Phenotypic profile and functions of T cell receptor-gamma delta-bearing cells from patients with primary immunodeficiency syndrome

TCR-gamma delta-bearing T cells have been reported to be increased in several immunodeficient patients. However, their functional role and phenotypic characterization have not yet been well documented. In this study we examined the surface phenotypes and functional properties of TCR-gamma delta+ cells from several patients with primary immunodeficiency syndrome. It was demonstrated that TCR-gamma delta+ cells detected by TCR-delta 1 mAb were increased in some of the patients, particularly in patients with Wiskott-Aldrich syndrome and severe combined immune deficiency. The TCR-gamma delta+ cells showed such a unique profile that more than 60% of the cells expressed delta-TCS1, which is normally present in a lesser amount, and that most of the cells lacked CD5 T lineage marker. TCR-gamma delta+ cells from the patients with primary immunodeficiency syndrome served as NK cells as observed in normal individuals, while displaying weak LAK and allogeneic cell-specific killer activities. The TCR-gamma delta+ cells were classified into several subpopulations according to their antigenic phenotype, then their NK activity of normal individuals and patients, lymphokine-activated killer and allo-specific killer activities of normal individuals were compared among the subpopulations. Delta-TCS1+ cells mediated almost the same killer activities as total TCR-gamma delta+ cells, whereas CD8+ TCR-gamma delta+ cells displayed stronger cytotoxic activities in both normal subjects and the patients with primary immunodeficiency syndrome.     

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.     

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)     

Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.     

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.     

Expansion of a maternally derived monoclonal T cell population with CD3+/CD8+/T cell receptor-gamma/delta+ phenotype in a child with severe combined immunodeficiency.

The diagnosis of severe combined immunodeficiency complicated by chronic graft-vs-host disease affecting liver and skin in association with engraftment of maternal T cells was established in a 5-mo-old boy. Detailed immunologic and molecular genetic studies were performed because a unique T cell phenotype was identified on initial evaluation. A major proportion of the patient's peripheral T cells expressed a CD8+ and TCR-gamma/delta+ phenotype while CD4+ T cells were virtually absent. Southern blot analysis of cell subpopulations isolated by fluorescence activated cell sorting indicated that approximately 50% of CD8+/TCR-gamma/delta+ cells were clonally related. Immunophenotyping and -genotyping also identified a clonal TCR-gamma/delta+ cell population in the child's mother. Clonal identity of these T cell populations in mother and child was demonstrated by studies using a clonspecific TCR-delta probe generated by polymerase chain reaction as well DNA sequence analysis. HLA typing and DNA fingerprinting confirmed that the child had acquired this clone diaplacentally from the mother. According to immunohistology and DNA analysis the clone was found to be virtually absent in the liver tissue suggesting that this clonal T cell population plays a minor role, if any, in the pathogenesis of the liver abnormalities in the patient. In the mother the CD8+/TCR-gamma/delta+ clone spontaneously declined to a level around 1% of PBMC several months later and has remained at this level since. We conclude that 1) a clonal expansion of TCR-gamma/delta T cells, triggered by yet unknown stimuli, may occur in otherwise healthy individuals, 2) respective T cells are able to cross the placental barrier, and 3) in an microenvironment precluding rejection, i.e., in severely immunocompromised patients, these cells may persist and even represent a significant proportion of circulating T cells.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

Immortalization of human T cells expressing T-cell receptor gamma delta by herpesvirus saimiri.

Herpesvirus saimiri (HVS) has recently been shown to immortalize human CD4+ and CD8+ T cells expressing T-cell receptor alpha beta (TCR-alpha beta) with the maintenance of their original phenotypes and functional properties. However, the immortalization of human T cells expressing TCR-gamma delta by HVS has not been successful. Here we report that HVS can also infect and immortalize human T cells expressing TCR-gamma delta. Two human TCR-gamma delta+ T-cell clones, which continuously proliferated in interleukin-2-containing culture medium without any exogenous stimulation or addition of feeder cells for more than 8 months, were established by HVS infection. Morphologically, the HVS-transformed TCR-gamma delta+ T-cell clones were granular lymphocytes which exhibited wide-range HLA-unrestricted cytotoxicity as untransformed TCR-gamma delta+ T cells. Their phenotypes and cytotoxic activities were not altered during long-term culture. The immortalization of human TCR-gamma delta+ T cells by HVS infection would be useful for functional analysis of this lymphocyte population, which is believed to play an important role in protection against various infectious diseases.     

Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes

Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2. This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth. Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2. Cells positive for TCR-alpha beta were barely detectable. If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred. From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals. TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth. Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed. This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells. Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8. Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected. From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55. The biologic significance of our findings is discussed.     

Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-alpha beta+, TCR-gamma delta+ T lymphocytes, and NK cells.

We have analyzed the effects of NK cell stimulatory factor/IL-12, on proliferation of PBL and their subsets. IL-12 synergizes with lectins and phorbol diesters to induce proliferation of CD4+ and CD8+ peripheral blood T lymphocytes. In the case of phorbol-diester-induced proliferation, the effect of IL-12 is in part mediated by induced IL-2 production, as suggested by the observation that IL-12 enhances IL-2 production in these cultures and that anti-IL-2 antibodies inhibit proliferation. IL-12 synergizes also with anti-CD3 antibodies and with allogeneic stimulation in MLC in inducing T cell proliferation. IL-12 alone is mitogenic for preactivated T and NK lymphoblasts. This mitogenic effect is observed with similar doses of IL-12 on NK lymphoblasts as well as on CD4+ and CD8+ TCR-alpha beta+ and on TCR-gamma delta+ lymphoblasts. On TCR-alpha beta+ T lymphocytes the effect of IL-12 is always additive to that of IL-2 over a wide dose range. The same effect is observed on highly activated, actively proliferating NK cells. However, on NK and TCR-gamma delta+ lymphoblasts reverting to a resting state after stimulation and on a TCR-gamma delta+ acute leukemia-derived T cell line, IL-12 inhibits significantly the proliferation induced by moderate to high doses (10 to 100 U/ml) of IL-2. This inhibitory effect is, at least in part, indirect, and depends on IL-12-induced production of TNF. Neutralizing anti-TNF antibodies, but not anti-IFN-gamma and anti-transforming growth factor antibodies, restore by more than 70% the inhibition of proliferation induced by IL-12 in these cultures. However, TNF alone cannot mimic the inhibitory effect of IL-12 on the IL-2-induced proliferation of NK and TCR-gamma delta+ lymphoblasts, suggesting the involvement of additional mechanisms. The relevance of these findings for the biology of lymphocyte subsets mediating MHC nonrestricted cytotoxicity is discussed.     

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.     

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.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

Introduction of T cell receptor (TCR)-alpha cDNA has differential effects on TCR-gamma delta/CD3 expression by PEER and Lyon-1 cells

A TCR heterodimer composed of a TCR gamma-chain and a TCR delta-chain was found to be expressed in association with CD3 by a small population of human peripheral blood T cells, thymocytes, and certain leukemic T cell lines. The leukemic T cell lines PEER and Lyon-1 express such a TCR-gamma delta/CD3 complex at the cell surface. In addition, PEER and Lyon-1 cells transcribe a productively rearranged TCR-beta gene. Introduction of TCR alpha-chain cDNA of human or murine origin resulted in cell surface expression of a TCR-alpha beta/CD3 complex on PEER and Lyon-1 cells. The expression of the TCR-gamma delta/CD3 complex on PEER cells was not affected by introduction of TCR-alpha cDNA. In contrast, introduction of a TCR-alpha cDNA and expression of the TCR-alpha beta/CD3 complex in Lyon-1 cells resulted in the disappearance of the TCR-gamma delta/CD3 complex. These data were confirmed by indirect immunofluorescence, at the protein level and by gene expression analysis. Triggering of the TCR-alpha beta/CD3 complexes by anti-CD3 mAb or anti-TCR mAb resulted in increased internal Ca2+ levels, indicating that these receptors were functional in signal transduction. These results indicate that, besides TCR gene rearrangements, membrane expression of TCR-alpha beta heterodimers may be important in regulating TCR-gamma delta cell surface expression.     

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.     

The expression of T cell receptor-associated proteins during T cell ontogeny in man

The expression of TCR-associated molecules was examined in human fetal and postnatal tissues. From gestational wk 7 onward in the fetal liver, putative prothymocytes have been identified with cytoplasmic CD3 positivity (cCD3+). These immature cells are TdT- and do not express membrane CD3 (mCD3-) or TCR beta identified by beta F1, but show CD7 and CD45 positivity without CD1, CD2, CD5, CD4, CD8, CD10, and class II Ag. Their high proliferative activity is indicated by greater than 85% Ki67 positivity. After the 10th wk, beta F1+, mCD3+ cells also appear in the liver and these are mostly Ki67- but no TCR gamma delta-bearing cells can be identified at such an early stage of extrathymic development. In the mCD3- TdT-fetal thymus (10 1/2 to 18th wk) cCD3+, mCD3- CD1-blasts proliferate (Ki67+) and lack TCR-beta or TCR-gamma delta. The TdT-, CD1+ cortical thymocytes develop into TCR-beta + and WT31-positive (TCR-alpha beta +) cells. Subsequently TdT-positive thymocytes become detectable around 19 to 20 wk, and in such glands the peak of proliferative activity is seen among TdT+, cCD3+ cells which appear to acquire, in a regular sequence, cytoplasmic beta F1 (TCR-beta), mCD3, and TCR-alpha beta (WT31 positivity) together with the loss of TdT and Ki67 positivity. A newly described transitional population of cells is TdT-, beta F1+ but exhibits no detectable WT31 positivity. These cells correspond to the CD1+, mCD3+ thymocytes and are probably the targets of thymic selection. The cells of the TCR-gamma delta lineage, detected by mAb TCR-delta-1 and delta TCS1, are rare (0.02 to 0.5%) among thymocytes from gestational wk 10 1/2 onward through the whole span of thymic development, but these cells include a proportion (18 to 59%) of cells expressing CD1 Ag, suggesting that these TCR-gamma delta cells differentiate in the thymus. Among the CD1+, TCR-gamma delta + thymocytes, no TdT positivity can be detected.     

Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.