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PubMed2000

The quantity of TCR signal determines positive selection and lineage commitment of T cells.

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.

Amino Acid Sequence↗

PubMed2005

Defective proximal TCR signaling inhibits CD8+ tumor-infiltrating lymphocyte lytic function.

CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3zeta, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells.

Actins↗

PubMed1990

Frequency of human B cells that differentiate in response to anti-CD3-activated T cells.

Activation of T cells by mAb to the CD3 molecular complex induces the differentiation of many more Ig-secreting cells (ISC) from resting human B cells in bulk cultures than do other modes of polyclonal B cell activation. In the current experiments, a limiting dilution assay was used to demonstrate that this increase in ISC generation reflects an increased frequency of responding B cells. Highly purified B cells were cultured at densities of between 1000 cells and 0.5 cell per microwell with fresh, mitomycin C-treated T cells (T mito) or T cell clones stimulated by immobilized mAb to CD3. After 5 days in culture, the number of wells containing ISC was determined, and the frequency of responding B cells was calculated. The proportion of B cells responding to anti-CD3-stimulated T cells was very large (10.7 +/- 2.8%) and greatly surpassed that induced by other polyclonal activators. B cells cultured with anti-CD3-stimulated T cell clones responded better than did those cultured with T mito. The addition of exogenous IL-2 or IL-6 to cultures supported by activated T mito enhanced the frequency of responding B cells, whereas IL-4 did not increase the generation of ISC and inhibited the augmentation of B cell responses induced by IL-2. Supplementation of cultures with mitomycin C-treated B cells as accessory cells had less of an effect. The addition of both accessory cells and IL-2 markedly increased B cell responsiveness, with precursor frequencies of 60 to 80% noted. In some experiments, cultures were carried out for 7 to 14 days and supernatants were analyzed for IgM, IgG, and IgA secretion. B cells activated by anti-CD3-stimulated T cells produced all three Ig isotypes. When the classes of Ig produced by single B cells were examined, it was observed that the stimulation of individual B cell precursors led to the production of multiple Ig isotypes, suggesting that isotype switching occurs in these cultures. These results demonstrate that under optimum culture conditions, T cells stimulated with immobilized anti-CD3 can activate the majority of human peripheral blood B cells to produce Ig and induce isotype switching by many.

Adult↗

PubMed2000

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Antibodies, Monoclonal↗

PubMed2001

Phosphatidylinositol 3-kinase inhibitors prevent mouse cytotoxic T-cell development in vitro.

To become competent killer cells, CD8(+) T cells require stimulation through signal transduction pathways associated with the T-cell receptor, costimulatory molecules such as CD28, and cytokine receptors such as the interleukin (IL)-2 receptor. We used wortmannin and LY294002, two inhibitors of phosphatidylinositol 3-kinase (PI3-K), to study the role of PI3-K in mouse cytotoxic T-lymphocyte (CTL) induction in response to mitogenic anti-CD3 antibody. Anti-CD3-induced CD8(+) T-cell proliferation and CTL development were inhibited dose dependently by both PI3-K inhibitors. IL-2 synthesis by anti-CD3-activated CD8(+) T cells was also diminished by PI3-K inhibition. PI3-K inhibition resulted in a modest decrease in anti-CD3-induced CD4(+) T-cell proliferation but failed to affect IL-2 expression by anti-CD3-activated CD4(+) T cells. PI3-K inhibition during CTL induction resulted in decreased levels of mRNAs coding for granzyme B, perforin, and Fas ligand. In addition, CTL induced in the presence of PI3-K inhibitors failed to conjugate normally with P815 target cells. Exogenous IL-2 did not reverse the effects of PI3-K inhibition on CD8(+) T-cell proliferation and CTL induction. These results support the conclusion that PI3-K activation is involved in T-cell receptor, CD28, and IL-2 receptor signaling of CD8(+) T cells. PI3-K is, therefore, an important component of multiple signal transduction pathways involved in CTL generation.

Androstadienes↗

PubMed1998

Positive selection through a motif in the alphabeta T cell receptor.

The two lineages of T cells, alphabeta and gammadelta, differ in their developmental requirements: only alphabeta T cells require major histocompatibility complex recognition, a process known as positive selection. The alphabeta T cell receptor (TCR), but not its gammadelta counterpart, contains a motif within the alpha-chain connecting peptide domain (alpha-CPM) that has been conserved over the last 500 million years. In transgenic mice expressing an alphabeta TCR lacking the alpha-CPM, thymocytes were blocked in positive selection but could undergo negative selection. Thus, the alpha-CPM seems to participate in the generation of signals required for positive selection.

Amino Acid Sequence↗

PubMed1989

Role of IL-2 in the generation of CD4+ suppressors of human B cell responsiveness.

The capacity of human T4 cells stimulated by immobilized monoclonal antibodies to the CD3 molecular complex (64.1 and OKT3) to induce and regulate B cell responsiveness was examined. T4 cells stimulated by low concentrations of immobilized 64.1 (3.0 ng/well) and all concentrations of immobilized OKT3 supported B cell proliferation and differentiation. High concentrations of immobilized 64.1 (200 ng/well) failed to stimulate help but rather induced suppression by T4 cells. Suppression was prevented by treating the T4 cells with mitomycin C. Suppression could not be accounted for by deprivation of IL-2. In contrast, induction of suppressor T4 cell activity was closely related to the amount of IL-2 produced by anti-CD3 stimulated T4 cells. Moreover, IL 2 appeared to facilitate the generation of suppressor T4 cell activity. Suppressor cell activity could be generated from unseparated T4 cells as well as from highly purified T4 cell subsets, including Leu 8-, CD45R+, and CD45R- T4 cells, after stimulation with immobilized 64.1. A primary action of suppressor T4 cells appeared to be the direct inhibition of B cell function, as evidenced by the finding that immobilized anti-CD3 activated T4 cells directly suppressed B cell responses stimulated by Staphylococcus aureus and IL-2. Anti-CD3 activated T4 cells did not inhibit initial B cell activation, but suppressed the capacity of the activated B cells to differentiate into ISC. The suppressive influence of anti-CD3 activated T4 cells was reversible as evidenced by the finding that removal of the activated T4 cells from the culture permitted B cell differentiation to proceed. Moreover, anti-CD3-activated T4 cells were able to stimulate initial B cell activation that became apparent when the T cells and B cells were separated. Inhibition of B cell responsiveness by 64.1-activated T4 cells was the result of a block at the G1-S interphase of the cell cycle. The data indicate that anti-CD3-stimulated T4 cells directly and reversibly suppress human B cell function. Moreover, IL 2 appears to play an important role in the differentiation of functionally effective suppressor cells from activated T4 cells.

Adult↗

PubMed1994

ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands.

Leukocyte activation is a complex process that involves multiple cross-regulated cell adhesion events. In this report, we investigated the role of intercellular adhesion molecule-3 (ICAM-3), the third identified ligand for the beta 2 integrin leukocyte function-associated antigen-1 (LFA-1), in the regulation of leukocyte adhesion to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the 38- and 80-kD fragments of fibronectin (FN40 and FN80). The activating anti-ICAM-3 HP2/19, but not other anti-ICAM-3 mAb, was able to enhance T lymphoblast adhesion to these proteins when combined with very low doses of anti-CD3 mAb, which were unable by themselves to induce this phenomenon. In contrast, anti-ICAM-1 mAb did not enhance T cell attachment to these substrata. T cell adhesion to ICAM-1, VCAM-1, FN40, and FN80 was specifically blocked by anti-LFA-1, anti-VLA alpha 4, and anti-VLA alpha 5 mAb, respectively. The activating anti-ICAM-3 HP2/19 was also able to specifically enhance the VLA-4- and VLA-5-mediated binding of leukemic T Jurkat cells to VCAM-1, FN40, and FN80, even in the absence of cooccupancy of the CD3-TcR complex. We also studied the localization of ICAM-3, LFA-1, and the VLA beta 1 integrin, by immunofluorescence microscopy, on cells interacting with ICAM-1, VCAM-1 and FN80. We found that the anti-ICAM-3 HP2/19 mAb specifically promoted a dramatic change on the morphology of T lymphoblasts when these cells were allowed to interact with those adhesion ligands. Under these conditions, it was observed that a large cell contact area from which an uropod-like structure (heading uropod) was projected toward the outer milieu. However, when T blasts were stimulated with other adhesion promoting agents as the activating anti-VLA beta 1 TS2/16 mAb or phorbol esters, this structure was not detected. The anti-ICAM-3 TP1/24 mAb was also unable to induce this phenomenon. Notably, a striking cell redistribution of ICAM-3 was induced specifically by the HP2/19 mAb, but not by the other anti-ICAM-3 mAb or the other adhesion promoting agents. Thus, ICAM-3 was almost exclusively concentrated in the most distal portion of the heading uropod whereas either LFA-1 or the VLA beta 1 integrin were uniformly distributed all over the large contact area. Moreover, this phenomenon was also observed when T cells were specifically stimulated with the HP2/19 mAb to interact with TNF alpha-activated endothelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1990

T cell regulation of in vitro IgM-rheumatoid factor production by normal human B cells. Differential effects of stimulation with immobilized monoclonal antibodies to CD3 and Staphylococcus aureus.

The roles of T cells in in vitro IgM-RF production were examined utilizing two different polyclonal B cell stimulators, Staphylococcus aureus Cowan I (SA) and immobilized mAb to the CD3 molecular complexes (64.1). In cultures stimulated with SA, T4 cells alone but not T8 cells alone could support IgM-RF production. This function of T4 cells could be replaced by IL2 alone or factors generated from mitogen activated T cells (TF). Moreover, when IL2 was supplemented to the cultures, T8 cells as well as T4 cells enhanced the IgM-RF production. Treatment of the T cell subsets with mitomycin C decreased or abrogated the helper function along with a decrease in IL2 production by the T cell subsets in SA stimulated cultures. By contrast, in cultures stimulated with immobilized anti-CD3, T4 cells that had been treated with mitomycin C could induce production of large amounts of IgM-RF, whereas control T4 cells as well as control T8 cells suppressed IgM-RF production. These results indicate that the regulatory functions of T cells in IgM-RF production might be different depending upon the nature of stimulation signals.

Adult↗

PubMed1992

Comparison of CD3 and CD2 activation pathways in T cells from young and elderly adults.

The ability of purified T cells to be activated by immobilized anti-CD3 and soluble anti-CD2 monoclonal antibodies (mAbs) was compared using cells from young and old donors. Purified T cells from elderly humans activated with immobilized anti-CD3 mAb incorporated less [3H]thymidine (58,780 vs 92,258 cpm; p < 0.02) into cellular DNA, and secreted less IL-2 into the culture supernatants than did T cells from young donors. In contrast, T cells activated with anti-CD2 mAbs displayed no age-related differences in proliferation or IL-2 production. Anti-CD2 stimulation resulted in equal IL-2 synthesis by cells from young and old donors that was comparable to the amount produced by cells from elderly donors stimulated with immobilized anti-CD3. Northern blot analysis of early cell cycle gene expression by anti-CD2 activated T cells demonstrated no age differences in the expression of p55 IL-2R or c-myc specific mRNA, although T cells from elderly individuals activated with immobilized anti-CD3 showed statistically significant decreases in both mRNAs. T cell receptor beta chain mRNA levels did not differ between cells from young or old donors after activation by either anti-CD3 or anti-CD2. The discordance in proliferative ability, IL-2 secretion, and specific mRNA expression between T cells from elderly donors activated through the CD3-TCR complex or by soluble anti-CD2 mAbs provides additional evidence for a multifactorial causation of age-related T cell proliferative defects, and may indicate that the difference in proliferative ability is, in part, attributable to responsiveness to secreted IL-2.

Adult↗

PubMed1998

Inhibition of TCR/CD3-mediated signaling by a mutant of the hematopoietically expressed G16 GTP-binding protein.

We have investigated the role of the hematopoietically expressed G16 GTP-binding protein on T cell activation. We constructed transfectants of Jurkat T cells that express a function-deficient mutant of G alpha 16 predicted to prevent activation of this G protein. Upon stimulation with anti-CD3 epsilon antibodies, mutant G alpha 16 transfectants display a profound defect in the production of IL-2 and IL-10, as well as in the expression of CD69. In contrast, the phorbol 12-myristate 13-acetate (PMA)-induced IL-10 production and CD69 expression, and the ionomycin plus PMA-induced IL-2 production are not affected. Consistent with the reduction in cytokine production is the inhibition of early signaling events in the mutant G alpha 16-expressing cells. There are significant reductions in anti-epsilon-induced tyrosine phosphorylation of zeta, epsilon, ZAP-70, and phospholipase C gamma 1, as well as in intracellular Ca2+ mobilization. In accordance with the effects on tyrosine phosphorylation is the reduction of TCR/CD3-mediated Fyn and Lck activities in G alpha 16 mutant cells. Even though the mechanism through which the G alpha 16 mutant mediates inhibition of T cell activation is not known, the data suggest a model where G proteins become activated upon TCR/CD3 engagement and regulate the activation of tyrosine kinases and subsequent downstream signaling events that lead to the activation of cytokine genes.

Alanine↗

PubMed1992

Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.

The capacity of human B cells to differentiate into high rate nondividing antibody-secreting plasma cells was investigated. Highly purified human peripheral blood B cells were stimulated with polyclonal B cell activators in the presence of a variety of recombinant cytokines (IL-2, IL-4, IL-6). Maximal production of Ig of all isotypes was observed when B cells were stimulated with intact T cells that were activated with mAb to the CD3 molecular complex. In these cultures, Ig production continued for more than 16 days. Moreover, differentiation to nondividing high rate Ig-producing cells was induced, as evidenced by a ninefold increase in the amount of Ig produced per Ig-secreting cell and the acquisition of resistance of ongoing Ig secretion to the inhibitor of DNA synthesis, hydroxyurea. To determine whether intact T cells were required for the entire culture period to achieve maximal Ig production, B cells were cultured with activated T cells for various lengths of time, reisolated and cultured with fresh activated T cells or various cytokines, then analyzed for Ig secretion. B cells preactivated for 6 days with anti-CD3-stimulated T cells required contact with intact T cells for continued Ig secretion. However, after 9 days of preactivation, dividing B cells responded maximally to anti-CD3-stimulated T cells, whereas cytokines were able to drive continued IgG secretion by nondividing B cells in the absence of intact T cells. IL-6 alone, or in combination with either IL-2 or IL-4, was the major cytokine driving ongoing Ig secreting by nondividing preactivated B cells. These results suggest that continued clonal expansion of Ig-secreting B cell blasts requires intact anti-CD3-activated T cells, whereas terminal differentiation of B cells into plasma cells after extensive clonal expansion is driven by cytokines, most notably IL-6.

Adult↗

PubMed1988

Monoclonal antibodies against human leucocyte antigens. II. Antibodies against CD45 (T200), CD3 (T3), CD43, CD10 (CALLA), transferrin receptor (T9), a novel broadly expressed 18-kDa antigen (MEM-43) and a novel antigen of restricted expression (MEM-74).

The specificities of several monoclonal antibodies described by us earlier were determined more exactly: (1) antibodies MEM-31 and MEM-32 are directed against CD8 and CD5 T cell antigens, respectively: (2) MEM-15 and MEM-18 react with the CD14 monocyte antigen; (3) MEM-28 recognizes the CD45 pan-leucocyte antigen. Several new monoclonal antibodies are described: (4) MEM-55 and MEM-58, reactive with subsets of CD45-related molecules; (5) MEM-43, recognizing a broadly expressed 18-kDa glycoprotein; (6) MEM-57, reacting with the CD3 antigen complex: (7) MEM-59 recognizing the CD43 leucocyte antigen; (8) MEM-74, which reacts with an unidentified antigen strongly expressed on several cell lines and many leukaemic cells but absent from most resting leucocytes; (9) MEM-75, directed against transferrin receptor, and (10) MEM-78, recognizing CD10 (CALLA) antigen.

Antibodies, Monoclonal↗

PubMed2000

Impaired expression of the CD3-zeta chain in peripheral blood T cells of patients with chronic myeloid leukaemia results in an increased susceptibility to apoptosis.

In patients with myeloid malignancies, cell-mediated immunity is often suppressed, being most profound in those with advanced disease. Such immune dysfunction, as demonstrated in many patients with chronic lymphocytic leukaemia (CLL) and myelodysplastic syndrome (MDS), may, at least in part, be due to altered expression of the CD3-zeta chain, which is an important component of the T-cell receptor (TCR). We speculated that impaired expression of the TCR-zeta chain would be evident in peripheral blood T cells of patients with chronic myeloid leukaemia (CML) and that such an abnormality would result in an increased ex vivo susceptibility to apoptosis. In this study, we demonstrated that, compared with normal controls, zeta chain expression was significantly downregulated in all of the T-cell subsets (P < 0.009) in more than 90% of CML patients. In addition, there was a significantly lower expression of the CD3-epsilon chain (P < 0.001) in patients than in controls. In those patients with abnormal zeta chain expression, the proportion of lymphocytes with spontaneous DNA fragmentation, as determined by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assays, was also significantly higher (P < 0.002) than controls. From all of the patients tested, it was possible to upregulate partially zeta chain expression and hence to reduce the susceptibility to apoptosis by cross-linking the T cells with interleukin (IL)-2, interferon (IFN)-alpha or immobilized CD3. In addition, such cross-linked T cells showed a significantly higher capacity to proliferate than the native CML T cells.

Adult↗

PubMed1988

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli.

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.

Animals↗

PubMed1991

A humanized monovalent CD3 antibody which can activate homologous complement.

The rat monoclonal antibody (mAb) YTH12.5, specific for the CD3 antigen complex on human T cells has been modified in order to improve its efficacy in human therapy. With the aim of rendering it less immunogenic, it has been humanized using the method of framework grafting. During this process sequence analysis of the YTH12.5 VL gene indicated that it was of the lambda subclass, however, it was markedly dissimilar from previously published rat and mouse V lambda gene sequences and may represent a new V lambda gene family. The humanization of this light chain represents the first successful reshaping of a lambda light chain V region. To improve the effector function of the antibody we have created a monovalent form (1 Fab, 1 Fc) using a novel method involving the introduction of an N-terminally truncated human IgG1 heavy chain gene into cells producing the humanized CD3 mAb. Comparison of the mono- and bivalent humanized mAb in a complement-mediated cell lysis assay revealed that the monovalent antibody mediated lysis of human T cell blasts whereas the bivalent form did not. The availability of a humanized, complement-fixing CD3 mAb may improve opportunities for human therapy, in the management of organ rejection, autoimmunity and the treatment of T cell lymphoma.

Amino Acid Sequence↗

PubMed1997

Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus.

Differentiation of thymocytes into mature single-positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), co-receptors (CD4 or CD8) and their appropriate major histocompatibility complex-encoded ligands. Precisely how these receptor/co-receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by co-ligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')2 antibodies to mimic TCR/ co-receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross-linking or targeting to Fc receptor-bearing cells. We show that TCR/CD3 co-ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co-ligation with CD8 also induces maturation of CD4 T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co-ligation of TCR and CD8 appear weaker than from co-ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single-positive cell differentiation.

Animals↗

PubMed2000

Characterization of the porcine gammadelta T-cell receptor structure and cellular distribution by monoclonal antibody PPT27.

The T-cell receptor (TCR) is the critical structure involved in antigen recognition of T lymphocytes. Although the pig has a large proportion of circulating T lymphocytes bearing the gammadelta TCR, their study has been impeded due to the lack of specific antibodies. Here a monoclonal antibody (mAb) PPT27 directed to gammadelta TCR is described. Flow cytometry analyses showed that the mAb recognized a subset of T lymphocytes of which the majority expressed no CD2, CD4 and CD8 whilst the minority bore CD2 and CD8. The mAb precipitated a protein of 86 000 MW under non-reducing conditions and a doublet of 43 000 MW under reducing conditions from peripheral blood T lymphocytes lysed in nonidet P-40 buffer, whilst it precipitated the CD3-TCR complex from the cells lysed in digitonin. Further analysis revealed that the antibody recognized the majority, but not all, of the gammadelta T cells, suggesting that there may be more isotypes of gammadelta TCR than currently believed. The antibody was unable to stimulate gammadelta T cells to proliferate in vitro, suggesting that these cells are activated by a different activation mechanism from that of alphabeta T cells.

Animals↗