Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic activity and lymphokine production of T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2 and HLA-A2 mutants. Recognition of TCR-gamma delta+ CTL clones is affected by mutations at positions 152 and 156

TCR-gamma delta+ CTL clones were generated from CD4-CD8- T cells that were stimulated twice with the cell line JY. Either IL-2 or IL-4 was used as growth factor. A number of TCR-gamma delta+ clones were found to lyse the stimulator cell line JY. Two of these clones secreted N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase activity after stimulation with JY cells. The cytotoxic activity of these two clones was blocked by a mAb specific for HLA-A2. Moreover, these two TCR-gamma delta+ clones selectively lysed human fibroblast line M1 and murine P815 cells transfected with DNA fragments encoding HLA-A2 but not those transfected with HLA-B7 encoding DNA, indicating that these clones recognize HLA-A2. Analysis of the recognition of HLA-A2 by using target cells transfected with mutated HLA-A2 encoding genes revealed that the nature of the amino acid at position 152 of the molecule is critical for recognition of the TCR-alpha beta+ as well as the TCR-gamma delta+ CTL clones since replacement of Val for Ala at that position resulted in abrogation of recognition of one TCR-gamma delta+ and one TCR-alpha beta+ clone and substitution of Val for Glu affected recognition of all clones. Substitution of Leu for Trp at position 156 abrogated recognition by one TCR-gamma delta+ and one TCR-alpha beta+ T cell clone, but recognition by the other clones was not changed. All clones were able to secrete IL-2, IFN-gamma, and GM-CSF but not IL-4 after activation.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T 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.     

Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.     

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.     

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.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.     

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.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

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.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Analysis of the donor-specific cytotoxic T lymphocyte repertoire in a patient with a long term surviving allograft. Frequency, specificity, and phenotype of donor-reactive T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ clones

In the present study the transplant specific CTL repertoire of a patient (HLA:A1,3, B8,18, Cw5,7 DR3, DQw2, DPw3) with a long term surviving HLA mismatched kidney graft (HLA: A1,24 B8,27 Cw2,7, DR3, w13 DQw2,6 DPw1,3) has been investigated. This patient was unable to generate specific cytolytic activity against donor-derived PHA-blasts in the MLC in which donor spleen cells or B lymphoblastoid cell line were used as stimulator cells. In addition, the CTL precursor frequencies against donor alloantigens were very low (1/67,000). The patient had otherwise normal immune responses in vivo and in vitro and no signs of transplant rejection. Transplant specific CTL clones were generated in high frequencies (1/195) from T cell bulk cultures activated by PHA in the absence of any sensitization by donor Ag in vitro. The repertoire of 14 donor-reactive CTL clones (12 TCR-alpha beta+ and 2 TCR-gamma delta+) was analyzed. Two TCR-alpha beta+ CD8+ clones were specific for B27. Ten TCR-alpha beta+ CTL clones directed against class II HLA Ag were isolated. Seven of these were CD4+ and recognized DRw13 (3), DQw6 (3), and DPw1 (1), whereas three of these clones were CD4-CD8+ recognizing DRw13 (1) and DQw6 (2). In addition, two donor-specific TCR-gamma delta+ CTL clones were obtained recognizing HLA-A9(23,24) and DQw6. Our data indicate that the precursors of CTL clones specifically directed against donor class I or II HLA Ag are not deleted from the repertoire and that part of this reactivity resides in the TCR-gamma delta+ fraction.     

IL-2 and IFN-gamma are two necessary lymphokines in the development of cytolytic T cells

A filler cell-free limiting-dilution microculture system has been developed for the expansion and differentiation of a high proportion of single CD4-CD8+ T cells into cytolytic T cell (CTL) clones. The stimulus used was PMA together with the calcium ionophore ionomycin. The growth and differentiation factors were rIL-2, together with either a Con A-stimulated spleen cell supernatant (CAS) or rIFN-gamma. CTL activity was monitored by an autoradiographic 111In-release assay. With CAS and rIL-2 present, 50% of all potential precursors (CTL-p) produced cytolytic clones. Substitution of rIFN-gamma for CAS gave a similar efficiency with up to 42% of CTL-p producing cytolytic clones. rIL-2 alone allowed only a small proportion (6%) of CD4-CD8+ T cells to become cytolytic clones. Addition of rIL-2 and rIFN-gamma at various stages of the culture demonstrated that IL-2 was required throughout, but exogenous IFN-gamma was required only during the early stages. It is concluded that for at least 40% of all CTL-p, the lymphokines IL-2 and IFN-gamma are essential and act in synergy to induce proliferation and differentiation into CTL.