Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.     

The role of Ca2+ and Mg2+ in the cytotoxic T lymphocyte reaction and in the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-serine esterase by human T cell clones

Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon Ag-specific activation. BLT-serine esterase secretion could also be induced by the Fc gamma+ target cell Daudi in the presence of mAb specific for the TCR/CD3 complex, CD2, or the T cell activation Ag Tp 103. In addition, anti-CD3 and a mitogenic combination of anti-CD2 mAb, induced secretion of BLT-serine esterase in the absence of target cells, whereas anti-Tp 103 failed to do so. The secreted BLT-serine esterase activity induced by the various ligands was inhibited by the serine esterase inhibitors PMSF and m-ABA, but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone. Significant BLT-serine esterase activity was induced by target cells or soluble anti-CD3 in the absence of extracellular Ca2+ ions, provided that extracellular Mg2+ ions were present. The cytotoxic activities by the human CTL clones were completely blocked under these conditions. All ligands that induced BLT-serine esterase secretion in the absence of extracellular Ca2+, induced a transient rise of intracellular Ca2+. Soluble anti-CD3 mAb did not induce a transient rise in intracellular Ca2+ or secretion of BLT serine esterase in CTL preincubated for 2 h with 5 mM EGTA. These findings indicate that mobilization of intracellular Ca2+ in human CTL clones is required for induction of secretion of BLT-serine esterase.     

A novel pathway of human T cell activation via a 103 kD T cell activation antigen

A novel triggering signal for human proliferating and cytotoxic T lymphocytes defined by a 103 kD T cell-specific activation antigen (Tp103) is described. Tp103 is expressed on all proliferating normal T cells but is not present, or present only in low amounts, on resting peripheral blood T lymphocytes. Cross-linking of T cell and Fc receptor-positive accessory or target cells by an antibody against Tp103 leads to activation of the T cell. The proliferative response is due to an autocrine IL 2-dependent mechanism and can be inhibited by antibodies against the IL 2 receptor or by Cyclosporin A. Resting Tp103-positive T cells also respond to anti-Tp103. Although Tp103 is not linked to the antigen receptor/T3 complex, triggering via Tp103 can be inhibited by modulation of the T3 molecule. Thus, Tp103 defines a new antigen-independent pathway of T cell activation that can be regulated via other T cell surface structures.     

CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4(+) T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8(-) murine lymphoma, 58 alpha(-)/beta(-). Immunofluorescent staining of TCR-transduced cells with human TCR V beta subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2(+) melanoma lines compared with T2 cells alone or HLA-A2(-) melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.     

T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP

TAP is a phosphatidylinositol-anchored Ly-6-encoded, glycoprotein that is involved in the activation of murine T-inducer cells. Anti-TAP antibodies can stimulate T cells directly and also synergize with Ag stimulation. To determine the relationship between TAP and TCR-mediated activation, we have derived TCR/CD3 loss variants of functional T hybrid clones. T-T hybrid variants that have lost TCR expression are not responsive to anti-TAP stimulation. Isolation and analysis of revertant clones, obtained from one of the TCR/CD3-negative mutant cell lines, demonstrate a concordant re-expression of the TCR/CD3 complex and responsiveness to anti-TAP stimulation. A similar loss of responsiveness to TAP stimulation is observed after antibody-induced modulation of the TCR/CD3 complex from the surface of T cell hybrids. In contrast, both the TCR/CD3 loss variants and TCR/CD3-modulated T-T hybrids remain fully responsive to stimulation with a calcium ionophore and phorbol esters. This structure-function correlation has been observed repeatedly in independent isolations of variants, and with TCR/CD3 modulated cells, from two different T-T hybridomas. Given the apparent functional interrelationship between TAP and the TCR/CD3 complex, we have also analyzed if these molecules were significantly associated on the T cell surface. Antibody-induced modulation of the TCR/CD3 complex does not affect the cell surface expression of TAP molecules. Moreover, the expression of these two sets of proteins is also independent as evidenced by the selective loss of either set of these proteins in the TCR/CD3 expression mutants.     

A regulatory role for the CD4 and CD8 molecules in T cell activation

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.     

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.     

Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.     

Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

Sarcoidosis is not associated with a generalized defect in T cell suppressor function

In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

Transient loss of MHC class I tetramer binding after CD8+ T cell activation reflects altered T cell effector function

Engagement of the Ag receptor on naive CD8+ T cells by specific peptide-MHC complex triggers their activation/expansion/differentiation into effector CTL. The frequency of Ag-specific CD8+ T cells can normally be determined by the binding of specific peptide-MHC tetramer complexes to TCR. In this study we demonstrate that, shortly after Ag activation, CD8+ T cells transiently lose the capacity to efficiently bind peptide-MHC tetramer complexes. This transient loss of tetramer binding, which occurs in response to naturally processed viral peptide during infection in vitro and in vivo, is associated with reduced signaling through the TCR and altered/diminished effector activity. This change in tetramer binding/effector response is likewise associated with a change in cell surface TCR organization. These and related results suggest that early during CD8+ T cell activation, there is a temporary alteration in both cell surface Ag receptor display and functional activity that is associated with a transient loss of cognate tetramer binding.     

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.     

Mechanisms of immune memory. T cell activation and CD3 phosphorylation correlates with Ta1 (CDw26) expression

The Ta1 (CDw26) Ag distinguishes a subset of circulating T lymphocytes that is the major population proliferating to recall Ag challenge. Unlike receptors for growth factors such as IL-2 and transferrin, the Ta1 Ag is present on T cell lines and clones irrespective of cell cycle. The appearance of Ta1 on T cells that respond to recall Ag allowed us to investigate activation requirements that may be associated with T cell immune memory. Ta1+ peripheral blood T cells were induced to proliferate by mAb recognizing either the invariant chains of the TCR, or by pairs of mitogenic antibodies directed to the CD2 molecule. In contrast, Ta1- cells were not stimulated by these antibodies. In addition, Ta1-cells did not proliferate maximally after addition of the phorbol ester PMA in combination with the calcium ionophore Ionomycin, suggesting that the intracellular targets of these agents may not be fully active. Anti-CD3-induced elevation of intracellular calcium levels was equivalent in the two subpopulations, suggesting that calcium mobilization mechanisms were intact. In contrast, PMA-induced phosphorylation of TCR CD3 chains was significantly greater in Ta1+ cells as compared to Ta1- T cells. Taken together, our results indicate that Ta1 expression, which is associated with T cell activation and memory, may be causally related to TCR and CD2-mediated activation mechanisms. The PMA inducible TCR phosphorylation in Ta1+ memory cells associated with their increased ability to proliferate after CD3/TCR or CD2 stimulation suggests that intracellular phosphorylation events may be causally associated with T cell immune memory.     

CD3- T cells with cis- or trans-acting mutations affecting expression of T cell receptor beta-chain mRNA.

Mutants of an untransformed T cell clone that no longer respond to TCR/CD3 stimulation have been derived using a selection procedure based on the loss of functional response to Ag. This functional selection gives rise to clones of several different phenotypes. We have previously described mutants with a TCR/CD3+ cell surface phenotype whose TCR are uncoupled from cellular responses. We describe six additional mutants that do not express TCR/CD3 at the cell surface. One of the CD3- clones contains a deletion in the successfully rearranged TCR-alpha gene, whereas another carries a deletion in the successfully rearranged TCR-beta gene. TCR/CD3 expression in these deletion mutants can be restored by transfection of TCR-alpha or TCR-beta DNA. Four other clones do not express TCR-beta mRNA, yet contain no obvious deletions or rearrangements in the TCR-beta genes. One of these clones does not transcribe TCR-beta chain mRNA. The mutation in this clone does not reside in the TCR-beta gene itself, but may instead reside in a trans-acting regulatory element affecting TCR-beta gene expression, because the TCR-beta mRNA-phenotype is complemented by fusion with a TCR-alpha-beta- cell line. TCR-beta chain regulatory mutants will be valuable in contributing to our understanding of how TCR expression is regulated.     

Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells

The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.