In vitro induction of HLA-restricted cytotoxic T lymphocytes against autologous Epstein-Barr Virus transformed B lymphoblastoid cell line

Cytotoxic T lymphocytes (CTL) against autologous EBV-transformed B lymphoblastoid cell line (LCL) were induced in vitro by culturing peripheral blood lymphocytes (PBL) of healthy donors together with mitomycin C-treated autologous LCL for 6 days. The cytotoxic cells developed only from the E-rosette-positive fraction but not from the negative fraction of PBL. These CTL killed autologous LCL but not PWM-stimulated autologous PBL. In addition, the CTL killed allogeneic LCL when at least 1 of the HLA-A antigens was identical with that of the LCL of CTL donor. However, identity of HLA-B and HLA-C antigens was not enough for a significant killing of allogeneic LCL. The specificity of the CTL was also confirmed by a cold target inhibition test. These results indicated that the CTL induced specifically recognized EBV-transformed cells with HLA restriction.     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the T cell-mediated cellular cytotoxicity during acute infectious mononucleosis

Primary infection with EBV during acute infectious mononucleosis (IM) is associated with a cytotoxic response against allogeneic target cells. C depletion with anti-CD3 (OKT3) and anti-CD8 (OKT8) mAb decreased the allogeneic cytolysis of two EBV-infected lymphoblastoid cell lines (LCL) by 96% and 89%, respectively. Complement depletion with the NK cell-specific mAb Leu-11b and NKH-1a resulted in only a slight decrease (less than 35%) in the lysis of these LCL. mAb inhibition studies with OKT3 and OKT8 inhibited the allogeneic lysis of two LCL by 87% and 82%, respectively. The alloreactive cytotoxic response was strongly inhibited by mAb specific for MHC class I determinants (W6/32, 65% inhibition and BBM.1, 58% inhibition). Acute IM lymphocytes lysed the allogeneic EBV-negative cell lines HSB2 (45%) and HTLV-1 T cell lines (16%). NK cell-depleted lymphocytes from an acute IM patient demonstrated preferential lysis of K562 transfected with human HLA-A2 (73%) compared with the K562 transfected control (20%). Cold target competition studies with allogeneic and autologous target and competitor LCL demonstrated no significant competitive inhibition between allogeneic and autologous cells. We interpret these results as evidence that 1) the acute IM-alloreactive cytotoxic response is mediated primarily by CTL; 2) these alloreactive CTL lyse allogeneic target cells irrespective of EBV antigenic expression; 3) MHC class I expression is sufficient for allogeneic recognition and lysis of target cells; 4) distinct effector CTL populations mediate lysis of autologous and allogeneic target cells; and 5) during acute IM, EBV infection results in the induction of both virus-specific and alloreactive CTL populations.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

Emergence of non-major-histocompatibility-complex-restricted lytic CD8+ cells in the blood of nasopharyngeal carcinoma patients

A large body of evidence has suggested that the Epstein-Barr virus (EBV) is strongly associated with undifferentiated nasopharyngeal carcinoma. Immunologically, this neoplasia is characterized by the absence of anti-EBV circulating cytotoxic T lymphocytes (CTL), despite a high number of peripheral activated CD8⁺ cells, as previously determined in our laboratory. In order to determine whether the absence of anti-EBV CTL is related to a reduced number of circulating anti-EBV effector cells, we attempted to expand these hypothetical specific T cells by induction of proliferation with recombinant interleukin-2 (rIL-2), in the, absence of any stimulator cells. Optimal conditions for stimulation of peripheral blood lymphocytes (PBL) of nasopharyngeal patients were obtained with 100 U/ml rIL-2 during 10 days of culture. PBL treated with rIL-2 induced a selective expansion of CD8⁺ cells and generated a potent cytotoxicity towards autologous or HLA-compatible lymphoblastoid cell lines, used as target cells in a chromium-release thest. However, this cytolysis was non-MHC-restricted, since, the monoclonal antibodies anti-(HLA class I) and anti-(HLA class II) were inefficient in inhibiting this cytotoxicity. Interestingly, purified CD8⁺ cells acquired the capacity for non-MHC-restricted cytolysis.This work was supported by grant MD7/91/FMT from La Fondation Nationale de la Recherche Scientifique Tunis, Tunisia.     

Propagation of cytotoxic effectors from chronic myeloid leukemia patients and cloning of cytotoxic T cells

Summary Cytotoxic cells (CTCs) generated from peripheral blood lymphocytes of 5 chronic myeloid leukemia (CML) patients in remission on stimulation with autologous leukemic cells and allogeneic lymphocytes (3-cell assay), were propagated in vitro in interleukin-2 (IL-2)-containing medium and periodic stimulation with autologous leukemic cells, for a period of 4 to 6 months. During this period, the cells were assessed for phenotype and for cytotoxic responses in a 4-h ⁵¹Cr release microcytotoxicity assay. The CTCs continued to show specific lysis of autologous leukemic cells and bone marrow (BM) cells. However, the nonspecific lysis of natural killer (NK) targets and the proportion of cells showing NK phenotype (HNK-1 antigen) increased progressively on cultivation in IL-2-containing medium. Therefore cells showing CD8 phenotype and specific cytotoxic function were segregated by cloning CTCs under the condition of limiting dilution in the presence of allogeneic feeder cells and IL-2-containing medium. Three cytotoxic T cell (CTL) clones expressing CD3+, CD8+, and HLA DR+ phenotypes were obtained from CTCs of 2 CML patients. These clonoid populations, maintained in IL-2-containing medium and periodic antigenic stimulation with autologous leukemic cells, showed specific lysis of autologous leukemic cells and BM cells even at lower (10:1) effector:target ratios. They did not kill K562 (erythroblastoid leukemic NK target cell line) cells and autologous phytohemagglutinin-induced blasts. These clones apparently functioned in an MHC-restricted manner as they did not lyse allogeneic CML cells which would also express a similar set of maturation antigens if sensitization was, as it appeared, against these antigens. Finally, interaction of autologous BM cells with CTL clones reduced the colony forming potential of BM cells only to the extent of 18%–30%. The results therefore indicate that such CTL clones can possibly be used in adoptive immunotherapy as they showed minimal BM toxicity.     

Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction

CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.     

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R₂₄ (γ3), specific for the G_D3 disialoganglioside on melanoma cell surfaces and I₂₄ (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R₂₄ when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R₂₄ occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R₂₄.     

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.     

Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.     

CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.     

Heterogeneity of allogeneic restriction of human cytotoxic T cell clones specific for Epstein Barr virus

Clones of human cytotoxic T cells (Tc) specific for Epstein Barr virus (EBV) were isolated from peripheral blood lymphocyte (PBL) cultures stimulated repeatedly with autologous EBV-transformed lymphoblastoid cell line (LCL) cells in vitro. The method employed to clone EBV-specific Tc was a limiting dilution technique utilizing T cell growth factor (TCGF). The EBV specificity of Tc clones was determined by showing that they were significantly cytotoxic for autologous LCL cells but not for either autologous PBL or (natural killer-sensitive) K-562 cells. Eight EBV-specific Tc clones derived from a single donor exhibited distinct cytotoxic patterns against allogeneic LCL targets. Two clones were cytotoxic to LCL targets sharing both HLA-A26 and B15 antigens with effectors, and killing by two other clones was strongly restricted to autologous LCL cells. The four remaining clones showed cytotoxicities against various allogeneic LCL targets irrespective of HLA antigen expression. Eight EBV-specific Tc clones derived from a second donor also exhibited a wide spectrum of cytotoxicity to allogeneic LcL targets. We conclude that EBV-specific Tc, induced in vitro, consist of a number of clones with respect to restrictions imposed by the major histocompatibility complex. The determinants regulating these restrictions may include not only private HLA antigenic determinants that are defined by the HLA serotyping, but also undefined HLA antigenic determinants.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.     

Enhancement of the recognition by cytotoxic T lymphocytes (CTL) of target membrane antigens after fusion with whole cells

Plasma membrane vesicles (R4-PM) prepared from mouse lymphoma cells (RDM4,H2k) were employed to investigate requirements for recognition of target cell membranes by allogeneic cytotoxic T lymphocytes (CTL). Using immunofluorescent staining and fluorescence microscopy, the R4-PM were tested for binding to CTL and were found to bind to these effector cells in a specific manner. However, this binding was very inefficient compared to the binding of whole RDM4 cells to CTL. The R4-PM were then attached to P388D1 cells (H-2d) in the presence of wheat germ agglutinin and polyethylene glycol (PEG), both under conditions which promote membrane fusion (40% PEG) and under conditions which do not (10% PEG). About 1 cell equivalent R4-PM becomes associated per P388D1 cell in both situations. In the cytotoxicity assays that were carried out, the P388D1 cells which had R4-PM attached under fusion conditions were lysed by CTL directed against H2k in a specific manner, while the P388D1 cells which had R4-PM attached under nonfusion conditions were not lysed above background levels by these CTL. These results suggest that recognition of target cells by allogeneic CTL such that lysis occurs requires more than presentation of the alloantigens as they are expressed in plasma membrane vesicles. However, fusion of these vesicles back into living cells apparently enhances the ability of the alloantigens to be recognized.     

Primary CD4+ T-cell responses provide both helper and cytotoxic functions during Epstein-Barr virus infection and transformation of fetal cord blood B cells

Most humans carry Epstein-Barr virus (EBV) in circulating memory B cells as a latent infection that is controlled by an immune response. When infected by EBV, B lymphocytes in fetal cord blood are readily transformed to lymphoblastoid cell lines (LCL). It is frequently assumed that this high efficiency of transformation is due to the absence of a primary immune response. However, cord blood lymphocytes stimulated with autologous LCL yield CD4+ T cells that can completely inhibit the growth of LCL by a major histocompatibility complex-restricted cytotoxic mechanism mediated by granulysin and granzyme B. Because EBV-transformed B cells maintain the phenotype of antigen-activated B-cell blasts, they can potentially receive inhibitory or helper functions from CD4+ T cells. To assess these functions, the effect of EBV-specific CD4+ T cells on the efficiency of virus transformation of autologous B cells was assayed. Paradoxically, although the cytotoxic CD4+ T-cell lines reduced EBV B-cell transformation at a high effector/target ratio of 10:1, they caused a twofold increase in B-cell transformation at the lower effector/target ratio of 1:1. Th1-polarized CD4+ T cells were more effective at inhibiting B-cell transformation, but Th2-polarized cell lines had reduced cytotoxic activity, were unable to inhibit LCL growth, and caused a 10-fold increase in transformation efficiency. Tonsil lymphoid follicles lacked NK cells and CD8+ T cells but contained CD4+ T cells. We propose that CD4+ T cells provide helper or cytotoxic functions to EBV-transformed B cells and that the balance of these functions within tonsil compartments is critical in establishing asymptomatic primary EBV infection and maintaining a stable lifelong latent infection.     

The role of HLA class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes. Evidence for shared tumor antigens

CTL lines were established in vitro by stimulating patient lymphocytes with autologous melanoma cells in the presence of IL-2. Resulting CTL lines lysed autologous melanoma and failed to lyse several allogeneic melanomas or K562. The mechanism of target cell recognition by autologous tumor-specific CTL was evaluated in this system, using several CTL lines: DT6, DT105, DT141, DT166, DT169, and DT179. Autologous melanoma lysis was inhibited by W6/32, mAb directed against HLA class I Ag, but not by L243, mAb directed against HLA class II Ag. CTL from DT6, DT141, DT166, DT169, and DT179 lysed fresh and cultured allogeneic melanomas, which shared the HLA-A2 Ag, but failed to lyse allogeneic melanomas, which shared B-region or C-region Ag, or shared no HLA class I Ag. CTL from DM141 lysed DM93, which shared A2 and Bw6, but failed to lyse DM105, which shared only Bw6. DM105 CTL failed to lyse allogeneic melanomas that shared HLA-A1, or that shared B or C region Ag, but they did lyse allogeneic melanoma DM49, which expressed an A region Ag that either was A10 or was serologically cross-reactive with A10. A T cell leukemia line, three EBV transformed B cell lines, and a pancreatic cancer line, all of which expressed HLA-A2, were not lysed by DM6 or DM179 CTL. Furthermore, HLA-matched nonmelanomas failed to inhibit autologous tumor lysis in cold target inhibition assays, whereas an HLA-A2+ allogeneic melanoma, DM93, inhibited autologous tumor lysis as effectively as the autologous tumor itself. HLA-A2, and possibly other HLA-A-region Ag, appear to function in HLA-restricted recognition of shared melanoma associated Ag by CTL.     

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.