Natural killer (NK) activity of cultured S100β-positive T-leukemia cells

In order to clarify the function of human S100β- positive T-cells, S100β-positive T-leukemia cells (S100β TLC) were examined in vitro. S100β TLC were obtained from the peripheral blood of a patient with S100β-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100β TLC expressing CD3 and CD8 antigens. Although S100β TLC expressed CD3 antigen, they were negative for the α/β and γ/δ T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and δ TCS-1, respectively. It was speculated that S100β TLC initially expressed α/β TCR but lost it during malignant transformation. When S100β TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100β TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100β TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100β TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 β TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition. These findings suggest that S100 β-positive T-cells are functionally similar to NK cells. We discuss the roles of S100 β-positive T-cells in the human immune system.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Expression of murine IL-2 receptor beta-chain on thymic and splenic lymphocyte subpopulations as revealed by the IL-2-induced proliferative response in human IL-2 receptor alpha-chain transgenic mice

Lymphocytes from the human (h) IL-2R alpha chain transgenic mice (TGM) constitutively express high affinity binding sites for hIL-2, consisting of transgenic h-IL-2R alpha and endogenous murine IL-2R beta, and therefore easily proliferate in vitro in response to hIL-2. Our study was undertaken to clarify the hIL-2-responsive lymphocyte subsets in the TGM, which should most likely reflect the normal distribution of m IL-2R beta expression. In both thymus and spleen, the majority of expanded cells by hIL-2 was CD3+CD4-CD8+ TCR alpha beta+ cells. The proliferation of CD4+ cells was not observed at all from either organ despite the expression of transgenic hIL-2R alpha. Potent cellular proliferation was also observed from the thymocytes that had been depleted of CD8+ cells, the expanded cells consisting of CD3- (15-40%) and CD3+ populations (60-85%). Among CD3+ cells, approximately the half portion expressed TCR alpha beta, whereas the other half was suggested to express TCR gamma delta. A variable portion (5-20%) of the CD3+ cells expressed CD8 (Lyt-2) in the absence of Lyt-3, and the CD3+CD8+ cells were confined preferentially to the TCR alpha beta- (TCR gamma delta+) population. In the culture of splenocytes depleted of CD8+ cells, however, the proliferated cells were mostly CD3-CD4-CD8-TCR-Mac1-, whereas a minor portion (10-30%) was CD3+CD4-CD8-TCR alpha beta- (TCR gamma delta+. Analysis of TCR genes at both DNA and mRNA levels confirmed the phenotypical observations. These results strongly suggested that IL-2R beta was constitutively and selectively expressed on the primary murine thymocytes and splenic T and NK cells, except for CD4+ cells in both organs.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.     

The antiviral efficacy of the murine alpha-1 interferon transgene against ocular herpes simplex virus type 1 requires the presence of CD4(+), alpha/beta T-cell receptor-positive T lymphocytes with the capacity to produce gamma interferon

Alpha/beta interferons (IFN-alpha/betas) are known to antagonize herpes simplex virus type 1 (HSV-1) infection by directly blocking viral replication and promoting additional innate and adaptive, antiviral immune responses. To further define the relationship between the adaptive immune response and IFN-alpha/beta, the protective effect induced following the topical application of plasmid DNA containing the murine IFN-alpha 1 transgene onto the corneas of wild-type and T-cell-deficient mice was evaluated. Mice homozygous for both the T-cell receptor (TCR) beta- and delta-targeted mutations expressing no alpha beta or gamma delta TCR (alpha beta/gamma delta TCR double knockout [dKO]) treated with the IFN-alpha 1 transgene succumbed to ocular HSV-1 infection at a rate similar to that of alpha beta/gamma delta TCR dKO mice treated with the plasmid vector DNA. Conversely, mice with targeted disruption of the TCR delta chain and expressing no gamma delta TCR(+) cells treated with the IFN-alpha 1 transgene survived the infection to a greater extent than the plasmid vector-treated counterpart and at a level similar to that of wild-type controls treated with the IFN-alpha 1 transgene. By comparison, mice with targeted disruption of the TCR beta chain and expressing no alpha beta TCR(+) cells (alpha beta TCR knockout [KO]) showed no difference upon treatment with the IFN-alpha1 transgene or the plasmid vector control, with 0% survival following HSV-1 infection. Adoptively transferring CD4(+) but not CD8(+) T cells from wild-type but not IFN-gamma-deficient mice reestablished the antiviral efficacy of the IFN-alpha 1 transgene in alpha beta TCR KO mice. Collectively, the results indicate that the protective effect mediated by topical application of a plasmid construct containing the murine IFN-alpha 1 transgene requires the presence of CD4(+) T cells capable of IFN-gamma synthesis.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.     

gamma delta T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium

We have investigated the expression of the alpha beta and the gamma delta T cell receptor (TCR) in the human intestine. By immunohistology we found that 39% of CD3+ intraepithelial lymphocytes (IEL) expressed the gamma delta TCR compared to 3% of CD3+ lamina propria lymphocytes (LPL). Cytofluorometric analysis of isolated cells revealed a significantly higher proportion of gamma delta T cells among CD3+ IEL compared to LPL and peripheral blood lymphocytes. This was due to an increase in both CD8+ (low density) and CD4-CD8- gamma delta T cells in IEL. Most alpha beta IEL expressed high-density CD8. About 30% of both IEL and LPL expressed CD25 (alpha-chain of the IL-2 receptor). HML-1 expression was detected on nearly all IEL and on 27% of LPL. CD25 and HML-1 were preferentially expressed on intestinal alpha beta and gamma delta T cells, respectively. Thus, human gamma delta T cells are located preferentially in the gut epithelium and are phenotypically different from alpha beta T cells, which constitute the majority of both LPL and IEL.     

Characterization of a monoclonal antibody which detects all murine alpha beta T cell receptors

Research on the specificities, functions, and maturation of T cells would be greatly aided by a collection of monoclonal antibodies which distinguishes different types of TCR. With this end in mind hamsters were immunized and tested for production of pan-reactive anti-mouse alpha beta TCR antibodies. In this report we describe the properties and uses of a mAb, H57-597, produced from one of these animals. The mAb reacts with surface receptors on all alpha beta TCR-bearing cells and does not react with receptors on gamma delta+ T cells. In an immobilized form, this antibody can directly activate T cells bearing alpha beta TCR. It can be used in immunoprecipitation reactions to precipitate receptor from the appropriate cell types. In combination with anti-CD3, the antibody can be used in cytofluorographic analyses to measure numbers of CD3+, alpha beta+, and CD3+, gamma delta+ cells in the thymus and periphery.     

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.     

Distribution of T cells bearing different forms of the T cell receptor gamma/delta in normal and pathological human tissues

Frozen sections from normal and pathologic human tissues were immunostained by the APAAP technique with three mAb directed against different epitopes of the TCR gamma delta; TCR delta 1 which binds to all cells bearing the TCR gamma delta; BB3 and delta TCS1 which, by immunoprecipitation studies, appear to react respectively with the disulfide-linked and nondisulfide-linked form of the TCR gamma delta. In normal thymus, TCR delta 1+ cells accounted for approximately 2% of the CD3+ thymocytes and were about three times more numerous in the medulla than in the cortex. TCR delta 1+ cells were mostly constituted by the delta TCS1 reactive subset (average ratio delta TCS1/BB3: 3.7). In the tonsil, the TCR delta 1+ cells (about 3% of CD3+ elements) were mainly located in the interfollicular area, where they frequently tended to arrange around high endothelium venules. In most samples, TCR delta 1+ cells were distributed beneath to the tonsil epithelium. Unlike thymus, the majority of TCR delta 1+ cells were usually constituted by the BB3-reactive subset (average BB3/delta TCS1 ratio: 2.0). A similar predominance of BB3+ over delta TCS1+ cells was also observed in normal peripheral blood. The spleen was the organ with the highest concentration of TCR delta 1+ cells that, like in the thymus, were mostly represented by delta TCS1+ elements. Noteworthy, the TCR delta 1+ cells were preferentially located in the splenic sinusoids while TCR alpha beta-bearing lymphocytes mostly occupied the periarteriolar sheaths of penicilliary arteries. The majority of neoplastic T cell proliferations studied lacked to express the TCR gamma delta. Two cases of beta F1-(TCR alpha beta-) T lymphoblastic lymphoma, however, were TCR gamma delta+ (delta TCS1+/BB3-). Both of them showed a stage II cortical phenotype, e.g., CD1+/CD3+/CD4+/CD8+/TCR delta 1+. Among inflammatory conditions, an increase of BB3+ cells was observed in close association with necrotic areas in cases of Kikuchi's and tuberculous lymphadenitis. The significance of this finding is under study.     

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.     

Interferon β enhances the natural killer activity of patients with bladder carcinoma

We have investigated the effect of interferon (INFß) on the natural killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) from patients with superficial and infiltrative transitional-cell carcinoma of the bladder (TCC) against both NK-sensitive and NK-resistant target cells. The normal NK activity found in PBMC from these patients can be significantly enhanced by short-term incubation (18 h) with INFß (P<0.05). The depressed NK cytotoxic activity found in PBMC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term incubation with INFß (P<0.05). In kinetic studies we found that the maximal levels of the INFß-promoted cytotoxic activity against NK-sensitive and against NK-resistant target cells in PBMC from TCC patients were reached after 18 h of culture. Short-term-INFß-incubated PBMC from patients with TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. The effector cells of the INFß-induced cytotoxic activity in PBMC from patients with TCC were CD16⁺ CD3^– NK cells. This cytotoxic inducer effect of INFß synergized with that of interleukin-2. In conclusion, INFß can enhance the NK activity of PMBC from patients with TCC of the bladder.     

Induction of lysis by T cell receptor gamma delta+/CD3+ T lymphocytes via CD2 requires triggering via the T11.1 epitope only

The requirements for activation of the lytic machinery through CD2 of TCR gamma delta+/CD3+ cells were examined, by utilizing bispecific heteroconjugates containing anti-CD2 mAb cross-linked to anti-DNP. Contrary to the CD2 activation requirements in TCR alpha beta+/CD3+ cells, cytotoxic activity in TCR gamma delta+/CD3+ clones and TCR-/CD3- NK cell clones can be induced by heteroconjugates containing a single anti-CD2 (OKT11.1) mAb. Activation of TCR gamma delta+/CD3+ cells via CD2 is independent of heteroconjugates binding to CD16 (Fc gamma RIII), because heteroconjugates prepared from Fab fragments induced equal levels of lysis. Moreover, anti-CD16 mAb did not inhibit triggering via CD2 in TCR gamma delta+/CD3+ cells. In TCR-/CD3- NK cells, however, induction of cytotoxicity via CD2 is co-dependent on interplay with CD16. Anti-CD3 mAb blocked the anti-CD2 x anti-DNP heteroconjugate-induced cytotoxicity of TCR gamma delta+/CD3+ cells, indicating a functional linkage between CD2 and CD3 on these cells. We conclude that induction of lysis via CD2 shows qualitatively different activation requirements in TCR gamma delta+/CD3+, TCR alpha beta+/CD3+ CTL and TCR-/CD3- NK cells.     

Patterns of T cell receptor gamma gene rearrangements in human CD3+ clones derived from WT31- or Leu7+ cells in relation to non-MHC-restricted cytotoxic activity

Clones were obtained from human peripheral blood WT31-, WT31-CD4-8-, CD4-8- or Leu 7+ cells in the presence of interleukin 2 and phytohaemagglutinin. Almost all clones were CD3+, about 50% were CD4-8- and all clones tested derived from WT31- remained WT31-, indicating that they were expressing a gamma/delta heterodimer in association with CD3. Some clones derived from CD4-8- cells expressing CD3 were WT31- and some were WT31+. All CD3+ clones had T cell receptor (TCR) gamma gene rearrangements; most also had their TCR beta genes rearranged, including all clones derived from Leu 7+ cells. TCR gamma gene rearrangements were noted involving all five known J segments. There was a tendency for V gene segments from the VII and VIII subgroups to be rearranged to J gamma 2 less often than those from the more 5' VI subgroup. Two clones definitely had one rearrangement to C gamma 1 and one to C gamma 2. When clones derived from WT31- cells were considered, the only obvious relationship which emerged was that all clones with both chromosomes rearranged to C gamma 2 had low or negligible cytotoxic activity against natural killer (NK)-sensitive and NK-resistant targets. Several of these clones were expressing CD8 on about 30% of cells. Most clones with rearrangements involving only C gamma 1 had high non-MHC-restricted cytotoxicity while those with at least one C gamma 1 rearrangement had either high or low activity. The only exceptions noted were a clone with a single V9JP rearrangement and a clone with a V9JP and a VI/IIIJP1 rearrangement, which both had low activity. A similar pattern was also found with most clones derived from Leu 7+ cells. The data are consistent with the participation of most types of disulphide-linked (C gamma 1) gamma/delta heterodimers in non-MHC-restricted cytotoxic activity mediated by CD3+ gamma/delta + T cell clones.     

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts.

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3+ phenotype and the alpha/beta T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56+. The evaluation of CD45 isoforms showed that both "naive" and "memory" cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.