CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1→S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

T-Cell Response to Anti-CD2 Monoclonal Antibodies in Down''s Syndrome

Peripheral blood mononuclear cells from 10 subjects with cytogenetically documented Down's syndrome (DS) and from 10 age- and sex-matched healthy controls were assayed for their ability to proliferate in response to phytohaemagglutinin, anti-CD3 (OKT3), or anti-CD2 (T11(2) plus T11(3] monoclonal antibodies. Interleukin 2 (IL-2) receptor expression and IL-2 production in mitogen-pulsed lymphocyte cultures was also investigated in parallel. DS cells responded poorly to all the blastogenic stimuli used in this study. Under certain experimental conditions (anti-CD3 or anti-CD2 antibody stimulation), the patients' lymphocytes expressed low levels of IL-2 surface receptors and failed to produce normal amounts of this lymphokine. Studies are currently in progress in our laboratories to determine whether these defects are due to an impairment of the early signalling events surrounding the complexing of CD3, CD2, or lectin receptors to their respective ligands.     

Mouse B Cell Activation is Inhibited by CD44 Cross-Linking

Lymphocyte activation and trafficking are indispensable to the immune system. CD44 is an adhesion molecule with known importance in T cell activation, lymphocyte trafficking, and tumor metastasis. Although CD44 has been shown to participate in the activation, rolling and adhesion, and homing of T cells, the role of CD44 on B cells is relatively unknown. The effects of CD44 cross-linking on murine B cell activation via CD40L was explored using the anti-CD44 mAbs RK3G9 and IM7. When immobilized on a plate, both RK3G9 and IM7 were found to strongly inhibit B cell proliferation and Ig production, especially at lower cell input concentrations. IgE inhibition was especially prominent. In contrast, soluble RK3G9 added to the B cell cultures had no effect. The inhibitory effect of anti-CD44 on B cell activation was not influenced by the addition of the anti-FcγRII, indicating that Fc cross-linking did not play a role in this inhibition. As Ig production requires several days for both B cell proliferation and differentiation to occur, the effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. Finally, B cells were activated by either LPS or anti-IgM F(ab′)₂. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. These results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.     

抗CD44抗体治疗急性髓系白血病的研究进展

CD44分子是一种广泛分布的细胞表面跨膜糖蛋白,在许多方面发挥着重要的生理和病理作用,其中之一就是CD44分子参与血细胞的生成,在血细胞分化成熟的过程中发挥着关键性作用。CD44与一些特异的抗体结合后能够逆转急性髓系白血病幼稚细胞的分化阻滞,从而逆转其恶性细胞的表型,最终使肿瘤细胞的抗原性、功能和细胞生物学特征与正常分化的骨髓系细胞相一致。     

Anti-CD44 Monoclonal Antibody Inhibits Heart Transplant Rejection Mediated by Alloantigen-primed CD4+ Memory T Cells in Nude Mice

Donor-reactive CD4⁺memory T cells threaten the survival of transplanted organs. In this study, we used anti-CD44 monoclonal antibody (mAb) to inhibit adoptively transferred B6-reactive CD4⁺memory Tcells (BALB/c origin) and to induce tolerance of B6 hearts in nude mice. The median survival time (MST) of the grafts was 6 days in the isotype group, and more than 100 days in the group treated with 8 doses of anti-CD44 at four-day intervals. Histological analysis revealed that the mean rejection level was Grade 3 in the isotype group, and Grade 0 or 1 in the multi-dose anti-CD44 treatment group. Compared with the isotype group, the multiply treated anti-CD44 group had significantly decreased IL-2 and IFN-γ expressions, while IL-10 and TGF-β were increased in the serum and the graft. Foxp3 in the graft was also increased. These data demonstrate that alloreactive CD4⁺ memory T cells mediate the destruction of allografts, and the adhesion molecule CD44 plays an important role in this course. Anti-CD44 mAb may promote the reduction of CD4⁺memory T cells and the production of regulatory T cells (Tregs). Furthermore, Tregs are maintained at a certain level while suppressing cellular immunity and inducing the grafts long-term survival in transplant recipients.     

Selection of Phage-displayed Peptides Recognised by Monoclonal Antibodies Directed against the Lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76–12G12 mAb, which is specific for LPS of all strains of Brucella, either A-or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66–4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66–2C8 and A15–6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

CD3-Induced T-Cell Proliferation and Interleukin-2 Secretion is Modulated by the CD45 Antigen

In the present study we have investigated the effect of CD45, CD45RA and CD45RO monoclonal antibodies (MoAbs) on the CD3 receptor-mediated proliferation of human T lymphocytes. It is shown that CD3-induced proliferation of purified resting T cells and quiescent T lymphoblasts (QTL) is promoted via all of the investigated CD45-associated epitopes. It is also shown that the CD45 molecules are required to be cross-linked for costimulation. The MoAbs enhance the interleukin-2 (IL-2) production of CD3-stimulated QTL. The elevation of the IL-2 production correlates with the increase in CD3-induced cell proliferation suggesting that the CD45-driven regulation of T lymphocyte activation is linked to the IL-2 pathway.     

T-Cell Activation. III. Attempts to Activate MHC Class I-Negative and Class I-Transfected EL4 T-Lymphoma Cells by Immobilized Anti-CD3 Antibody

The aim of this study was to examine whether the unresponsiveness of MHC class I-negative subclones of the EL4 thymoma to CD3 cross-linking can be restored by transfection of class I genes into the H-2-negative cells. Cell activation experiments with selected MHC class I-negative subclones and H-2b- and H-2Ld-positive transfectants showed that these cells are equally capable of secreting interleukin 2 (IL-2) after exposure to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and ionomycin. In contrast, only the parental H-2-positive EL4 cells are capable of responding to treatment with immobilized anti-CD3 antibody with IL-2 secretion and IL-2 receptor expression. Measurements of intracellular free Ca2+ (Ca2+i) following anti-CD3 antibody-induced cross-linking of parental EL4 cells and H-2-negative and H-2b gene-transfected subclones showed that the parental cells and two of the class I transfectants, one H-2-positive and one H-2-negative, responded with a slow rise in Ca2+i, whereas one H-2-positive transfected cell clone was completely refractory to CD3 cross-linking. Modulation experiments using parental EL4 cells, H-2-negative subclones and H-2-positive transfectants demonstrated that the CD3 and class I molecules of these different cells are modulated to the same extent after exposure to specific antibodies. The present findings thus indicate that the unresponsiveness of H-2-negative EL4 subclone cells to CD3 cross-linking is not functionally associated with a lack of class I surface expression.     

The Induction of T Cell-Mediated Cytolysis by Monoclonal Antibodies Against the T Cell Receptor/CD3 Complex

Antigen-induced T cell death is an important regulatory mechanism in the peripheral immune system. Evidence suggests that this process depends on T cell growth-inducing lymphokines such as IL-2 and occurs in proportion to the degree of T cell receptor occupancy. Strong T cell receptor stimulation leads to the synthesis of death molecules such as Fas ligand and tumor necrosis factor that cause T cell suicide. We propose that T cell death under these circumstances is the culmination of a feedback control mechanism termed propriocidal regulation or autocrine feedback death that regulates the expansion of specific T cell clones under conditions of high lympho-kine and antigen load. In a quasi-stochastic system such as the antigen receptor repertoire, feedback information may be essential for the appropriate regulation of peripheral immune responses. Our understanding of this feedback mechanism affords a means to manipulate antigen-specific T cell death in vivo. The application of this approach to the therapy of T cell-medicated immunological diseases is discussed.     

B-Cell Activation In Vitro by Helper T Cells Specific to a Protein Region that is Recognized Both by T Cells and by Antibodies

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.     

B-Cell Activation In Vitro by Helper T Cells Specific to a Protein Region that is Recognized Both by T Cells and by Antibodies

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.     

Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but not Ehrlichia chaffeensis, Ehrlichia sennetsu, Ehrlichia canis, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.     

The Stimulation of T Cells with Anti-CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B-Cell Responses but does not Enhance the Activation of Antigen-Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.     

Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by the Cryptococcus neoformans Capsule

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, a life-threatening infection of particular importance in patients with deficiencies in cellular immunity, most notably patients with the AIDS. The yeast is surrounded by a polysaccharide capsule that is composed primarily of glucuronoxylomannan (GXM), which has a linear (1→3)-α-d-mannopyranan backbone bearing β-d-xylopyranosyl, β-d-glucopyranosyluronic acid, and O-acetyl substituents (3, 9, 54). The cryptococcal capsule occurs as four major serotypes (A, B, C, and D) and is an essential virulence factor for the yeast.One of the most striking features of the cryptococcal capsule is its ability to activate the alternative complement pathway. Incubation of encapsulated cryptococci in normal human serum (NHS) leads to the deposition of 10⁷ to 10⁸ C3 fragments on the yeast (28, 56). The C3 is deposited at the surface and throughout the capsule (30). Available evidence indicates that the amount of anti-GXM antibodies found in NHS is not sufficient to initiate the classical pathway (24); consequently, activation and binding of C3 to the cryptococcal capsule are mediated entirely by the alternative complement pathway (29, 30, 55). One of the hallmark features of alternative pathway deposition of C3 onto encapsulated cryptococci is a delay of 5 to 8 min before readily detectable amounts of C3 are found on yeast cells incubated in NHS (29, 55). Once past the initial lag, C3 fragments rapidly accumulate on the yeast cells as incubation proceeds for an additional 10 min.Recently, there has been interest in antibody-mediated resistance to cryptococcosis. Monoclonal antibodies (MAbs) have been proposed for treatment of cryptococcosis (7), and immunization with GXM-protein conjugates has been suggested for prevention of cryptococcosis (6, 12, 13). However, it is becoming increasingly clear that anti-GXM MAbs may have distinct specificities and biological activities. Anti-GXM MAbs which differ in (i) reactivities with GXM of the four major serotypes (2), (ii) apparent binding sites in the cryptococcal capsule (32, 37), and (iii) abilities to provide protection in a murine model of cryptococcosis (32, 37) have been described. Some differences in biological activity are related to differences in the epitope specificities of the various MAbs (32, 37).One means by which antibodies could enhance resistance to cryptococcosis is through accelerated deposition of opsonic C3 fragments via the action of the classical pathway. Such an acceleration would reduce or eliminate the 5- to 8-min lag that occurs during alternative pathway-mediated deposition of C3 fragments. The objectives of our study were to evaluate the effects of anti-GXM MAbs on the kinetics and sites for deposition of C3 fragments into the cryptococcal capsule. We examined several well-characterized antibodies that differed in the epitope specificity of the MAbs. The results showed that MAbs with different isotypes and epitope specificities had distinctly different effects on activation and binding of C3 via the classical and alternative pathways; many antibodies markedly suppressed C3 binding, some antibodies accelerated C3 binding, and other antibodies had little or no effect.     

CD3-mediated T cell activation is inhibited by anti-CD44 monoclonal antibodies directed to the hyaluronan-binding region.

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1-->S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

Production of Mouse Monoclonal Antibodies Directed Against the Oolemma of Human and Hamster Oocytes by Intra-splenic Injection of Oocytes

PROBLEM: To develop an additional approach for the study of oolemmal surface moieties involved in gamete interactions, we decided to obtain monoclonal antibodies by intrasplenic injection of human and hamster oocytes in Balb/c mice. METHOD: Two Balb/c males were injected three times intrasplenically at 15-day intervals with approximately 40 zona-free hamster and 3–5 zona-free human oocytes. After the third injection, spleen cells were fused and hybridomas developed. We used a novel screening system based upon the use of sections of frozen human and hamster eggs, tested by means of indirect immunofluorescence. The antibodies that we produced were evaluated for their ability to interfere with the zona-free hamster eggs penetration by human spermatozoa. The B2B5 antibody was also developed as ascitic fluid and further characterized. RESULTS: Seven antibodies reactive with hamster oocytes were produced. Six of them also reacted with human oolemmas. The binding was confined to the oolemma, and no staining of the zona nor the cytoplasm was present. One of these antibodies reduced the penetration of zona-free hamster eggs by human spermatozoa. This antibody, B2B5, an IgM kappa, was confirmed to interact with the oolemma by means of indirect immunofluorescence of fresh eggs and Covasphere binding. B2B5 did not react with other human or hamster tissues except capacitated human spermatozoa. The reactivity with the oolemma of hamster oocytes was not lost after egg penetration by human sperm. CONCLUSIONS: Intrasplenic immunization using zona-free human and hamster oocytes allows the production of anti-oolemma antibodies. A system of screening based upon the use of sections of frozen eggs also allows an easy and quick scoring of many supernatants. B2B5 monoclonal anti-oolemma antibody deserves further studies in that is able to interfere with fertilization and its antigen appears to be confined to the gametes surface.     

Failure to Synthesize the Human T-Cell CD3-ζ Chain and Its Consequence for the T-Cell Receptor-CD3 Complex Expression

The T-cell antigen receptor is composed of two variable chains (alpha and beta, termed TcR) which confer ligand specificity, and four constant chains (gamma, delta, epsilon, and zeta, collectively termed CD3) whose functions are not fully understood. To explore the role of the individual CD3 components, the human T-cell tumour line Jurkat was chemically mutagenized followed by negative selection with F101.01 (a monoclonal antibody against the TcR-CD3 complex), and cloning. Growing clones were analysed for TcR-CD3 expression by immunofluorescence. One clone, J79, was found to express greatly diminished levels of TcR-CD3. This clone produced all the TcR-CD3 components except the CD3-zeta, as demonstrated by metabolic labelling and immunoprecipitation followed by one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These data indicate that the CD3-zeta determines the normal intracellular fate of the TcR-CD3 complex, and that the CD3-zeta is necessary for the intracellular transport and expression at the cell surface of the TcR-CD3 complex.