anti-TCR Antibody Research Articles

Identification of a Putative G Protein-Coupled Receptor Induced during Activation-Induced Apoptosis of T Cells

During development, self-reactive immature thymocytes are clonally deleted in the thymus, a phenomenon which establishes T cell tolerance (negative selection). It has been shown that the deletion of self-reactive immature T cells in the thymus is mediated by apoptosis upon T cell receptor (TCR) engagement. Apoptosis of immature thymocytes mediated by the TCR requires the expression of a new set of genes. To define which genes are required during the TCR-mediated death of immature thymocytes, we sought to identify genes whose expression is increased during TCR-mediated cell death. Using the technique of differential mRNA display, we have identified a novel gene, TDAG8, which encodes a putative G protein-coupled receptor. The expression of TDAG8 is greatly induced upon activation of T cells by anti-TCR antibody or by phorbol 12-myristate 13-acetate plus ionomycin. The treatment of T cells with glucocorticoids also greatly induces the expression of TDAG8. In mice, TDAG8 is predominantly expressed in thymus and spleen. The tissue-specific expression of TDAG8 and the induction of its expression during cell death of T cells mediated by the TCR or glucocorticoids suggest that it may have a role in activation-induced cell death or differentiation of T cells.

Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: Analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of cyclic GMP, PDE4 activity predominated (∼80% total) but in the presence of low (10 μM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (∼80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity ( ic 50 ∼ 65 nM). PDE2 was dose-dependently activated ( ec 50 ∼ 1 μM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) ( ic 50 ∼ 4 μM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effects were seen in the absence of cyclic GMP with Hill coefficients of ∼0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (∼83%) in PDE-4 activity and in PDE2 activity (∼40%). Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

Isolation of newly expressed surface T cell antigen receptor complexes by serial precipitation with anti-TCR antibodies and immobilized streptavidin

Expression of the multisubunit T cell antigen receptor (TCR) complex is an enigmatic process requiring coordinated regulation of at least six different gene products (α, β, γ, δ, ε, and ζ ), the ordered pairing of partner chains within the endoplasmic reticulum (ER), and intracellular transport of complete, but not incomplete, TCR complexes to the cell surface. Movement of nascent TCR glycoproteins from the ER to the Golgi compartment is easily studied using various lectins and/or glycosidases specific for oligosaccharide modifications that occur within the Golgi system. In contrast, cell surface transport of TCR complexes is relatively difficult to assess, since this requires physical separation of intracellular complexes from surface TCR complexes. In the current report we describe a method for the isolation of newly transported surface TCR complexes which utilizes metabolic and surface labeling techniques in conjunction with serial precipitation methods. Specifically, we describe the use of anti-TCR antibodies and immobilized streptavidin to isolate nascent TCRα proteins localized on the plasma membrane. This technique is rapid, specific, and provides a novel approach for studying the intracellular transport of nascent immune receptor molecules to the cell surface.

Abnormal signal transduction through CD4 leads to altered tyrosine phosphorylation in T cells derived from MRL-lpr/lpr mice.

CD4+ T cells play a crucial role in the development of lupus in MRL-lpr/lpr mice: incomplete deletion/silencing of self-reactive CD4+ T cells leads to T cell activation, which causes both polyclonal B cell activation and T cell infiltration of multiple organs. Furthermore, anti-CD4 antibody therapy ameliorates disease and prolongs survival. Because CD4 is normally involved in both tolerance induction and T cell activation, we questioned whether signaling through CD4 was normal among T cells in this strain. For this purpose, signal transduction in CD4+ T cells derived from MRL-lpr/lpr and normal mice were compared, using an autoreactive CD4+ T cell clone and freshly isolated CD4+ T cells derived from mice of varying ages. Tyrosine phosphorylation was similar among MRL and normal CD4+ T cells after cross-linking with either anti-TCR antibody or anti-CD3 antibody, and following co-culture with Con A. In constrast, cross-linking of surface CD4 resulted in deficient tyrosine phosphorylation of cellular proteins in MRL T cells. By comparison, lck protein expression in MRL CD4+ T cells was found to be lower than normal. However, following stimulation with Con A, lck enzyme activity, as detected by autophosphorylation of lck, was comparable in MRL and normal T cells. The observed differences were present in the autoreactive T cell clone as well as in T cells isolated from both pre-diseased and diseased mice, and they could not be explained by variation in surface density of CD4. These results raise the possibility that abnormal signaling through CD4 may contribute to impaired tolerance and expansion of autoreactive T cells exhibited in MRL-lpr/lpr mice.

Cross-linking the TCR complex induces apoptosis in CD4+8+ thymocytes in the presence of cyclosporin A.

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD+8+ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.

Characterization of a Single-chain Antibody to the β-Chain of the T Cell Receptor

In this report the VH and VL genes of the anti-T cell receptor (TCR) antibody KJ16, which recognizes the TCR V beta 8.1 and V beta 8.2 regions in mice, were cloned and expressed as a single-chain antibody (scFv) in Escherichia coli. A 29-kDa protein was obtained after renaturation from inclusion bodies. The KJ16 scFv had a relative affinity for the native TCR that was slightly higher than KJ16 Fab fragments. The scFv and Fab fragments of the KJ16 antibody, together with monovalent forms of two other anti-TCR antibodies, were evaluated as antagonists of the T cell-mediated recognition of a peptide-class I complex or of a superantigen, Staphylococcus enterotoxin B (SEB) bound to a class II product. Each of the anti-TCR antibodies was efficient at inhibiting the recognition of the SEB-class II complex. In contrast, only the clonotypic antibody, which binds to epitopes on both V beta and V alpha regions, inhibited the recognition of peptide-class I complex. We conclude that the TCR binding site for the SEB-class II ligand encompasses a larger surface area than the TCR binding site for the peptide-class I ligand.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Multiphasic modulation of signal transduction into T lymphocytes by monoiodoacetic acid as a sulfhydryl reagent.

Actions of monoiodoacetic acid (MIA) as a sulfhydryl reagent on the different stages of the T cell receptor (TCR)-mediated signal transduction were examined. MIA (1 mM) prevented anti-TCR (CD3) monoclonal antibody (mAb)-induced energy-dependent receptor capping but at the same time promoted the anti-CD3 mAb/mitogen-induced tyrosine phosphorylation of the T cell activation-linked cellular proteins of 120, 80, 70, 56, and 40 kDa. Relatively low concentration (0.01 mM) of MIA further promoted anti-CD3 mAb-induced transcription of c-fos, production of IL-2, and cell surface expression of IL-2 receptors. The MIA-promoted TCR-mediated IL-2 production actually required signal transduction that could be inhibited by cyclosporin A, genistein, or H-7. In contrast, the same concentration of MIA as promoted the signal transduction for cell activation severely inhibited the anti-CD3 mAb-triggered signal delivery for cell proliferation, selectively at its early stage. We conclude from these results that MIA differentially affects various steps of signaling into T lymphocytes, suggesting that there exist multiple sites of MIA-sensitive or redox-linked control in the signal cascade.

Amelioration of acute GVHD disease and reestablishment of tolerance by short term treatment with anti TCR antibody

We investigated whether tolerance can be reestablished in mice with graft-versus-host disease (GVHD) by using a short-term, T-celldepleting treatment with an anti-TCR-αβ mAb. GVHD was induced in 950-rad—irradiated AKR mice (H-2k, Mls-1a) by injecting 5 × 106 T celldepleted bone marrow cells together with either 107 or 2 × 106 lymph node (LN) cells from BALB/c mice (H-2d, and Mls-1b). AKR mice that received 107 LN cells exhibited a severe form of acute GVHD, in which all mice died by day 60. In this severe form of GVHD, treatment with anti-TCR-αβ-mAb completely ameliorated the induction of GVHD when initiated on day 0 (a total of 800 μg/mouse administered on days 0, 5, and 10). When the same protocol was begun on day 10, it had no therapeutic effect. However, this delayed treatment with anti-TCR-αβ mAb was very effective in reversing a less severe form of GVHD that was induced by the injection of 2 × 105 donor LN cells. Recipient mice given prophylactic antiTCR-αβ treatment achieved host-specific tolerance in association with clonal deletion of host Mls-1a-reactive Vβ+T cells. In contrast, spleen cells from recipient mice that recovered from the mild form of GVHD as a result of the delayed anti-TCR-αβ treatment contained a considerable proportion of Vβ6+T cells, despite the healthy appearance of these mice. A MLR assay showed that the spleen cells from these mice responded well to Mls-1a Ag but not to H-2k Ag, in contrast with the apparent responses of spleen cells from untreated GVHD controls to both Ags. In addition, cells from the anti-TCR-αβ treated mice exhibited a specific reduction in cytotoxicity against AKR blasts. Collectively, these data indicated that a short-term treatment of mice having GVHD with an anti-TCR-αβ mAb, starting even after disease onset, can re-establish host-specific tolerance, at least to the host-histocompatibility Ag. (Copyright 1994. The Journal of Immunology.)

Limited T cell antigen receptor repertoire in tumor-infiltrating lymphocyte and inhibition of experimental lung metastasis of murine melanoma by anti-TCR antibody.

We analyzed the variability of T cell Ag receptor in tumor-infiltrating lymphocytes in primary and metastatic melanomas. Using a very sensitive inverse/double step PCR, we found the preferential V alpha usage of TCR in tumor-infiltrating lymphocytes in which some TCR sequences were homogenous. However, the profile of TCR V alpha expression was different in primary and metastatic melanomas. V alpha 8+, V alpha 2+, V alpha 4+, and V alpha 3+ TCR were the most dominant repertoires in primary melanoma, whereas V alpha 3+ and V alpha 4+ TCR dominated metastatic melanoma. Depletion of V alpha 3 T cells by the injection of tumor-bearing mice with anti-V alpha 3 Ab resulted in protection against experimental lung metastasis, indicating that V alpha 3+ regulatory T cells exist in the tumor site and help metastatic tumor growth.

Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77.

Engagement of T-cell receptors (TCRs) on immature thymocytes by self-antigen-major histocompatibility complexes causes the death of self-reactive thymocytes via apoptosis, a phenomenon that establishes T-cell tolerance. Similarly, treatment of thymocytes with anti-TCR antibodies leads to TCR-mediated apoptosis, which can also be induced in T-cell hybridomas. TCR-mediated apoptosis in immature thymocytes and T-cell hybridomas requires the expression of a new set of genes. In particular, it has recently been shown that the expression of Nur77, a transcription factor which is a member of the steroid/thyroid receptor superfamily, is required for TCR-mediated apoptosis in T-cell hybridomas and perhaps in thymocytes. Cyclosporin A (CsA), an immunosuppressive drug, has been shown to interfere with clonal deletion of self-reactive T cells in vivo, partly by blocking TCR-mediated apoptosis. We report here that CsA inhibits the TCR-mediated activation of Nur77 protein in T-cell hybridomas by blocking the DNA binding activity of Nur77 protein rather than its de novo synthesis. We also show that CsA mediates its negative effects on the Nur77 DNA binding activity through the N-terminal region of the protein. This complete inhibition of Nur77 protein DNA binding activity may explain how CsA interferes with TCR-mediated apoptosis.

Functional significance of CD23- on CD23-transfected Th2 clone

CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression has been shown to be transiently induced in vivo and in vitro on some CD4 + T cells [1–4] and in vivo on CD8 + T cells by IgE-secreting hybridoma tumors [5]. To investigate the functional role of CD23 on T cells, we inserted a CD23 construct into an expression vector driven by a CD2 promoter and transfected it into a murine Th2 clone D10.G4.1 (D10). We stimulated the transfected D10 cells (D10.3M.24) with anti-TCR antibody in the presence or absence of IgE, and measured IL-4, IL-5 and IL-6 production in the culture supernatants. Activation of D10.3M.24 cells by anti-TCR antibody induced greater levels of IL-4, IL-5 and IL-6 production, when the TCR and CD23 were co-crosslinked by TNP anti-TCR and IgE anti-TNP antibodies. IgG anti-TNP antibody did not enhance lymphokine production by D10.3M.24 cells. The enhanced lymphokine production by IgE was blocked by monoclonal anti-CD23 antibody. IgE anti-TNP antibody did not enhance lymphokine production by the wild-type D10 cells induced by TNP anti-TCR antibody. These studies show that when co-crosslinked with the TCR, CD23 can modulate the lymphokine production in activated Th2 cells. Since CD23 binds to IgE and also binds to CD21 [6], a complement receptor commonly expressed on B cells, T-cell CD23 could play an immunoregulatory role during cognate T-B cell interaction and during IgE antibody responses.

Autoregulation of Tcr V region epitopes in autoimmune disease.

Normal individuals possess low levels of autoantibodies specific for certain peptide defined regions of T-cell receptor (Tcr) variable regions, particularly CDR1 and Fr3. These regions are predicted to be exposed on the surface of the native molecule and, by analogy and comparison with immunoglobulins, correspond to public idiotype determinants. The anti-Tcr idiotype antibodies appear to be ubiquitous and we propose that they play a role in the regulation of T-cell function. To delineate the parameters of expression of these antibodies, we characterized anti-Tcr antibody activity in normal individuals, in those suffering from the autoimmune diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and in patients with non-autoimmune arthritis (osteoarthritis) as a disease control. There were significant increases in autoantibody levels in the autoimmune patients. There was also variation in isotype and the particular variable regions recognized. IgM autoantibodies directed against a few peptide defined determinants were elevated in RA, whereas SLE patient sera showed high levels of IgG binding to a broad spectrum of Tcr peptides.

Impaired CD28-mediated co-stimulation in anergic T cells.

We have investigated a CD28 co-stimulation in anergic T cells in staphylococcal enterotoxin B-inoculated mice by stimulating the cells with a plate-coated anti-TCR antibody in the presence or absence of an anti-CD28 antibody. CD28 co-stimulation increased the levels of IL-2 and IL-4 mRNAs in naive CD4+V beta 8+ T cells. However, it did not increase the levels of IL-4 mRNA at all and only partially increased those of IL-2 mRNA in anergic T cells. It was demonstrated that CD28 co-stimulation was impaired so that it no longer stabilized cytokine mRNAs in anergic cells. The levels of IL-4 mRNA in response to TCR stimulation were higher in anergic T cells than those in naive T cells in spite of the defective CD28 co-stimulation in the former cells. Anergy induction and generation of a Th2-type immune response in vivo are discussed.

CD4+ and CD8+ T cell expansions using selected TCR V and J gene segments at the onset of giant cell arteritis.

To investigate T cell receptor (TCR) V alpha/V beta (and in selected cases, J beta) usage in CD4+ and CD8+ peripheral blood lymphocytes of patients with giant cell arteritis (GCA), before and after treatment, as well as to analyze the HLA types of these patients. Flow cytometry, with 10 anti-TCR V-specific monoclonal antibodies (MAb), was used. To analyze J beta usage by cell populations expressing certain V beta, we used the polymerase chain reaction (PCR) technique, with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabeled J beta-specific probes. HLA typing was performed using the microlymphocytotoxicity technique. Seven of the 9 GCA patients had increased anti-TCR V MAb reactivities (interpreted as T cell expansions), which in many cases, correlated with clinical signs of disease. A strict preference for particular J beta segments was found in 3 of 3 expanded CD4+ T cell populations. T lymphocytes expressing specific antigen receptors are implicated in the pathogenesis of GCA.

Amelioration of acute graft-versus-host disease and re-establishment of tolerance by short-term treatment with an anti-TCR antibody.

We investigated whether tolerance can be re-established in mice with graft-vs-host disease (GVHD) by using a short-term, T cell-depleting treatment with an anti-TCR-alpha beta mAb. GVHD was induced in 950-rad-irradiated AKR mice (H-2k, Mls-1a) by injecting 5 x 10(6) T cell-depleted bone marrow cells together with either 10(7) or 2 x 10(6) lymph node (LN) cells from BALB/c mice (H-2d, and Mls-1b). AKR mice that received 10(7) LN cells exhibited a severe form of acute GVHD, in which all mice died by day 60. In this severe form of GVHD, treatment with anti-TCR-alpha beta mAb completely ameliorated the induction of GVHD when initiated on day 0 (a total of 800 micrograms/mouse administered on days 0,5, and 10). When the same protocol was begun on day 10, it had no therapeutic effect. However, this delayed treatment with anti-TCR-alpha beta mAb was very effective in reversing a less severe form of GVHD that was induced by the injection of 2 x 10(6) donor LN cells. Recipient mice given prophylactic anti-TCR-alpha beta treatment achieved host-specific tolerance in association with clonal deletion of host Mls-1a-reactive V beta 6+ T cells. In contrast, spleen cells from recipient mice that recovered from the mild form of GVHD as a result of the delayed anti-TCR-alpha beta treatment contained a considerable proportion of the V beta 6+ T cells, despite the healthy appearance of these mice. A MLR assay revealed that the spleen cells from these mice responded well to Mls-1a Ag but not to H-2k Ag, in contrast with the apparent responses of spleen cells from untreated GVHD controls to both Ags. In addition, cells from the anti-TCR-alpha beta-treated mice exhibited a specific reduction in cytotoxicity against AKR blasts. Collectively, these data indicate that a short-term treatment of mice having GVHD with an anti-TCR-alpha beta mAb, starting even after disease onset, can re-establish host-specific tolerance, at least to the host-histo-compatibility Ag.

Both high and low avidity antibodies to the T cell receptor can have agonist or antagonist activity

Anti-TCR antibodies can activate or block the activation of T cells. In the present experiments, we have shown that different monoclonal antibodies to the same TCR can have either agonist or antagonist activity, and we have examined the relationship between these functional effects and the avidity of the antibody for the TCR. We show here that it is not the avidity of an anti-TCR antibody that determines whether it acts as an agonist or an antagonist. Moreover, we show that monovalent Fab fragments of agonist antibodies produce detectable changes in T cell behavior. These data suggest that T cell activation may involve not just aggregation of the TCR but also some induced change in individual ligated receptors, and that agonists may produce this change while antagonists do not. We argue that similar effects may apply to peptide-MHC ligands as well.

Expression of Major Histocompatibility Complex Class II but Not of CD8 Molecules by Lectin-Stimulated Peripheral CD4 + T Cells in LEC Mutant Rats

LEC rats show a congenital maturational arrest from CD4 +8 + to CD4 +8 - but not to CD4 -8 + cells in the thymus. However, some CD4 + cells exist in peripheral lymph nodes. In normal F344 rats, a part of CD4 + T cells expressed CD8 molecules upon concanavaline A (Con A) stimulation. The percentage of CD4 +8 + cells and the level of CD8 expression in F344 rat CD4 + cells were enhanced by the glucocorticoid hormone dexamethasone. In contrast, although LEC rat CD4 + cells induced DNA synthesis normally upon Con A stimulation, expression of CD8 was significantly reduced. Furthermore, addition of dexamethasone did not restore the inhibition of CD8 expression. However, LEC rat CD4 + cells could induce CD8 expression, when stimulated by T cell receptor (TCR) cross-linking with anti-TCR monoclonal antibody, suggesting that the TCR-mediated signal is normally transduced in LEC rat CD4 + cells. On the other hand, LEC rat CD4 + cells showed normal expression of major histocompatibility complex (MHC) class II antigens, when stimulated by either Con A or TCR cross-linking, indicating that signals for the induction of MHC class II expression are normal in LEC rat CD4 + cells. Activation of peripheral CD4 + cells has been reported to induce simultaneous expression of CD8 and MHC class II molecules in rats. However, our results using LEC rat CD4 + cells suggest that Con A-induced signaling pathways for CD8 and MHC class II expression can be separable.

Fine antigen specificity of human gamma delta T cell lines (V gamma 9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium, Streptococcus sanguis.

We have established human gamma delta T cell lines specific for Streptococcus sanguis (S. sanguis) KTH-1 present in normal oral cavity flora. The CD4-CD8-CD3+V gamma 9+V delta 1-CD45RO+ CD25+ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen-presenting cells without apparent evidence of HLA restriction. The proliferative response of the gamma delta T cell lines was completely blocked by anti-TcR gamma delta monoclonal antibody (mAb) and anti-HLA class I mAb (W6/32), whereas anti-HLA classical class Ia mAb (B-H9; anti-HLA-A,B,C), anti-HLA class II mAb (anti-DR, anti-DQ, and anti-DP) and anti-CD4 mAb did not have any inhibitory effects. Surprisingly, the gamma delta T cell lines showed the proliferative response against the original bacterial Ag KTH-1 exclusively, and exhibited no cross-reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH-3. Moreover, cytokine production of the gamma delta T cell lines was similar to the Th1 pattern [interferon-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta]. They also produced interleukin-8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH-1 specific gamma delta T cell line and its subclones is rather limited. It is suggested that gamma delta T cells with canonical TcR could preferentially respond to KTH-1 Ag. Thus, in addition to a broad or cross-reactivity of gamma delta T cells against phylogenetically conserved stress/heat-shock protein, which is well characterized by others, some peripheral blood gamma delta T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.

The degree of CD8 dependence of cytolytic T cell precursors is determined by the nature of the T cell receptor (TCR) and influences negative selection in TCR‐transgenic mice

Although much has been learned about CD8 structure-function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non-dependence to CD8+ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)-transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen-specific cytolysis by CD8+ T cells from two TCR-Tg mice expressing respectively the TCR (Tg) from a "CD8-dependent" and from a "CD8-independent" CTL clone, which were both reactive against the H-2Kb alloantigen and originated from H-2k mice. The results indicate that the property of the Tg+CD8+ cells from H-2k TCR-Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg+CD4+ cells could also differentiate into H-2Kb-specific CTL when originating from the "CD8-independent", but not from the "CD8-dependent" Tg-TCR. The influence of the property of "CD8 dependence" on negative selection occurring in TCR-Tg H-2k/b mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the "CD8-independent" TCR-Tg mice; (ii) in the periphery, Tg+(hi) cells with low to negative CD8 expression were present for the "CD8-dependent" Tg-TCR, whereas only Tg+CD4-CD8- cells with low surface Tg-TCR and CD3 expression were found for the "CD8-independent" Tg-TCR, indicating that Tg+CD4-CD8- cells are susceptible to tolerance induction involving TCR/CD3 surface down-modulation. Furthermore, different in vitro conditions led to H-2Kb-induced stimulation of Tg+CD4-CD8- cells to differentiate into CTL detected in an anti-TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin-2 of H-2k/b Tg+CD4-CD8- cells was sufficient to induced CTL activity in the "CD8-independent" model, whereas stimulation with cells which overexpressed H-2Kb was required in addition to interleukin-2 to induce CTL differentiation in the "CD8-dependent" model. These data suggest that peripheral Tg+CD4-CD8- cells present in a situation of in vivo tolerance to H-2Kb can still be triggered by H-2Kb with a sensitivity correlated with the degree of CD8 dependence.