Characterization and expression of the human alpha beta T cell receptor by using a framework monoclonal antibody

A monoclonal antibody (mAb) with framework reactivity against the T cell receptor (TCR) alpha beta complex is characterized. The mAb, beta Framework 1 (beta F1) is capable of immunoprecipitating the TCR alpha beta complex from 125I-labeled human T cell tumors, immunocompetent T cell clones, and peripheral blood lymphocytes (PBL). beta F1 recognizes the separated TCR beta subunit in Western blotting. Because it does not bind to the surface of viable T cells but does react with the plasma membrane form of the TCR after treatment with membrane solubilizing agents, the beta F1 mAb reacts with a "hidden" determinant on the TCR beta subunit. After solubilization with 70% ethanol, the TCR alpha beta complex is shown to exist on greater than 92% of T3+ human PBL, whereas 2 to 8% of T3+ PBL do not react with the mAb. The beta F1 mAb demonstrates the existence of differently glycosylated surface 125I-labeled TCR alpha-chains (alpha, alpha', alpha") in association with a common TCR beta-chain on the HPB-MLT T cell leukemia. Reactivity of the beta F1 mAb on thymus tissue sections is similar to that of anti-Leu-4 (anti-T3). The beta F1 mAb should prove useful as a research tool for both the immunochemical characterization and isolation of virtually any alpha beta T cell receptor, whether from individual T cell clones or polyclonal populations of T lymphocytes. Recognition of T cell receptors in histologic tissue sections suggests that the beta F1 mAb may be useful in the clinical diagnosis of T cell lineage neoplasms. In failing to recognize all T3+ lymphocytes, it allows the identification of novel populations of T3+ lymphocytes that may express non-alpha, non-beta T cell receptors.     

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

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

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.     

A combined immunodeficiency with oligoclonal CD8+, V beta 3-expressing, cytotoxic T lymphocytes in the peripheral blood.

The diagnosis severe combined immunodeficiency was made in a male infant at the age of 18 wk. Known causes of severe combined immunodeficiency were excluded. The activity of total 5'-nucleotidase (E.C. 3.1.3.5) in the PBMC was found to be strongly decreased. Analysis of the peripheral blood revealed a lymphocytosis, mainly of CD8+ T cells. These lymphocytes expressed high levels of CD29, CD38, CD45RA, and MHC class II molecules but no CD25, CD26, CD27, or CD28 Ag. The cells proliferated poorly to all T cell stimulants tested and no helper activity for IgM secretion could be induced. In contrast to the poor proliferative responses, high levels of TCR-induced cytolytic activity, without lymphokine-activated killer-cell outgrowth, were induced by CD3 mAb. Analysis of TCR-beta gene rearrangements indicated that two clonal populations constituted the majority of the E-rosette+ peripheral blood fraction. Moreover, the vast majority of the CD8+ cells were found to react with a mAb to V beta 3. Polymerase chain reaction on cDNA from peripheral blood cells with primers that amplify TCR V beta elements showed, in agreement with the fluorescence data, an overrepresentation of V beta 3 but absence of usage of approximately 50% of the other V beta elements. Thus, in a severe combined immunodeficiency patient, CD8+ T cells with limited T cell receptor usage and restricted effector functions were found. The observed alterations in the 5'-nucleotidase levels may be secondary to the outgrowth of this population.     

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

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

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Analysis of a monoclonal rat antibody directed to the alpha-chain variable region (V alpha 3) of the mouse T cell antigen receptor

Three rat mAb, RR3-15, RR3-16, and RR3-18, were established by fusing spleen cells from a rat immunized with the male Ag-specific cytolytic T cell clone, OH6, to mouse myeloma cells. The mAb was identified by their capacity to focus the cytolytic activity of the OH6 CTL clone on nonspecific target cells via FcR-FcR interaction. That all three mAb recognized the OH6 TCR was confirmed by immunoprecipitation studies in which each antibody precipitated a 90 kDa disulfide-linked heterodimer characteristic of the TCR. Surface immunofluorescence staining of a panel of T cell lines and splenic T cell populations showed that RR3-16 reacted not only to the OH6 T cell clone but also to a minor fraction of normal T cells. This reactivity was found to be due to the expression of a gene in the V alpha 3 family. However, RR3-16 did not react with all T cell lines and clones known to express genes from the V alpha 3 family. cDNA sequences of three independent RR3-16+ T cell hybridomas analyzed by polymerase chain reaction were identical to the previously published V alpha 3 sequence of the CTL clone C9. Thus, the mAb RR3-16 is specific for a single member of the TCR V alpha 3 gene family. Analysis of the expression of RR3-16+ TCR in CD4+ and CD8+ subsets of peripheral T cells demonstrated preferential expression on CD8+ T cells, suggesting regulated expression of this particular TCR V alpha gene.     

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

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

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

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

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

Requirement for CD4+ T cells in V beta 4+CD8+ T cell activation associated with latent murine gammaherpesvirus infection.

A CD8+ T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8+ T cells of mice expressing different MHC haplotypes express V beta 4+ TCR. Identification of the ligand driving the V beta 4+CD8+ T cell activation remains elusive, but there is a general correlation between V beta 4+CD8+ T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4+ T cells in the V beta 4+CD8+ T cell expansion has been addressed. The results show that CD4+ T cells are essential for expansion of the V beta 4+CD8+ subset, but not other V beta subsets, in the peripheral blood. CD4+ T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-gamma. Assessment of V beta 4+CD8+ T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4+ T cells control levels of the stimulatory ligand that drives the V beta 4+CD8+ T cells. As V beta 4+CD8+ T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4+ T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4+ T cell-mediated help for V beta 4+CD8+ T cells.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.     

Analysis of expression of CD2, CD3, and T cell antigen receptor molecules during early human fetal thymic development

To define early stages of T cell maturation during human fetal thymic development, we have used mAb reactive with CD2, CD3, and TCR molecules in indirect immunofluorescence assays on a series of early human fetal thymic specimens. Using a technique of quantitating the relative proportions of fluorescent-positive cells present in tissue sections, we found at 8.5 wk of gestational age after arrival of CD7+ T cell precursors into the thymic rudiment, 60% of thymic CD7+ cells were CD2+, 4% were CD3+ and none was TCR-delta+ or TCR beta+. Moreover, cells reactive with anti-CD2 antibodies against T11(2) and T11(3) epitopes of CD2 as well as thymic stromal cells expressing the CD2 ligand, lymphocyte function associated Ag-3, were also present at 8.5 wk. From 9.5 wk to birth TCR beta+ cells increased to include greater than 90% of all CD7+ cells while TCR-delta+ cells fell from a peak of 11% of CD7+ cells at 9.5 wk to 1% of CD7+ cells at birth. These data suggest that epitopes of CD2 molecules are expressed early on during fetal thymic development. Moreover, these data suggest that CD7+, CD2+, cytoplasmic CD3+ T cell precursors in man give rise to both TCR-delta+ T cells as well as to T cells expressing TCR-alpha beta.     

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

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

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex.

Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.