping sub-regions of OMP(49-61) were identified, capable of activating a single T cell clone independently.

The results are discussed in the context of a three- Binding of peptides to MHC class II molecules that are affinity purified o r on the surface of cells has been studied. In neither case do the experimental conditions resemble those in endosonies where peptides bind to class I1 molecules after removal of the invariant (Ii) chain.

We have developed an experimental system that allows us t o more closely approximate conditions for peptide binding to DR4Dw4 as they occur in endosomes. CHO cells expressing DR4Dw4 (SE24 cells) were transfected with DNA coding for a truncated form of the li chain lacking the first 20 amino acids (SE24A20 cells). Similar to fibroblasts expressing DRlDwl and the truncated li chain (Roche EMBO I. 11: 2841;1992), we observed that the li chain is expressed at the cell surface and that the majority of DR4Dw4 molecules is complexed with the Ii chain.

SE24 cells present peptides [two derived from the human immunoglobulin K chain (amino acids 145-159 and 188-203) and one from the HLA-A2 heavy chain (amino acids 52-70)) efficiently to their corresponding DR4Dw4 restricted T cell hybridomas. whereas SE24A20 cells present peptides very poorly.

After exposure of SE24A20 cells to pH5.5 in the presence of EDTA and cysteine they became more efficient in presenting peptides.

I t was also found that this treatment preferentially removed the invariant chain from the cell surface and that li chain loss did not occur when a mixture of protease inhibitors was present during treatment.

Data will be presented to show the effect of individual protease inhibitors on invariant chain removal. thus allowing the identification of the classes of protease responsible for this. Intracellul;; processing of viral proteins produces peptich which associate with class I MHC and are presented to host cytotoxic T cells (CTL). Such virus specific CTL are crucially important in the resistance to many virus infections, and are thought to be important in controlling human infection with papillomavirus type 16 (HPV 16), a virus associated strongly with cervical cancer. We have studied mouse CTL response to the transforming proteins E6 and E7 of HPV 16.

Mice were immunised with recombinant vaccinia E7 (vac E7) and T cells were boosted in vitro with E7 transfectants or vac E7 infected stimulators. H-Zb, H-2d and H-2k mice immunised with vac E7 developed E7-specific CTL which killed vac E7 infected targets. An epitope was mdpped by CTL of H-2b mice to a region between amino acids 46 to 65 using transfectants contain E7 N-and C-terminal half constructs and a set of overlapping peptides spanning E7. The boundary of the epitope in H-2b mice was defined to E7 amino acids 49-57. This epitope binds to H-2Db, but has only a weak H-2Db motif. E7 peptide 21-28 contains a strong Kb binding motif, but is not recognized by E7 specific CTL from H-2b mice.

We are currently mapping epitopes recognised by E7 specific H-2d and H-2k restricted CTL. To date, none of the overlapping peptides have been recognised, raising the possibility that a host cell protein is involved as a target antigen.

LYMPHOCYTES. Andrew, D.P., Berlin, C., Hamaan, A. and E.C. Butcher. Department of Pathology, Stanford University School of Medicine, Stanford CA 94305.

In this report we examined expression and function of a4p7 on lymphocytes using a panel of mAbs to this integrin. We looked at the blocking activity of this panel of anti-a4P7 mAbs in several in vim adhesion assays and also mapped the epitopes recognized by the panel of mAbs in competition studies. From the results we conclude that there are unique although overlapping epitopes on a4p7 for each of its various adhesive interactions with MAdCAM-1, fibronectin, VCAM-1 and an as yet uncharacterized ligand on lymphocytes for a4p7. Using a novel mAb to a combinatorial epitope on a4p7 we further show that a4p7 is the major homing receptor on lymphocytes for homing to mucosal lymphoid organs.

In terms of expression p7 proved a useful marker in T cell development, been expressed on mature single positive CD4 and CD8 thymocytes but been absent from double positive CD4+vCD8+ve thymocytes, although a minor subset of double negative CM-VeCD8~ve thymocytes expressed p, . The majority of P7Qe thymocytes expressed p7 as a& while a minor non overlapping subpopulation of thymocytes expressed a4P7 at low levels. In the bone manow expression of 87 could be used to subdivide the HSC population Finally, in the periphery, p7 was expressed at high levels on subsets of memory (activated) memory CD4 lymphocytes and using LSelectin a4P7 and a4pI expression it is evident that there are at least 4 subsets of memory CD4 lymphocytes which will differ in their Rafficking properties due to the differences in the homing receptors that they express VLA molecules (0, integrins) are cell-surface ap heterodimers that bind to extracellular mamx (ECM) proteins such as fibronectin and merosin. We demonstrate that the avidity between VLAs and their ligands is down-regulated during T lymphocyte maturation in the thymus. Mouse thymocytes were fractionated into immature and mature populations based on their expression of the heat stable antigen (HSA), recognized by the J1 Id mAb. Virtually all thymocytes expressed VLA-4, -5 and -6.

However, predominantly immature (HSA') thymocytes exhibited ECMbinding capacity in vitro. This constitutive binding was down-regulated at the HSA' to HSA transition stage. The low binding of mature HSAcells was not due to decreased levels of VLAs on the cell surface or permanent protein modification since HSA-cells were able to become adherent cells after stimulation with both PMA and ionomycin. Binding to fibronectin was inhibited by RGD and CS-1 peptides, suggesting that both VLA-4 and VLA-5 are thymocyte receptors for fibronectin. Binding to merosin was inhibited by antibodies recognizing VLA-a, or -PI, suggesting that VLA-6 is a merosin receptor. The constitutive binding of immature HSA' thymocytes to ECM was inhibited by protein kinase C (PKC) inhibitors, indicating that PKC plays an important role on regulation of thymocyte adhesion during T cell development. We postulate that a down-regulatory mechanism is responsible for the decrease ECM-binding of mature HSA-thymocytes, and that this mechanism may play an important role in allowing mature thymocytes to leave the thymus and emigrate to the periphery. The molecular mechanism involved in this down-regulation will be discussed. Migration of lymphocytes into sites of inflammation is known to be mediated by adhesive interactions. Additionally, it has long been suspected that a chemoattractant secreted at sites of inflammation might also play a role in recruiting lymphocytes from the bloodstream into the inflammatory lesion. However, the role of chemoattractant~ in lymphocyte transendothelial migration into inflammatory sites has been poorly characterized. We have utilized a novel transendothelial lymphocyte chemotaxis assay to identify and purify a lymphocyte chemotactic factor in supernatants of mitogen-stimulated peripheral blood mononuclear cells (PBMC). The factor was purified by heparin-Sephai-ose, size exclusion, and HPLC chromatography. Amino acid sequence analysis revealed it to be identical to monocyte chemoattractant protein-1 (MCP-I), a chemoattractant of the C-C chemokine family that had previously been described a\ monocyte specific. We confirmed that recombinant MCP-1 is chemoattractive for purified T lymphocytes and for CD3+ lymphocytes i n peripheral blood lymphocyte (PBL) preparations. Furthe]-more, we showed that the majority of T lymphocyte cbemotactic activity in mitogen-stiinulated PBMC supernatants is neutralized by antibody to MCP-I. Phenotyping of chemoattracted T lymphocytes ahowa that they are an activated. memory subset, expressing the antigens CD26, CD4SRO, and increased levels of CD29. The chemoattracted population is depleted of cells expressing L-selectin and CD4SRA. The T lymphocyte response tn MCP-I is dose-dependent, with maximal chemotaxis occurring at SO ng/ml. The response is chemotactic, rather than chemokinetic. as demonstrated in a checkerboard assay. Furthermore, the response to MCP-I by T lymphocytes is not dependent on the endothelium preaenc in our chemotaxis assay system since the response can be duplicated using uncoated filters. We conclude that MCP-I is the nia.joi lymphocyte chemoattractant secreted by mitogen-activated PBMC. MCP-1 is capable of acting as a potent T lymphocyte, as well a\ monocyte, chemoattractant and this may help explain why monocyteh and T lymphocytes of the memory subset ai-e always found together at sites of antigen-induced inflammation.

The p1 or VLA integrins are ap heterodtmeric cell surface receptors that play an important role in T cell function by mediating adhesion to extracellular matrix proteins such as fibronectln (FNI and cell surface counter-receptors such as VCAM-1. A critical but poorly understood mechanism of p l lntegrin regulation involves the rapid upregulation of the functional activlty of p l integrins by T cell activation without corresponding changes in p l integrin cell surface expression. Although studies of pZ integrins have implicated the p2 cytoplasmic domain as a target for intracellular signals that upregulate PZ integrin activity, similar analysis of p1 Integrin activity has not been conducted. Like peripheral T cells, activation of the Jurkat T cell line with PMA or mAb crosslinking of either CD3. CD2 or CD2S results in increased Jurkat adhesion to FN via the a4pl (VLA-41 and a5pl (VLA-51 integrins. Using y-irradiation and multiple selections by panning for mutants that have lost the ability to bind to FN upon PMA stimulation, we have isolated a Jurkat derivative. designated Al, that lacks expression of the p l chain. FACS analysis of mutant A1 reveals that loss of cell surface p1 expression also results in complete loss of cell surface expression of the a3. a5 and a6 chains.

Compared to parental Jurkat cells. cell surface expression of a4 is reduced but not eliminated in mutant Al. presumably due to asscciation of a4 with p7. Functional studies show that mutant Al. either unstimulated or PMA-stimulated. is completely unable to bind to either FN or VCAIv-1. lmmunoprecipitation studies using metabolically labeled lysates also show no expression of the p1 chain but normal levels of a4 expression. Northern blotting analysis reveals reduced levels of pl mRNA in muLant A1 compared to parental cells. ReintroducLion by DNA-mediated gene transfer of a full-length p l cDNA results in reexpression of the p l chain on Lhe cell surface. although not to levels seen in parental Jurkat cells. However, adhesion studies show that these trandectants are now able to bind to FN or VCAM-1 and this adhesion can be upregulated by PMA stimulation or CD3 crosslmking. Thus, mutant A1 represents a unique cellular reagent for the analysis of T cell p 1 integrin structure and function. To allow in uiuo studies, which would provide more insight into CD27 function, we have identified murine CD27 (mCD2T) at the cDNA and protein level. The mCD27 gene was mapped to mouse chromosome 6, tighly liked to Ly-4, Tnfr-2 and Hcphl. mCD27 is 65% identical to human CD27 (hCD27). Highest homology of 80% is found in the amino terminal cysteine rich, ligand binding domain, and the carboxy terminal part of the cytoplasmic domain. The structure and expression of mCD27 was compared to that of 4-1BB. another lymphocyte specific member o f the NGF-R family. mCD27 and 4-1BB are 399:

identical in the ligand binding domain and have a high degree o f homology in the carboxy terminal part of the cytoplasmic tail. mCD27 mRNA was detected in spleen and thymus, in CD3-/dUiI as well as CD3br'ght thymocytes, but not in non lymphoid tissues; mCD27 mRNA expression was found in resting T cells and was upregulated upon activation. 4-1BB was detected exclusively in activated T cells, where it was expressed with different kinetics than mCD27. Peptide antisera identified murine CD27 as a 45 kDa protein on thymocytes and activated T cells and 4-1BB as a 3540 kDa protein on activated T cells. Mouse complement receptor 1 and 2 (MCR1 and MCR21 are receptors for mouse C 3 and are considered to be the 6 lymphocyte homologues of human CRl and CR2. MCRl also acts as a cofactor for factor I mediated cleavage of C3, a process necessary to generate the C3d ligand form of C 3 which then binds CR2 with high affinity. A s opposed to human CR1 and CR2, however, which are products of unique genes, MCRl and MCRZ are the alternatively spliced products of a common mRNA. MCRl shares with MCR2 the COOH-terminal 15 short consensus repeats (SCRI but has six additional NH,-terminal SCR. To further determine the relationship between human and mouse CR1, and identify C 3 binding and factor I cofactor activity sites, we analyzed mousehuman chimeras in which the C 3 binding domain of human CR2 has been replaced by different regions within the first eight SCR of MCRl . Rosette analysis of our chimeras with erythrocytes bearing different mouse C3 fragments revealed a weak C3b binding site within SCR 1-2 of MCRl . There is no independent C 3 b binding domain within SCR 3-6, but their presence enhances C3b rosette formation to chimeras containing SCR 1-2. 8C12. a mAb which partially blocks C3b interaction with MCRl, binds only chimeras containing SCR 3-4. There is no C3d binding area within the first six SCR, but our data confirm previous studies demonstrating the presence of an additional C3blC3d binding region within SCR 7-8 of MCRl (SCR1-2 of MCR21. Cofactor activity for C3 cleavage is in the first four SCR of MCR1. In summary, like human CR1, MCRl contains two independent C3b binding sites and has unique cofactor activity. These results further support our hypothesis that these two molecules are functional homologues of human CR1 and CR2 despite their pronounced structural differences.

Callebaut and A.G Hovanessian, Institut Pasteur, Unite de Virologie et Immunologie Cellulaire (UA CNRS 1157), 28, rue du Dr. Roux 75015 Paris France

The external or surface (SU) and transmembrane (TM) envelope glycoproteins of HIV, are involved in the mechanism of HIV entry into cells and when expressed on the membrane of infected cells have the capacity to trigger cell death by apoptosis through interactions with cell-surface CD4 molecules. Recently, we demonstrated that entry of HIV requires the presence and functioning of the T cell activation antigen, CD26. Here we suggest that CD26 may also be involved in the mechanism of initiation of apoptosis by the SU/TM complex in CD4 expressing cells.

Previous reports from several laboratories have indicated that CD26 may contribute to T cell activation, and that its dipeptidyl peptidase IV activity may be required during this process. Accordingly, stimulation of CD26 with a specific monoclonal antibody results in a comitogenic effect on T cell activation. This and the observation that CD26 is found to be associated with CD45, suggest that the CD26/CD45 complex through tyrosine kinase-dependent transduction pathways may play a regulatory role in T lymphocyte activation. The HIV envelope SU glycoprotein, through the interaction of its V3 loop with CD26, could probably interfere with the normal functioning of CD26 during the T cell activation process and thus result in an abnormal signaling which is associated with apoptosis. Several reports have shown a selective decrease in the proportion of CD26 expressing CD4+ T lymphocytes in HIV-1 infected individuals. This latter is consistent with the requirement of CD26 for HIV entry, and the fact that HIV producing cells in vitro cultures die by apoptosis. Taken together, these observations favor the hypothesis that CD26 is implicated in the mechanism of triggering apoptosis in CD4 expressing cells by the SU/TM complex of HIV. Ly-49 is a recently identified cell surface molecule expressed on a subpopulation of NK cells and certain T lymphomas. It has been suggested, based on g e n e transfection and antibody blocking studies, t h a t Ly-49 is a negative regulator of NK lytic activity, possibly t h r o u g h an i n t e r a c t i o n w i t h t a r g e t cell class I molecules. W e have found t h a t T lymphomas expressing Ly-49 bind isolated class I MHC molecules b u t n o t class I1 molecules immobilized on plastic. Adhesion t o class I molecules was n o t found with T lymphomas lacking Ly-49 expression. T h e Ly-49 expressing EL4 lymphoma bound Dd and Dk b u t not K d and Kk, demonstrating a restricted p a t t e r n of class I adhesion t h a t is consistent w i t h t h e specificities previously suggested for Ly-49 in NK lytic assays. The observed cell adhesion is class I density d e p e n d e n t and is extensively inhibited b y t h e A1 monoclonal antibody directed against Ly-49. These results provide direct evidence for Ly-49 serving as a receptor for a subset of class I MHC molecules. L-selectin is the principal homing receptor that directs the homing of blood lymphocytes to peripheral lymph nodes via high endothelial venules. It is involved to a lesser degree in lymphocyte homing to Peyer's patches. We have demonstrated rapid changes in L-selectin epitope expression early (10 minutes) after activation of blood B cells in vitro. Using three different monoclonal antibodies specific for the L-selectin molecule (TQ1, Leu& and FMC46) we found that most freshly isolated blood B cells express TQ1 and FMC46, whereas only an average of 60% express Leu8. After activation with pokeweed mitogen in vitro we observed a rapid increase in the number of B cells expressing Leu8, almost to the same numbers as for TQ1 and FMC46. This suggests that some B cells express an alternative form of L-selectin, not recognized by the Leu8 antibody, and that this can be rapidly modified upon activation, possibly by a conformational change. The number of B cells positive for FMC46 and TQ1 remained almost constant at 95% during the first 24 hours post activation, whereas the number of B cells expressing the Leu8 epitope of L-selectin after 24 hours were only 40%. Thus, the activation-induced up-regulation of the Leu8 epitope of Lselectin is transient. Our data indicate that previously described functionally different subsets of B cells based on Leu8 expression are 1) not reflecting L-selectin expression and cannot be reproduced using other L-selectin antibodies, and 2) are reversible, and may reflect the activation status of a given cell. It has been demonstrated that in purified B cells IgE synthesis is induced by a T-cell derived cytokine, IL-4 and CD40 ligand (or anti-CD40). Since these stimulants cause a strong aggregation of B cells, we examined the expression of adhesion molecules such as ICAMl(CD54), LFA-I(CDlldCD18p) and CD43. The incubation of purified B cells from tonsils with anti-CD40 plus IL-4 strikingly enhanced the expression of ICAMl and CD43 but modestly affected the expression of LFA-1 by FACS analysis. The addition of anti-ICAM1 inhibited its aggregation and, suprisingly, enhanced IgE synthesis by such stimulated B cells at 5-10 fold. This enhancing effect of anti-ICAM1 on IgE synthesis was achieved by the increase in the expression of germline transcript of CE gene. However, the mechanism through which anti-ICAM1 affected the germline expression remained to be determined. Wiscott-Aldrich syndrome (WAS), is a X-linked immunodeficiency, in whose patients a decreased level of platelets and a defect of Ig production to T-independent antigen are charcteristic features. In the patients, a molecular disturbance of CD43, gplb or cytoskelton perhaps due to an abnormal glycosylation has been shown. But how these defects lead to immune disfunction is unknown. Since in the patients with WAS generally serum level of IgE is increased, CD43 as well as LFA-1 are ligands to ICAMl and since it is reported that the avidity change (an increase of the binding capacity) of LFA-1 to ICAMl may occur by its interaction with cytoskelton, we tested the effect of anti-ICAMI on IgE synthesis by B cells from two patients with WAS. In contrast to the enhancing effect of anti-ICAMI on IgE synthesis by normal B cells, the addition of antiGICAM1 could not enhance IgE synthesis nor augment the expression of germline transcript of CE gene by B cells with WAS, suggesting that in vivo physical association of ICAMl with CD43 or LFA-1 molecule may be defected and its interaction regulates IgE production. The binding capacity of LFA-1 and CD43 in lymphocytes from the patients to ICAMl is now being determined and the molecular characterization of this defect will be discussed. Antigen recognition by mature T cells is associated with a class-specific bias in MHC molecule recognition, with CD4+ T cells responding to MHC class II-and CDW T lymphocytes to MHC class Iassociated antigen. We have recently identified a region in the MHC class II 0-chain that is critical for functional interactions with CD4 (Konig et al., Nature 356: 796, 1992). Here, I show that the a-chain of MHC class II molecules participates in interactions with CD4, too. The threedimensional structure of the class II MHC molecule HLA-DR1 (Brown et al., Nature 364: 33, 1993) suggests that HLA-DRI exists as a dimer of class II afi heterodimers. Also, mutational analyses of MHC class II interaction sites on CD4 reported by other laboratories suggest that CD4 may interact with two MHC class II molecules simultaneously.

The data reported here corroborate this hypothesis. Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China Intestinal IEL locate between and beneth the epithelial cells covering the intestine lumen, and are probably the first group of lymphocytes that encounter antigens and pathogens passing through the intestine. composition of ilEL is more complicated than that of peripheral T cells, as shown by the presence of CD4+8+ and aaCDB+ cells. Among the aaCD8+ cells, one fouth to half are ap TCR+ whereas the rest are yS TCR+ cells. To understand function(s) of CD8 ilEL subpopulations, we have developed purification scheme to separate apCD8+ and aaCD8+ iIEL, and characterized them in comparison with lymph node (LN) CD8 cells. We found that the expression pattern of Ly6C and CD44 molecules are different among freshly isolated LN and ilEL CD8 subpopulations. When stimulated with apTCR-specific mAb and peritoneal macrophages in the presence or absence of IL2, apCD8+ ilEL proliferate and produce IL2, interferon-y, and tmor necrosis factor(s), whereas aaCD8+ ilEL are negative for these responses even with addition of anti-CD2 mAb. However, the expression patterns of Ly6C and Thy-I molecules of aaCD8+ ilEL are different before and after stimulation. Cytotoxic T lymphocytes (CTL) are generally specific for class 1 major histocompatibility complex (MHC) proteins plus antigen and express CD8 co-receptor molecules. The effector function of some CTL can be blocked by antibodies to CD8 (CD8 dependent CTL), whereas that of others is resistant to blocking (CDS independent CTL). This difference in sensitivity to antibody-mediated inhibition is assumed to reflect variations in affinity of particular T cell receptors (TCR) for antigen. However, with a panel of T cell hybridomas from CTL clones which have different susceptibility to blocking with anti-CDS antibody, we have found that a major difference between CD8 independent and CDX dependent T cells lies in their sensitivity to stimulation, the former responding to lower concentrations of anti-CD3 antibody than the latter. Thus the contribution to cell signalling provided by the co-association of p561ck and CD8 is particularly relevant for CD8 dependent cells. These data challenge the notion that the affinity of an individual TCR for antigen is related to the sensitivity of a cell to anti-CD8

antibodies. Indeed we can show that antibodies to co-receptor molecules have several effects on T cell activation, only some of which may be related to T cell affinity. Parkhouse#, Chris Goodnow*, and Maureen Howard, DNAX Research Institute, Palo Alto CA 94304, 'Dept. of Micro. and Immunol., Stanford, CA, and #Div. of Immunol., Insr. for Animal Health, Pirbright Laboratory, Surrey, England.

Murine CD38 is glycoprotein present on the surface of many different hematopoietic cell types. CD3X possesses a unique enzymatic activity which allows the conversion of NAD to ADP-ribose and cyclic ADP-ribose. In addition, Abs to CD38 induce upregulation of cell surface molecules as well as B cell proliferation in conjunction with additional costimuli.

We now demonstrate that signalling through CD38 is impaired in two immunodeficient subsets of B cells; XID B cells and anergic B cells derived from the double transgenic mouse model of tolerance developed by Goodnow er al. We have observed that while B cells from both of these mice are competent to receive and respond to signals from T cells, cytokines, and mitogens, they do not proliferate in response to triggering through either the Ag receptor or CD38. Although anergic B cells do not proliferate to these stimuli, these cells can be activated by CD38 and antigen as evidenced by the increases in class II, ICAM-I or other activation markers. This is distinct from the XID B cells where the CD38 response is completely abrogated, suggesting that the block in CD38 signal transduction is at different points in B cells from these two types of mice. The expression and enzymari examined in both types of B cells nnd appear were tyrosine phosphorylated after anti-CD3R triggering were examined.

was found to overlap with rylated after Ig receptor XID and anergic B cells. We have also examined proteins which are either directly or indirectly associated with CD38 in an attempt to understand CD3R signal transduction. Our results demonstrate that by a variety of criteria, the CD3X signalling pathway is functionally similar but not identical to the Ig receptor pathway, and therefore, CD3X may play an important role in a variety of Ag driven responses.

Malek and Tony J. Fleming, Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136. Cross-linking of Ly-6 molecules on T lymphocytes leads to IL-2 production that is dependent upon expression of the TCR C chain and the GPI-anchor of Ly-GNE. More recently we found that co-stimulation of T cells via L y W E and the TCR inhibits 11-2 secretion by a pathway independent of TCR < chain expression. This effect was specific for Ly-6NE in as much as anti-Thy-1 did not block anti-CDSinduced 11-2 production. The present study was initiated in order to determine whether there were unique structural requirements at the level of the Ly-6 molecule for its capacity to activate or block IL-2 production. Functional studies using EL4 cells transfected with various Ly-6 hybrid molecules demonstrated that direct activation of IL-2 secretion or inhibition of anti-CD3induced 11-2 production was independent of whether anti-Ly-mAbs reacted with NH,-or COOH-terminal epitopes of Ly-6. In addition, stimulation of mouse Ly-6 proteins expressed in Jurkat cells antagonized PMNOKT3induced IL-2 production suggesting that the L y 5 inhibitory pathway is operative in human cells. EL4 cells were also transfected with a hybrid construct in which the GPI anchor of Ly-6E was replaced by the transmembrane and a portion of the cytoplasmic tail of H-2Db. Anti-Ly-6NE mAb also blocked anti-CD3E-induced 11-2 production for these cells even through anti-Ly6E failed to directly induced IL-2 secretion. Thus, anti-Ly-6NE blockade of IL-2 production is independent of the GPI anchor of Ly-6. This finding suggests that there may be aspects of signalling via Ly-6 that are solely dependent upon the extracellular amino acid sequence of this protein. with co-immoiilized FN and substimulatory amounts of anti-TcR has a synergistic effect and rolongs the increased phosphorylation past 40 minutes. 8reliminarGxperiments s u y t that this increase in phosphorylation by is inhibited by GD, a seqhuen~ found on many extracellular matrix proteins that forms t e binding site for some inte rin receptors. Consistent with this, Pronectin, a recombinant &-like protein consisting of RGD repeats stimulates even hi her, but still transient, levels of phosphorylation of p120. b e are in the process of trying to identify this tar et molecule of phozhorylation. A likely candidate is foc3 adhesion kinase FA which is a 125kd kinase induced in fibroblast cells after binding to fibronectin. The experiments to determine if p120 is FAK are in progress. LAG-3 is a membrane molecule belonging to the lg superfamily, which is expressed selectively on activated T and NK lymphocytes. This molecule is closely related to CD4 in protein structure, sequence and chromosomal location, suggesting both these two molecules may have shared a common ancestor. Like CD4, LAG-3 molecules associate with class II MHC molecules, however the function of LAG-3 molecule is still unknown. Because this molecule is expressed not only on activated CD4+T cells but also on activated CDI+T cells, yS T cells and NK cells, LAG-3 molecule may have several immunological roles in addition to the interaction with class II MHC molecule. Moreover, so far as we have examined the expression of LAG-3 molecule in mice, it is expressed also on some populations of thymocytes. This suggests that this molecule may be involved in T cell ontogeny in the thymus. The best way to determine the exact function of LAG-3 molecule in vivo is to disrupt this molecule in mice by gene targeting. After cloning cDNA and genomic DNA of LAG-3 gene, homologous recombination was carried out in ES cells. The efficiency of homologous recombination was very high; about one in six neo resistant clones was targeted clone. Injected targeted clones were well transmitted into germ line. As Littman et al reported recently, some helper-T cell function is saved in CD4 knock-out mice and it is possible that this helper function may be compensated by LAG-3 molecules. Therefore, in the mean time, we are disrupting both CD4 and LAG-3 molecules using double knock-out method. We will present preliminary results of analysis of the knock-out mice. Adhesion molecules play a major role in generating chronic inflammatory infiltrates by regulating leucocyte migration and local retention. First, they mediate adhesion to endothelium (EC). Second, they increase tissue retention by enhancing binding to extracellular matrix. Third, they promote homolheterotypic intercellular adhesion which is crucial in antigen responses. Finally, they deliver costimulatory signals for cell activation. CS are potent immunosuppressants affecting many immune functions including cytokine production; the relative importance of these effects is unclear. Since CS induce blood leucocytosis in vivo and reduce cellular infiltration in inflamed tissues, an ability to inhibit cell adhesion and migration may be important. We examined whether preincubation of resting or PMA-activated MNC with CS could inhibit 1) adhesion to IL-1 stimulated EC, measured as % of "Cr-labelled MNC binding to an EC monolayer, 2) formation of cell clusters, assessed by visual scoring on an arbitrary scale of 0-5 (after Rothlein et al) and 3) expression of LFA-I and CD2 adhesion proteins, measured as mean intensity of fluorescence (mif). First, pretreatment with CS was able to inhibit the binding to IL-1 stimulated EC of both unstimulated (22.8% vs 15.9%) and activated MNC (26.6% vs 14.1%). Second, although the initial PMA-induced aggregation was not affected by CS, CS-pretreated MNC had largely disaggregated by 72h (mean aggregation score 1 compared to 4 for non-CS-treated cells). Third, the expression of LFA-1 (155.7 vs 600.2) and CD2 (116.7 vs 261.3) was reduced in CS-pretreated compared to CS-untreated activated MNC. All these effects were mediated through the glucocorticoid receptor since they were inhibited by the receptor antagonist RU-486. In conclusion, CS-pretreated MNC were less able to adhere to EC and to form stable aggregates; consonant with this, LFA-1 and CD2 expression was decreased. We are currently investigating whether these effects are mediated by a direct action of CS on transcription of adhesion protein genes via their glucocorticoid regulatory elements (GREs) or indirectly, via decreased production of cytokines important for regulating adhesion, We have examined the relative roles of CD40, CD54 and MHC I1 signalling in the induction of B cell proliferation and responsiveness to IL-2. We used a non-cognate Thl helper system that is contact as well as IL-2 dependent. Paraformaldehyde-fixed, activated Thl cells induced expression of IL-2Ra. IL-2Rp and B 7 , and upregulated both MHC I1 and CD54 expression on B cells. Both the T-dependent induztion of Ig secretion, and B cell phenotypic chanyes were inhibited by anti-CD54 and MHC I1 mAbs and by a CD8-CD40-L/gp39 construct. We compared this to the effects generated through crosslinking of tnese molecules on B cells. Crosslinking CD40 upregulated expression of MHC 11, CD54 and B 7 , analogous to the effect of fixed Thl cells. Cocrosslinking of MHC I1 and CD54 generated comparable effects. Crosslinking of MHC I1 and CD54 (in the presence of IL-5) induced expression of a functional IL-2R on B cells, but did not induce proliferation. By contrast CD40 ligation induced B cell proliferation, but did not induce IL-2X expression or IL-2 responsiveness. Our data confirm the importance of the CD40:CD40L/gp39 interaction during delivery of T help for B cells, but show that CD40 ligation is not sufficient for B cell differentiation. CD54 and MHC I1 signalling is complementary to that of CD40 in the generation of T-dependent B cell responses to IL-2. We are presently investigating Protein Tyrosine Kinase activation that accompanies CD54 and MHC I1 ligation with a view to defining biochemical signalling pathways critical for these events. Supparted by MRC-Canada. CD-L' I cells are actilated t n signals transduced vid the T cell receptor, CD3 complex in the presence of accessory cells which deliver necessary costimulatory signals. We investigated the costmulator)-role of diverse extracellular matrix (ECMIproteins such as various collagen types. fihronectin. laminin m d undulin for resting and preactivated murine L U 4 + I cells. IIighl!, purified CIM+ 'I cells, treated with plate-hound anti-CDS antibodies, were not induced to IL-L secretion, 11-2 receptor upregulation and proliferation by coimmohilized ECM-proteins. Hower, J. contamination of the CD4+ T cell population with small amounts of accessory cells resulted in enhanced IL-L secretion and proliferation induced by coimmohilized coilagens or fibronrrtin. Irradiated splenocytes induced a significantly highcr lecel 01 7 cell proliferation. suggesting that signal\ &li

Stimulation of the T lymphocyte antigen receptor complex (TCR-CD3) elevates [Ca2+li, an important step in the immune activation of ma re T cells. Paradoxically, TCR-CD3 signaling also involving

Although antigen receptor occupancy with a peptide in association with major histocompatibility complex antigen initiates T cell activation, a second co-stimulatory signal is necessary the optimal proliferation and lymphokine secretion.CD28 is a homodimeric membrane glycoprotein expressed on most of the T cells and the signal generated by the interaction with its natural ligand, B7, has been implicated as a costimulatory signal. While the CD28 signal in concord with the TCR signal induces proliferation and lymphokine secretion by T cells, a notable activation event is nonexistent by the CD28 signal alone and very little is known about CD28 signaling pathway. In this presentation we would like to report that CD28 ligation activates sphingomyelinase thereby triggering the sphingomyelin (SM) signaling pathway. Activation of this pathway resulted in the induction of ceramide driven kinases specific for serine and threonine. Importantly the sphingomyelinceramide signaling pathway is also known to couple to receptors for interleukin 1 and tumor necrosis factor which are costimulatory to T cell activation. The data demonstrate the pivotal role of SM signaling pathway to conduct co-stimulatory signal in T cells. Activation of antigen specific T-cells requires more than signalling through the TCR/CD3 complex. A second or costimulatoty signal is also required. Triggering through the Tcell receptor (TCR) without simultaneous costimulation induces non-responsiveness in T-cells under certain conditions. The best known receptor-ligand interaction through which second signals can be transduced is formed between CD28 on T-cells and B7 on APC's. Nevertheless, some B7-negative cell lines appear capable of providing second signals under certain conditions. For instance, the peptide transporter defective T-lymphoma RMA-S does not express 87, but can effectively induce peptide specific CTL responses in vitro, once loaded with an exogenous source of antigenic peptide. Such responses cannot be blocked by anti-87 reagents (i.e. anti 8 7 or CTLA4-Ig), suggesting that B7 independent costimulatoty pathways may exist. This notion was further investigated in various models. First, RMA-S can support clonal expansion of purified CD4 or CD8 Tcells from unprimed mice activated with immobilised anti-CD3. In fact, also the parental RMA cell line, as well as at least one other T-cell lymphoma (i.e., EL4), can aid in such anti-CD3-induced clonal expansion in a dose dependent fashion. This expansion is accompanied with IL2 production. Secondly, also costimulation of antigen-specific T-cell proliferation of both class I and class II restricted T-cell clones can be provided by B7-negative Tlymphoma cells: non-responsiveness induced by pre incubation of T-cells with ECDl fixed APC plus antigen can be rescued by RMA-S or RMA. The costimulatory signals induced by Tlymphoma lines are destroyed by ECDl fixation, in analogy to the CD28-B7 dependent pathway. The results thus far indicate a novel pathway able to costimulate T-cell responses. Characterisation of the molecules involved is in progress. Proteins produced and secreted by tumor cells may be involved in regulation of the immune response to the tumor. One example of such a protein is immunoregulin, a 90kD glycoprotein which was originally identified by reactivity with a monoclonal antibody raised against conditioned medium of human mammary tumor cells. This protein enhances the generation of natural killer (NK) and lymphokine activated killer (LAK) activity in PBL from apparently healthy donors. To elucidate the mechanism of action of immunoregulin, we have examined its effect on cytokine production by mitogen-stimulated PBL.Addition of immunoregulin to human PBL stimulated with the T-cell mitogen Concanavalin A caused an increase in the levels of IL-2 produced in a 48 hour culture. This increased response was dependent on the presence of C o d , and was most apparent at suboptimal doses of the mitogen. Under the same conditions, production of the accessory cell-derived cytokines IL-1 and IL-6 was also increased. In contrast to the IL-2 response, immunoregulin alone (i.e., without ConA) was sufficient to stimulate production of IL-6 by PBL, suggesting that the effect of the molecule is at the level of the accessory cell rather than the T-cell. Nylon wool-enriched T-cells did not produce lymphokines in response to ConA, as expected for this accessory cell-dependent mitogen. The addition of immunoregulin failed to restore the ability of the T-cells to respond to ConA, providing further evidence that immunoregulin does not act directly on T-cells. We propose a model in which immunoregulin activates accessory cells, which indirectly leads to T-cell activation, resulting in increased production of IL-2 and other T-cell-derived cytokines. The result of this cascade is enhanced cell-mediated immunity, such as NK and LAK, which may lead to rejection of tumor cells or virus-infected cells in vivo.We asked whether different types of TCR-mediated [Ca +Ii signals could explain the different responses of mature and immature T cells. Using a digital fluorescence imaging system, we measured [Ca2+Ii of individual CD4 and CD8 thymic and splenic cells following app ication of CD3-c mAb. An unexpected result was that the peak more lynchronized than those of ymocytes. This variable temporal coupling d y not depend on ~~5 9 % " .shown by examination of cells from pp59 Y"-deficient mice (Appleby el al., 1992, Cell 70751). Instead, p~5 9~~" d ficiency affected the amplitude and probability of CD3-mediated [Ca"]. responses of CD4+CD8+ and CD4+CD8thymocytes. Herbim;cin A and pervanadate application showed t at immature cells contain additional tyrosine ki ses involved in [Ca Ii regulation. In addition, the average peak [Ca 1. attained by CD3responsive cells increased with maturiy;. T p s e :esults demonstrate that the coupling of TCR-CD3 to [Ca during development. In particular, the timing of peak [Ca li , relative to other TCR-CD3-derived signals, such as those involving ras, PI3 kinase, and tyrosine kinases, might explain the different effects of TCR-CD3 ligation during T cell development. 

Carbone, and Michael J. Bevan. Department of Immunology, HHMI. University of Washington, Seattle, WA 98 103 Positive selection of T lymphocytes occurs when an immature T cell interacts with a complex of self peptide plus MHC on the surface of a thymic epithelial cell. We have used organ culture of fetal thymic lobes from p2M KO mice to study the critical role of peptides in this process. In these mice, CD8+ T cells are not positively selected in the thymus. This process can be restored by the addition of class I binding peptides along with P2M to organ cultures. We have now applied this approach to TCR transgenic mice.Mice were generated which have a transgenic TCR isolated from a CD8+ cytotoxic T cell specific for the peptide ovalbulmin 257-264 in the context of Kb. This receptor is positively selected on an H-2b background (Kb specifically) as demonstrated by the skewing of thymocytes and peripheral T cells to the CD8 lineage. On an H-2b p2M KO background, the receptor is not selected as indicated by the absence of TCRhi CD4-CD8+ T cells and the resultant inabiltiy of these cells to respond to antigen. However, when fetal thymic lobes from such mice were cultured with various MHC binding peptides plus exogenous p2M, several peptides with the ability to induce the appearance of TCRhi CD4-CD8+ T cells were identified. These cells have the same phenotype. as mature CD8+ T cells. More importantly, treatment of the lobes with these peptides restores the ability of the p2M KO thymocytes to respond to antigen.Only certain Kb binding peptides were capable of mediating this selection. Two naturally occuring self-peptides had no effect on differentiation. Additionally, a peptide composed of only serine residues at the TCR contact sites had no effect. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E l , induced positive selection on the p2M KO background, but negative selection on the wild-type. H-2b background. These results argue that the process of positive selection is highly specific, and support an efficacy model of T cell differentiation. We previously showed that variants of an antigenic peptide can act as clone specific TCR antagonists for cytotoxic T cells (CTL), inhibiting T cell activation in response to antigen/ MHC (J. Exp. Med. 1773541). Further experiments were performed using a TCR transgenic mouse bearing a receptor from an OVA/Kb specific CTL. Variants at several residues in the OVA peptide acted as antagonists for this receptor. Most were substitutions at TCR contacts, but a few were variants in regions of the peptide buried in the MHC groove, suggesting an effect on TCR binding to MHC contact residues. The response of unprimed TCR transgenic T cells to stimulators pre-pulsed with OVA peptide is effectively blocked by antagonist peptides, indicating that naive cells as well as long term lines and clones are susceptible to this form of inhibition. Furthermore, antagonists blocked CTL mediated lysis not only of OVA peptide coated target cells but also of cells expressing OVA as an endogenous antigen. This implies that antagonists can inhibit T cell responses to naturally presented antigens.

Our data indicate that mature T cells fail to respond directly to these peptides. In contrast, we have evidence that immature T cells do respond to antagonists: Using fetal thymic organ culture, we have demonstrated that certain antagonist peptides induce positive selection of TCR transgenic T cells. The effects of these antagonists on various aspects of thymocyte activation is described.