Heterogeneity of protein kinase C isoenzyme gene expression in human T cell lines. Protein kinase C-beta is not required for several T cell functions

An early consequence of stimulation of T cells via their Ag receptor is the activation of protein kinase C (PKC). It has recently been shown that PKC activity resides in a family of homologous proteins. Inasmuch as T cells are phenotypically and functionally heterogeneous, we examined the possibility that this heterogeneity may be reflected in differential expression of message for PKC isoenzyme genes. RNA from six leukemic T cell lines was probed for PKC-alpha, -beta, and -gamma message before and after activation. These studies revealed significant differences among these lines. None expressed mRNA for PKC-gamma. Whereas all cells possessed message for PKC-alpha, there was consistent variability in the level expressed. The greatest heterogeneity was seen with PKC-beta. Two cell lines, HUT 78 and HPB-ALL, did not hybridize with the beta probe under any conditions tested. We subsequently used these PKC-beta negative cells to study the role of this isoenzyme in mediating some of the effects seen with phorbol esters that directly bind to and activate PKC. Our results indicate that PKC-beta, which is expressed in some T cells, is not necessary for PMA-induced CD3 or CD4 internalization, IL-2 production, or acquisition of the p55 chain of the IL-2 receptor.     

Response of Jurkat T cells to phorbol ester and bryostatin. Development of sublines with distinct functional responses and changes in protein kinase C activity.

T lymphocyte activation is initiated as a result of the interaction between the TCR complex and Ag as seen in the framework of a membrane-bound MHC molecule. Receptor stimulation results in a rise in free intracellular Ca2+ and the activation of protein kinase C (PKC). Bryostatin (Bryo) and phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA] are PKC activators with somewhat different immunologic effects. We compared the effect of Bryo and TPA on the T cell tumor line Jurkat and derivatives of Jurkat cells grown in media supplemented with 100 nM Bryo ("BR100" cells) or 100 nM TPA ("TP100" cells). In untreated Jurkat cells, there is a dose- and time-dependent decrease in proliferation, compared to media controls, after the administration of as little as 10 nM TPA. This can be reversed in a dose- and time-dependent manner by Bryo. Interestingly, the expression of the transferrin receptor parallelled this effect on proliferation. Furthermore, Jurkat cells grown continuously in 100 nM TPA regained full proliferative capacity after several weeks in culture and transferrin receptor expression returned to near the level seen in untreated Jurkat cells. The chromatographic separation of PKC activity in these three cell lines showed that total PKC activity was dramatically decreased in both the TP100 and BR100 cells when compared to untreated Jurkat cells. However, in the TP100 cells there exists a peak of activity that is activated by Bryo, but not TPA. Western blots of whole cell lysates of the three cell lines showed that PKC-alpha and PKC-beta II were both down-regulated in BR100 and TP100 cells compared to untreated Jurkat cells. PKC-gamma was not detected in any of the cell lines. Therefore, the Bryo-specific peak seen in TP100 cells may be PKC-delta, -epsilon, -zeta, -eta, or a novel PKC isoform. This could provide the basis for a molecular characterization of the differences in PKC activation between phorbol esters and Bryo.     

Evidence for protein kinase C-dependent and -independent activation of mitogen-activated protein kinase in T cells: potential role of additional diacylglycerol binding proteins

Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to stimulate the T cells. Extracellular signal-regulated kinase (ERK) activation in CTL stimulated with soluble cross-linked anti-CD3 is completely inhibited by the PKC inhibitor bisindolylmaleimide (BIM). In contrast, only the later time points in the course of ERK activation are sensitive to BIM when CTL are stimulated with immobilized anti-CD3, a condition that stimulates CTL degranulation. Surprisingly, MAPK activation in response to immobilized anti-CD3 is strongly inhibited at all time points by the diacylglycerol (DAG)-binding domain inhibitor calphostin C implicating the contribution of a DAG-dependent but PKC-independent pathway in the activation of ERK in CTL clones. Chronic exposure to phorbol ester down-regulates the expression of DAG-responsive PKC isoforms; however, this treatment of CTL clones does not inhibit anti-CD3-induced activation of MAPK. Phorbol ester-treated cells have reduced expression of several isoforms of PKC but still express the recently described DAG-binding Ras guanylnucleotide-releasing protein. These results indicate that the late phase of MAPK activation in CTL clones in response to immobilized anti-CD3 stimulation requires PKC while the early phase requires a DAG-dependent, BIM-resistant component.     

Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL

Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells.     

Direct cross-priming by th lymphocytes generates memory cytotoxic T cell responses

Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.     

Phosphorylation of a 15- to 17-kDa protein correlated with lytic function in cytotoxic T lymphocytes

CTL are activated to lyse their targets through the interaction of the CTL-R and the appropriate Ag on the surface of the target cell. Experiments with tumor-promoting phorbol esters have suggested that the activation and translocation of protein kinase C (PKC) to the CTL membrane may be important in the activation process. We have studied the functional role of PKC in lytic signal transduction by correlating the phosphorylation of a set of CTL membrane proteins bound by the lectin Con A with lytic function in CTL clones. The data obtained indicate that the phosphorylation of a 15- to 17-kDa polypeptide in this subset is associated with the translocation of PKC to the membrane and the stimulation of lytic function. This suggests that the 15- to 17-kDa protein may be a physiologically relevant substrate for PKC translocated to the membrane as a result of Ag-specific perturbation of the CTL-R.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.     

IL-12 augments antigen-dependent proliferation of activated T lymphocytes.

Ag-dependent T cell activation requires multiple transmembrane signals including activation of Ag-specific T cell receptor in combination with signals delivered through cytokine receptors. IL-12 is a heterodimeric cytokine involved in the regulation of NK and T lymphocyte responses. In examining the role of IL-12 in T cell activation, we found a direct relationship between Ag stimulation and IL-12-induced proliferation. Unlike IL-2, which induced proliferation of CTL either in the presence or absence of a CD3/TCR co-signal, IL-12 mediated proliferation of CTL only when the cells were recently co-stimulated with alloantigen or solid-phase anti-CD3 antibody. After culture in the absence of alloantigen or anti-CD3 for 7 to 14 days, these CTL lost the ability to proliferate to IL-12 alone. Under these conditions, however, IL-12 synergized with low-dose IL-2 to induce CTL proliferation. Restimulation with alloantigen or solid-phase anti-CD3 restored the ability of the CTL to proliferate to IL-12 alone. Not all Ag signals resulted in IL-2 independent proliferation to IL-12. For example, CTL with specificity for influenza matrix peptide proliferated best when co-cultured with peptide Ag presented on self MHC and a combination of IL-2 and IL-12. This evidence suggests that IL-12 may be useful in expanding an Ag-specific T cell population, as the culture of CTL with IL-12 and low-dose IL-2 leads to proliferation only in response to an Ag co-signal.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

Helper-independent CD8+ cytotoxic T lymphocytes express IL-1 receptors and require IL-1 for secretion of IL-2

The purpose of this study was to examine the role of IL-1 on the activation of CD8+/CD4- class I-restricted helper cell-independent cytolytic T cell (HITc) clones known to produce IL-2 and proliferate in vitro after Ag stimulation with a Friend retrovirus-induced leukemia (FBL). The functional role of IL-1 in Ag-specific proliferation and IL-2 secretion was assessed by stimulating the T cell clones with FBL either in the presence or absence of macrophages (M phi), rIL-1, or rIL-2. Resting cloned HITc cells, purified from residual accessory cells, failed to proliferate in response to FBL alone, but proliferated in response to FBL plus M phi, rIL-1 or rIL-2. Stimulation with FBL alone in the absence of M phi or IL-1 was sufficient for induction of IL-2R expression, and rendered cells responsive to IL-2, but M phi or IL-1 were also required to induce production of IL-2. The activity of IL-1 was further examined by measuring the binding of [125I]rIL-1 alpha, which demonstrated that resting cloned HITc cells expressed IL-1R that increased in number after activation with Ag. This expression of IL-1R and requirement for IL-1 by CD8+ HITc was surprising because previous studies examining T cell populations after mitogen stimulation have not detected IL-1R on the CD8+ population. Therefore, the role of IL-1 in the activation of CD8+ CTL that do not secrete IL-2 after activation was assessed. By contrast to HITc, CD8+ CTL required exogenous IL-2 to proliferate in vitro and did not express IL-1R. These data demonstrate that the subset of CD8+ T cells responsible for IL-2 production express IL-1R and that triggering this receptor with IL-1 after Ag stimulation results in the production of IL-2 and subsequent proliferation.     

Tyrosine phosphorylation of the human T cell antigen receptor zeta-chain: activation via CD3 but not CD2

TCR stimulation by Ag or anti-receptor antibodies in murine T cells results in the activation of two independent protein kinases, protein kinase C (PKC) and a protein tyrosine kinase. Similarly, stimulation of murine Thy-1 or Ly-6 with mAb also results in activation of both of these kinase pathways. Tyrosine phosphorylation in all cases occurs on the TCR zeta-chain. It is known that Ag and anti-receptor antibodies activate PKC in human T cells. In this study we demonstrate that mitogen or anti-CD3 antibodies activate tyrosine phosphorylation of the human TCR-zeta-chain. PMA, which activates PKC, does not result in zeta-chain tyrosine phosphorylation. Stimulation of human T cells by antibodies that bind the CD2 molecule is an alternate mode of inducing T cell proliferation. These antibodies surprisingly do not induce tyrosine phosphorylation of the zeta-chain. Thus, different methods of cellular activation can result in distinguishable patterns of receptor-mediated biochemical signaling events.     

Adhesion of epithelial cells to fibronectin or collagen I induces alterations in gene expression via a protein kinase C-dependent mechanism

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

A human T cell-specific molecule is a member of a new gene family

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.     

Functional expression of HLA-C blank antigens on human blood lymphocytes

The surface expression of two HLA-C blank Ag (Cb-1 and Cb-2) on PBL was investigated with Cb-1- and Cb-2-specific CTL clones generated by the stimulation of the HLA-C blank Ag on transfected Hmy2CIR cells. The Cb-1- and Cb-2-specific CTL clones could lyse EBV-transformed B cells and PHA-induced T cells from which the HLA-C blank genes were derived. Furthermore, the reactivity of these CTL clones with PHA-induced T cells was blocked by HLA class I monomorphic mAb. These results demonstrated that the HLA-C blank Ag are expressed on the surfaces of PBL. Thus, despite the fact that the HLA-C blank Ag are expressed on normal PBL, they are incapable of generating corresponding alloantibodies. On the other hand, the present study demonstrated that these Ag on normal PBL are able to induce specific CTL and that the capacity of these Ag to induce allogeneic CTL is almost identical to that of HLA-B Ag, indicating that they may function as alloantigens in vivo and play a significant role in the rejection of organ grafts and in the graft-versus-host reaction in bone marrow transplantation.