Human CTL Clones Research Articles

CAR NK and CAR T cells versus CTL: factors limiting the sensitivity and efficiency of engineered cytolytic effectors

Abstract Factors limiting highly sensitive and efficient responses of virus- and cancer-specific CTL have been extensively studied. However, the sensitivity and effectiveness of T cells transduced with CAR specific for target molecules on cancer cells have been less characterized. We endowed NK92 cells and T cells purified from human cord blood with second generation CAR specific for High Molecular Weight Melanoma Associated Antigen (HMW-MMA) and compared the ability of CAR-NK92 and CAR-T cells to kill skin melanoma and uveal melanoma cell lines in in vitro cytolytic assay. We also evaluated the ability of established human virus-specific CTL clones to kill the same target cells presenting cognate pMHC ligands. Skin melanoma cells were more sensitive to specific lysis than uveal melanoma cells. The extent of the specific lysis strongly depended on the level of HMW-MMA expression. CTL clones appeared to be more superior effectors than CAR-NK92 or CAR-T cells as they lysed the melanoma cells that displayed much lower levels of cognate pMHC ligands. CAR-NK92 cells proliferated more vigorously than NK92 cells and maintained the level of CAR expression in culture. In contrast, CAR-CD8+T cells exhibited lower proliferative capacity than the control T cells and were losing cell surface CAR expression. We are currently evaluating the extent of TCR vs CAR clustering and co-clustering of CAR with TCR at the cell surface. We are also comparing Ca2+ flux and granule release by CAR-NK92, CAR-CD8 T cells and CTL in response to CAR or TCR stimulation. Disparities in these parameters could account for differential sensitivity and efficiency of engineered and natural cytolytic effectors.

Long-Peptide Cross-Presentation by Human Dendritic Cells Occurs in Vacuoles by Peptide Exchange on Nascent MHC Class I Molecules.

Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.

Anti-CD8 antibodies can trigger T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining (113.28)

Abstract Cytotoxic T lymphocytes (CTLs) recognize foreign peptides presented at the cell surface bound to major histocompatibility complex class I (MHCI) molecules. Antigen recognition involves the binding of both T-cell receptor (TCR) and CD8 co-receptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates an effect on antigen sensitivity. Anti-CD8 antibodies have been used extensively in previous studies to examine the role of CD8 in CTL activation. However, it is unclear from the literature whether anti-CD8 antibodies per se are capable of inducing effector function. Here, we report on the ability of seven monoclonal anti-human CD8 antibodies to activate six human CTL clones with a total of five different specificities. Six out of seven anti-human CD8 antibodies tested did not activate CTLs. In contrast, one anti-human CD8 antibody, OKT8, induced effector function in all CTLs examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 antibodies to trigger T-cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of antibody-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CTL signalling.

Conditional Superagonist CTL Ligands for the Promotion of Tumor-Specific CTL Responses

Abstract Although it has been demonstrated that CTLs can be raised against tumor-associated self-antigens, achieving consistent and effective clinical responses has proven challenging. Superagonist altered peptide ligands (APLs) can often elicit potent antitumor CTL responses where the native tumor-associated epitope fails. Current methods have identified a limited number of superagonist APLs, including the prototypic 27L mutant of MART-1. However, more comprehensive screening strategies would be desirable. In this study, we use a novel genetic screen, involving recombinant technology and class I Ag cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant by surveying the effectiveness of virtually every single amino acid substitution mutant of 27L to activate human Ag-specific CTL clones recognizing the wild-type MART-126–35 epitope. We identify three novel mutant epitopes with superagonist properties that are functionally superior to 27L; however, the ability of a given analogue to act as superagonist varies among patients and suggests that a given superagonist APL may be ideally suited to different patients. These findings endorse the use of comprehensive methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients.

Distinct CD8+ T Cell Repertoires Primed with Agonist and Native Peptides Derived from a Tumor-Associated Antigen

Heteroclitic peptides are used to enhance the immunogenicity of tumor-associated Ags to break T cell tolerance to these self-proteins. One such altered peptide ligand (Cap1-6D) has been derived from an epitope in human carcinoembryonic Ag, CEA(605-613) (Cap1). Clinical responses have been seen in colon cancer patients receiving a tumor vaccine comprised of this altered peptide. Whether Cap1-6D serves as a T cell agonist for Cap1-specific T cells or induces different T cells is unknown. We, therefore, examined the T cell repertoires elicited by Cap1-6D and Cap1. Human CTL lines and clones were generated with either Cap1-6D peptide (6D-CTLs) or Cap1 peptide (Cap1-CTLs). The TCR Vbeta usage and functional avidity of the T cells induced in parallel against these target peptides were assessed. The predominant CTL repertoire induced by agonist Cap1-6D is limited to TCR Vbeta1-J2 with homogenous CDR3 lengths. In contrast, the majority of Cap1-CTLs use different Vbeta1 genes and also had diverse CDR3 lengths. 6D-CTLs produce IFN-gamma in response to Cap1-6D peptide with high avidity, but respond with lower avidity to the native Cap1 peptide when compared with the Cap1-CTLs. Nevertheless, 6D-CTLs could still lyse targets bearing the native epitope. Consistent with these functional results, 6D-CTLs possess TCRs that bind Cap-1 peptide/MHC tetramer with higher intensity than Cap1-CTLs but form less stable interactions with peptide/MHC as measured by tetramer decay. These results demonstrate that priming with this CEA-derived altered peptide ligand can induce distinct carcinoembryonic Ag-reactive T cells with different functional capacities.

Modulation of CD103 Expression on Human Colon Carcinoma-Specific CTL

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-gamma, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.

In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones.

Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8beta-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E7(11-20)-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E7(11-20)-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.

A reversible functional defect of CD8+ T lymphocytes involving loss of tetramer labeling.

We have observed that human CTL clones lose their specific cytolytic activity and cytokine production under certain stimulation conditions, while retaining an antigen-dependent growth pattern. These inactive CTL simultaneously lose their labeling by an HLA-peptide tetramer, even though the amount of TCR-CD3 at their surface is not reduced. The tetramer-negative cells recover tetramer staining and cytolytic activity after stimulation with tumor cells in the presence of a supernatant of activated lymphocytes. Our results suggest the existence of a new type of functional defect of CTL. They also indicate that tetramers may fail to reveal some CTL bearing the relevant TCR, even when such functionally arrested CTL retain the potential to participate in immune responses because their defect is reversible.

A Novel Approach to Antigen-Specific Deletion of CTL with Minimal Cellular Activation Using α3 Domain Mutants of MHC Class I/Peptide Complex

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using α3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy.

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to IL-2 and Ca++ fluxes were observed only at peptide concentrations > 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least 20-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.

Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.

HIV-1 envelope protein is expressed on the surface of infected cells before its processing and presentation to class II-restricted T lymphocytes.

T lymphocytes are activated upon binding of their Ag receptors to a complex of Ag-derived peptides and MHC class I or class II molecules expressed on the surface of APC. It is now well established that APC degrade exogenous Ag in acidic endosomal compartments, and that Ag fragments bind to class II molecules moving through these compartments on their way to the surface of the APC. Although peptides derived from some endogenous Ag can also bind to class II molecules and subsequently be recognized by class II-restricted T cells, the intracellular trafficking pathways that enable endogenous proteins to be processed for association with class II molecules remain controversial. We have analyzed the mechanism by which the envelope (env) protein of the HIV-1 is processed in infected cells for recognition by class II-restricted T cells. A large number of env-specific class II-restricted human CTL clones were shown to lyse B-lymphoblastoid cell lines expressing the env. A novel dilutional assay proved that A novel dilutional assay proved that recognition of endogenous env protein was not a consequence of release and re-uptake of the env protein and subsequent processing by the standard class II-restricted pathway. Processing of endogenous env protein required that the protein be co-translationally translocated into the endoplasmic reticulum (ER) and then exit the ER, since the class II-restricted CTL did not recognize env protein localized to the cytosol or retained in the ER of target cells. Under these conditions, however, class I-restricted recognition was readily demonstrated. Finally, class II-restricted recognition was strikingly dependent upon the steady state level of surface env protein, since extracellular reagents that removed intact env protein from the surface of target cells inhibited recognition. This inhibition operated at the Ag-processing level rather than at the level of subsequent Ag recognition. These results provide the first direct evidence that endogenously synthesized membrane proteins enter the class II-restricted Ag-processing pathway after expression on the cell surface in an intact form.

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.

Interleukin-6 is constitutively produced by human CTL clones and is required to maintain their cytolytic function

Maturation of cytolytic T lymphocytes from nonlytic precursors requires cytokines in addition to IL2. Interleukin-6 is the principal cytokine that cooperates with IL2 in the induction of CTL differentiation from murine and human thymocyte precursors. However, a cytotoxic differentiation factor (CDF) role of IL6 for mature T cells is challenged by data indicating that IL2 alone is sufficient for CTL generation. The aim of this study was to identify a model system in which IL6 acted as a CDF for human peripheral T cells. We noted that IL6 was endogenously produced by CTL clones in the course of their expansion with APC, lectin, and IL2. The majority of several hundred T-cell clones, both CD4+ and CD8+, produced IL6 in response to relatively high doses of IL2. Other experiments that compared the cytolytic function of CTL clones cultured in the presence of IL6 with that of the same clones cultured in the absence of IL6 demonstrated that IL6 contributes to the cytolytic ability of the majority of human CTL clones. Our data suggest that IL6 acts in an autocrine fashion to support CTL differentiation in human T-cell clones.

DISCRIMINATION OF HLA-B5 CROSSREACTIVE GROUP ANTIGENS BY HUMAN ALLOSPECIFIC CTL CLONES

Human CTL clones discriminating serologically closely related HLA-Bw52, B51, and B35, which belong to HLA-B5 crossreacting group (CREG), were established from peripheral blood lymphocytes by repeated in vitro stimulations. Five HLA-Bw52-specific CTL clones from an individual with HLA-B5 CREG antigens and four CTL clones from another individual with HLA-B51 were generated. The specificity of these CTL clones was ascertained by their lysis of EBV-transformed B cells with HLA-Bw52, but not those with HLA-B51 or B35, and Bw52-transfected Hmy2CIR cells but not HLA-B51 or B35 transfectants. Conversely HLA-B51-specific clones were generated from the HLA-B5 CREG-negative individual, as well as another individual with HLA-Bw52. Their specificity was determined in a similar fashion. Since HLA-B51 differed from HLA-Bw52 only by two amino acid substitutions on the alpha helical region of the alpha 1 domain, these results demonstrated that allospecific CTLs can be produced and discriminate the epitopes formed by the subtle difference in the structure of these HLA class I molecules. Furthermore, three HLA-B35-specific CTL clones were generated from the HLA-B5 CREG-negative individual that discriminated HLA-B35 from HLA-Bw52 and B51. Taken together these results demonstrated that human CTL clones could definitively discriminate the three serologically related HLA-B5 CREG specificities.

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads.

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.

The role of Ca2+ and Mg2+ in the cytotoxic T lymphocyte reaction and in the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-serine esterase by human T cell clones.

Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon Ag-specific activation. BLT-serine esterase secretion could also be induced by the Fc gamma+ target cell Daudi in the presence of mAb specific for the TCR/CD3 complex, CD2, or the T cell activation Ag Tp 103. In addition, anti-CD3 and a mitogenic combination of anti-CD2 mAb, induced secretion of BLT-serine esterase in the absence of target cells, whereas anti-Tp 103 failed to do so. The secreted BLT-serine esterase activity induced by the various ligands was inhibited by the serine esterase inhibitors PMSF and m-ABA, but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone. Significant BLT-serine esterase activity was induced by target cells or soluble anti-CD3 in the absence of extracellular Ca2+ ions, provided that extracellular Mg2+ ions were present. The cytotoxic activities by the human CTL clones were completely blocked under these conditions. All ligands that induced BLT-serine esterase secretion in the absence of extracellular Ca2+, induced a transient rise of intracellular Ca2+. Soluble anti-CD3 mAb did not induce a transient rise in intracellular Ca2+ or secretion of BLT serine esterase in CTL preincubated for 2 h with 5 mM EGTA. These findings indicate that mobilization of intracellular Ca2+ in human CTL clones is required for induction of secretion of BLT-serine esterase.

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen.

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.