Frequent coexpression of cytolytic activity and lymphokine production among human T lymphocytes. Production of B cell growth factor and interleukin 2 by T8+ and T4+ cytolytic clones

These studies were designed to determine the degree of overlapping between cytolytic function and lymphokine production among peripheral blood human T lymphocytes. T8⁺ and T4⁺ cells were obtained by sorting purified T cells using the fluorescence-activated cell sorter (FACS) and cloned at 0.25 cells/well in a microculture system that allows clonal growth of every T cell. 49/49 T8⁺ clones but only 5/102 T4⁺ clones displayed cytolytic activity, as assessed by a lectin-dependent assay. Cytolytic clones were further analyzed for their ability to release interleukin 2 (IL 2) and B cell growth factor(s) (BCGF) upon 24 h stimulation with phytohemagglutinin. Fifteen percent and 18% of T8⁺ cytolytic clones were producing IL2 and BCGF, respectively; in addition 3 out of 5 T4⁺ cytolytic clones released both lymphokines. Although the majority of cytolytic clones producing IL2 were also producing BCGF, 5 clones released either BCGF or IL2. The present studies indicate that the coexpression of cytolytic and helper/inducer functions is relatively common among peripheral blood T lymphocytes, irrespective of their T8⁺ or T4⁺ phenotype.     

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction

Fas was recently demonstrated to be the major target molecule engaged by CD4⁺ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4⁺ or CD8⁺ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4⁺ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4⁺ CTL and a CD8⁺ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4⁺ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.     

T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function

A new anti-p58 monoclonal antibody (mAb), termed CH-L, has been used to characterize a minor subset of T lymphocytes co-expressing p58 and CD3 molecules. In two-color immunofluorescence analysis, CH-L⁺CD3⁺ cells represented 0.5 to 6% of the peripheral blood lymphocytes (in 20 healthy donors). Clonal analysis showed that most CD3⁺CH-L⁺ T cell clones expressed the CD8⁺4^? T cell receptor (TcR) α/β⁺ phenotype, while only a few were CD8^?4⁺ TcR α/β⁺, CD8^?4^? TcR α/β⁺ or CD8^?4^? TcR γ/δ⁺. Western blot analysis indicated that the CH-L mAb identifies the same 56-58-kDa diffuse band in both T and natural killer cell (NK) clones. A minority of T cell clones also expressed other NK-related markers such as CD16, CD56 and CD94 and two clones also reacted with the anti-p58 mAb EB6. Interestingly, most clones displayed cytolytic activity in an anti-CD3 mAb-triggered redirected killing assay against the Fcδ receptor⁺ P815 target cells and NK-like activity against K562 and Raji cells. In contrast, the IGROV-1 ovarian carcinoma cell line was resistant to cytolysis by all of these clones. Since p58 molecules have previously been shown to exert regulatory functions on NK-mediated lysis, we investigated whether anti-p58 mAb could also influence cytotoxicity mediated by CD3⁺p58⁺ T lymphocytes. Lysis of P815 target cells, triggered by anti-CD3 mAb, could be inhibited by anti-p58 mAb in 8 out of 12 cytolytic clones tested, while 4 clones were not inhibited. In addition, anti-p58 mAb enhanced the cytolytic activity of 3 clones against IGROV-1 and of 4 other clones against Raji target cells. Taken together, these data indicate that p58⁺ T cells express heterogeneous phenotypes and different forms of TcR and, in most instances, display cytolytic functions. Perhaps more importantly, the p58 molecule appears to modulate the cytolytic activity triggered via the CD3/TcR complex.     

Ultrastructural characteristics of human T cell clones with various cytolytic activities

Fifteen T cell clones with different (specific, antibody-dependent or natural killer-like) cytolytic activity were derived from mixed lymphocyte culture activated T cells and analyzed for their morphological characteristics and, in some instances, for their surface markers. All of the five cytolytic clones analyzed by electron microscopy possessed numerous electron-dense granules and in some instances multivesicular bodies, with or without an electron-dense matrix, that are the putative precursors of the granules. In addition, light microscopy examination of semithin sections of other ten cytolytic clones showed that the large majority of the cells in each clone had numerous toluidine blue-stained cytoplasmic granules. It is of note that nine clones without detectable cytolytic activity analyzed by electron microscopy did not possess granules and presented the features of large agranular blasts. Ten cytolytic clones were analyzed for different surface markers including rosette formation with sheep erythrocytes (E rosettes), receptors for the Fc portion of IgG or IgM (FcγR and FcμR) and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8 or OKT4. All the clones were Erosette⁺, Ia⁺ and 4F2⁺. Expression of FcγR was restricted to the clones active in antibody-dependent cell-mediated cytotoxicity. Expression of OKT8 and OKT4 antigens was variable; in particular, five clones were OKT8⁺/OKT4^?, whereas the other five expressed the OKT8^?/OKT4⁺ phenotype. It is noteworthy that the ultrastructural features of cytolytic T cell clones are similar to those of large granular lymphocytes, known to be the only lymphoid cells in normal peripheral blood which possess cytolytic activity. Thus, it is possible that the presence of electron-dense granules may represent a morphological marker for all human cytolytic lymphocytes.     

Tumour-specific cytotoxic T lymphocyte activity in Th2-type Sézary syndrome: its enhancement by interferon-gamma (IFN-γ) and IL-12 and fluctuations in association with disease activity

Sézary syndrome (SzS) is the leukaemic variant of cutaneous T cell lymphoma (CTCL), whose malignant T cells are of the Th2 type in most cases. In this study we investigated the tumouricidal activity of cytotoxic T lymphocytes (CTL) present in peripheral blood of a patient with Th2-type SzS, focusing on the effect of IL-2, IFN-γ and IL-12 on their cytotoxic activity, and the relationship between their lytic capacity and the patient's clinical course. At four different time points during a 2-month clinical period, CD4⁺ CD7^? Sézary cells and CD8⁺ cells were separated from the patient's circulating cells. CD8⁺ cells were cultured with chemically attenuated, purified Sézary cells in the presence of IL-2 to develop specific cytotoxicity. The CD8⁺ cells thus cultured exhibited lytic activity against autologous Sézary cells. Concomitant addition of IFN-γ or IL-12 exerted a synergistic cytolytic effect with IL-2 on the tumour cells. Cytotoxicity inhibition studies using MoAbs revealed that the cytotoxicity operated in MHC class I-, CD8- and αβ T cell receptor-dependent manners. Furthermore, eight CD8⁺ T cell clones generated from cultured CD8⁺ cells exhibited a strong cytotoxicity against Sézary cells in an MHC class I-restricted fashion. During the clinical course, the activity of generated CTL and the number of CD8⁺ cells were inversely correlated with disease activity as assessed by the serum level of lactate dehydrogenase. These findings suggest that CTL down-regulate the growth of malignant T cells in this long-standing disease. Since Th2 cytokines such as IL-4 down-modulate CTL activity, CTL are assumed to be usually suppressed in SzS, whose malignant T cells are of Th2 type. It is likely that the administration of IFN-γ normalizes this Th2-skewing state, activates CTL, and thus exerts the therapeutic effectiveness in the treatment of CTCL.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.     

T cell clones from a Sjögren's syndrome salivary gland biopsy produce high levels of IL-10

Sjögren''s syndrome (SS) is characterized by a focal periductal salivary gland infiltrate consisting mainly of T and B lymphocytes. Most of the T cells bear the memory of CD4⁺ Th-1-like phenotype and express high levels of class II, though CD8⁺ cells are also present. We have studied 17 labial salivary gland and 15 peripheral blood T cell clones from a patient with primary SS. The tissue clones were 71% CD8⁺ and 29% CD4⁺, and the peripheral blood-derived clones were 60% CD8⁺ and 40% CD4⁺. The CD4⁺ T cell clones from both the salivary gland and autologous peripheral blood were of the Th1 phenotype, in that they produced interferon-gamma (IFN-γ) and IL-2 but very little IL-4 after 24 h stimulation with phorbol myristate acetate and anti-CD3 antibody. The salivary gland-derived CD4⁺ clones produced 15 times more IL-10 (7·92 ng/ml) than peripheral blood-derived CD4⁺ clones (0·52 ng/ml, P≤0·02). The tissue CD8⁺ clones produced 1·2 times (P<0·04) more IFN-γ and CD4⁺ clones produced 3·5 times less IL-2 (P<0·02) than the respective PBM-derived clones. The accumulation of Th1-type cells producing high levels of IL-10 in the salivary gland suggests a specific immunoregulatory function at the site of inflammation in SS.     

Factors contributing to the potency of CD8+ T cells

CD8⁺ cytotoxic T lymphocytes (CTLs) play a crucial role in targeting virus-infected and cancer cells. Although other cytotoxic lymphocytes such as CD4⁺ T and natural killer (NK) cells, as well as chimeric antigen receptor (CAR)-T cells, can also identify and destroy aberrant cells, they seem to be significantly less potent based on available experimental data. Here, I contemplate the molecular mechanisms controlling the sensitivity and kinetics of granule-mediated CD8⁺ T cell cytolytic responses. I posit that the clustering of MHC-I molecules and T cell receptors (TCRs) on the cell surface, as well as the contribution of the CD8 co-receptor, are major factors driving exceptionally potent cytolytic responses. I also contend that CD8⁺ T cells with known specificity and engineered TCR-T cells might be among the most efficient cytolytic effectors for treating patients suffering from viral infections or cancer.     

γδ T cells represent a major spontaneously cytotoxic cell population in the chicken

Natural killer cells in the chicken are mainly confined to the intestine, while only small frequencies are detectable in spleen, lung and blood. Here, we compared the spontaneous cytotoxicity of lymphocytes isolated from blood, spleen and intestine using a flow cytometric based cytotoxicity assay. There was no spontaneous cytotoxicity detected in chicken blood preparations. In contrast, freshly prepared splenocytes exhibited a spontaneous cytotoxicity of up to 50% and intestinal epithelial lymphocytes of up to 85%. This cytotoxicity was observed against the RP9 but not against the chicken CU24 target cell line. The observed cytotoxicity was MHC unrestricted since B²B² derived effector cells killed RP9 target cells (B²B¹⁵) equally well compared to MHC mismatched 2D8 targets (B¹⁹B¹⁹). The cytotoxicity of splenocytes was enhanced by preincubation with IL-2 or strongly increased with IL-2 plus IL-12. By cell sorting, we identified the CD8⁺γδ T cell subset as the major effectors, whereas both CD8⁻γδ T cells and CD8⁺αβ T cells had only low cytolytic potential. Within intestinal lymphocyte CD45⁺cells displayed cytotoxicity as well as sorted γδ T cells and NK cell. In conclusion, the chicken γδ T cells represent a major cytotoxic lymphocyte subset that can lyse target cells in a MHC unrestricted manner.     

CD4 T Cell Clones Producing both Interferon-γ and Interleukin-10 Predominate in Bronchoalveolar Lavages of Active Pulmonary Tuberculosis Patients

The pattern of cytokine production in T cell clones derived from bronchoalveolar lavages (BAL) of active pulmonary tuberculosis (TB) patients was analyzed in clones obtained by limiting dilution procedures which expand with high efficiency either total T lymphocytes, independently of their antigen-recognition specificity, or Mycobacterium tuberculosis-specific T cells. BAL-derived clones, representative of CD4⁺ cells from five patients with active TB, produced significantly higher amounts of IFN-γ than BAL-derived CD4⁺ clones from three inactive TB donors or four controls (with unrelated, noninfectious pathology). Average IL-4 and IL-10 production did not differ significantly in the three groups. Although these data suggest a predominant Th1 response to M. tuberculosis infection in the lungs, the majority of BAL-derived CD4⁺ clones produced both IFN-γ and IL-10 and the percentage of clones with this pattern of cytokine production was significantly higher in clones derived from BAL of active TB patients than from controls. Only rare clones derived from peripheral blood (PB)-derived CD45RO⁺ CD4⁺ T cells of both patients (nine cases) and controls (four cases) produced both IFN-γ and IL-10; instead, the IL-10-producing clones derived from PB T cells most often also produced IL-4, displaying a typical Th2 phenotype. Higher average amounts of IFN-γ and IL-10 were produced by BAL-derived CD8⁺ clones of four active TB patients than of four controls, although the frequency of CD8⁺ clones producing both IFN-γ and IL-10 was lower than that of CD4⁺ clones. The M. tuberculosis-specific BAL-derived T cell clones from three active TB patients were almost exclusively CD4⁺ and produced consistently high levels of IFN-γ often in association with IL-10, but very rarely with IL-4. Unlike the BAL-derived clones, the M. tuberculosis-specific clones derived from PB CD45RO⁺ CD4⁺ T cells of three different active TB patients and two healthy donors showed large individual variability in cytokine production as well as in the proportion of CD4⁺, CD8⁺, or TCR γ/δ⁺ clones. These results indicate the predominance of CD4⁺ T cells producing both the proinflammatory cytokine IFN-γ and the anti-inflammatory cytokine IL-10 in BAL of patients with active TB.     

Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: Heterogeneity in cytotoxic activity and cytokine secretion levels

Protective immunity against mycobacteria is dependent on antigen-specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium-specific CD4⁺ CTL are readily detectable among primary peripheral T cells but what proportion of CD4⁺ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4⁺ T cells are identical to or distinct from those that produce interferon (IFN)-γ is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4⁺ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium-pulsed target cells and to release IFN-γ and interleukin (IL)-4. Our results show that the vast majority of CD4⁺ T cell clones are able to lyse mycobacterial antigen-pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN-γ, but low or nondetectable levels of IL-4. In contrast, control tetanus toxoid-specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL-4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN-γ release. Interestingly one T cell clone was able to lyse only mycobacterium-pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4⁺ T helper type 1 (Th1)-like cells that display cytotoxic activity, and release high levels of IFN-γ but no or little IL-4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL-4.     

hsBAFF enhances activity of NK cells by regulation of CD4(+) T lymphocyte function

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T cell activation. Recently, we have demonstrated that human soluble BAFF (hsBAFF) potently elevates NK cell activity of murine splenic cells in vivo. In this study, we further show that hsBAFF markedly enhanced NK cell activity of in vitro mouse splenic cells. Purified NK cell activities treated with hsBAFF in the presence of IL-2 or IFN-γ, but not with hsBAFF alone were obviously elevated. hsBAFF strongly increased the activity of NK cells co-incubated with mixed T and B lymphocytes or T lymphocytes alone, but not with B lymphocytes alone. We also found that culture supernatants, from mixed T and B lymphocytes or T lymphocytes alone, but not from B lymphocytes alone post-hsBAFF treatment, effectively increased NK cell activity, and noticed that IL-2 and IFN-γ levels significantly increased in the culture supernatants of hsBAFF-treated T lymphocytes. Of importance, we pinpointed that hsBAFF elicited robust population of CD4⁺ T lymphocytes in vitro, and further demonstrated that hsBAFF induced significantly high IL-2 and IFN-γ secretion of CD4⁺ T lymphocytes. These findings indicate that hsBAFF results in the elevation of NK cell activity by regulation of CD4⁺ T lymphocytes contributing to IL-2 and IFN-γ generation.     

Induction of immunoglobulin synthesis by CD4 T cell clones

The study of mechanisms by which CD4⁺ T cells induce Ig synthesis has been greatly enhanced by the availability of CD4⁺ T cell clones with restricted cytokine profiles. We have demonstrated with in vitro and in vivo studies that both Th1 (T helper cell 1) and Th2 clones can provide MHC restricted help and induce primary as well as secondary antibody responses under cognate antigen driven conditions. In addition, we have shown that both types of clones, utilizing distinct cytokines, can effect B cell memory and affinity maturation of the Ig response, although the precise mechanisms by which this occurs are not yet clear. Using Th1 and Th2 clones, we have also shown that the pathways for IgG1 synthesis are redundant, in that induction of IgG1 synthesis in secondary responses in which B cells have already switched from IgM to 1gG1, can occur via several pathways, one involving IL-4 and IL-5, the other involving IL-2. In contrast, IgE and IgG2a synthesis require specific cytokines for synthesis in both primary and secondary B cells. Finally, the cytokines produced by Th1 and Th2 clones can 'neutralize' each other, when both types of clones are present during the induction of primary Ig responses. As an exception however, the induction of IgA synthesis is greatly augmented by the presence of both types of clones.     

Regulation of Human Cytotoxic T Lymphocytes Development by the Synergistic Effect of IL-7 and sCD23

The present study was undertaken to investigate the interaction of IL-7 and sCD23 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were stimulated with mitogen plus IL-2 and subcultured for 7 days with IL-7 and/or sCD23. The combination of IL-7 and sCD23 synergistically enhanced the proliferation of both CD4⁺and CD8⁺T cells. CD8⁺T cells, however, were usually more responsive to IL-7 and sCD23. This synergy was observed on both subsets of T cells. Furthermore, these cytokines synergistically augment the CTL activity of CD8⁺T cells in both mitogen- and antigen-activated T cells. MAbs anti-IL-2 or anti-IL-2R (CD25) and anti-IL-12 had no effect on T cell proliferation and CD8⁺cytotoxic activity induced by IL-7 and sCD23. We analyzed the effect on IFN-γ induction by CD8⁺T cells and found that IL-7 alone was incapable of inducing detectable levels of IFN-γ production, but together with sCD23 it enhanced the production of IFN-γ. We also found that IFN-γ was not required for enhanced CTL activity of CD8⁺T cells, because rabbit anti-IFN-γ did not block the synergistic effects of either cytokine. The data demonstrate that the synergistic stimulatory activity of IL-7 and sCD23 may be of significance in the human CTL development and provide an alternative mechanism of stimulating T cells for use in immunotherapy.     

Co-receptor-independent signal transduction in a mismatched CD8+ major histocompatibility complex class II-specific allogeneic cytotoxic T lymphocyte

The contribution of co-receptors in signal transduction upon T cell receptor (TCR)-mediated recognition of major histocompatibility complex (MHC) class II antigen by mature T lymphocytes expressing TCR derived from the apparently co-receptor-independent, I-A^k-specific allogeneic CD8⁺ CTL clone QM11 has been examined. Mature double-negative, CD8⁺ and CD4⁺ bulk T cell lines and clones expressing TCRQM11 were developed from TCRQM11 transgenic mice. All these T cells, irrespective of co-receptor expression, showed specific lytic activity on cells expressing I-A^k. Furthermore, co-receptorless mutants were obtained from a CD4⁺ and CD8⁺ clone. The responses of these co-receptorless mutants upon specific recognition of the alloantigen, as judged by cytolytic activity, granule exocytosis, lymphokine production, proliferation, and tyrosine phos-phorylation of the ξ chain, were comparable to those of the original clones. Thus, the results proved the co-receptor independence of the recognition of I-A^k by TCRQM11 and further indicated there is no indispensable unique signal transduced by co-receptors. However, when the amount of the available antigen was limited by anti-I-A^k antibody, the CD4⁺ T cell clone showed a remarkable resistance to the inhibition whereas the mismatched CD8⁺ clone was readily inhibitable. The anti-I-A^k-resistant component of the CD4⁺ clone showed dependency on the CD4 molecule. Taken collectively, the results indicate that the role played by a co-receptor molecule in mature T cells is purely quantitative amplification of the signal through the formation of a TCR/MHC/co-receptor ternary complex, and also indicate that the role of co-receptor molecules as TCR-independent adhesion molecules is at best minimal.