The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

CD4 Dependence of Activation Threshold and TCR Signalling in Mouse T Lymphocytes

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4^? mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4^? mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4^? clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56^lck or p59^fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4⁺ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56^lck-TCR co-precipitation.     

Butyrophilin 3A (BTN3A,CD277)‐specific antibody 20.1 differentially activates Vγ9Vδ2 TCR clonotypes and interferes with phosphoantigen activation

Phosphoantigens (PAgs)‐like HMBPP ((E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate) and butyrophilin 3 (BTN3A, CD277)‐specific monoclonal antibody 20.1 induce TCR‐mediated activation of Vγ9Vδ2 T cells. Here, we compared murine reporter cells transduced with Vγ9Vδ2 TCRs G115, D1C55, and MOP for the activation in culture with human RAJI cells and PAgs or mAb 20.1 and its single‐chain (sc) derivative. All transductants responded readily to PAg but only TCR MOP γ‐chain‐expressing cells responded to mAb/sc 20.1. Furthermore, both antagonist and agonist mAb and sc of the agonist mAb inhibited the PAg response of TCR‐transduced murine reporter cells. These findings suggest that, in contrast to stimulation by physiological stimulators (PAg), the responsiveness to mAb 20.1 depends strongly on CDR3 sequences of the TCR, and that mAb 20.1 can interfere with the PAg‐response. Mouse or human origin of reporter cells might affect the mAb 20.1 response since all three TCR‐mediated mAb 20.1‐induced activation of TCR‐transduced Jurkat cells. The pronounced differences between PAg and mAb 20.1‐induced activation observed here help to understand the often contradictory published data. This study provides novel perspectives on the physiological mechanism of Vγ9Vδ2 T‐cell activation, and highlights the complex mode of action of BTN3A‐specific antibodies as agents in cancer immunotherapy.     

C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8⁺ and TCR α β⁺. About half of them expressed a CD94^bright phenotype, whereas the remaining were CD94^dim. Only the CD94^bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94^bright and CD94^dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94^bright, but not by CD94^dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca²⁺]_i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vβ repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vβ22 expression. Most importantly, sequence analysis showed that they all expressed identical Vβ22 TCR rearranged with Jβ2.1 and Cβ2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.     

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.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

p40, a novel surface molecule involved in the regulation of the non-major histocompatibility complex-restricted cytolytic activity in humans

Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions (“p40” molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the “inhibitory” receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double-immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti-p40 mAb. p40^bright cells were homogeneously HLA-DR-positive, p40^medium cells were HLA-DR-negative but co-expressed CD56 antigens, while p40^dull cells were all CD3⁺. Anti-p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti-p40 mAb strongly reduced the anti-CD16 mAb-induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti-CD3 or anti-T cell receptor mAb-mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40-induced inhibition of the NK cytotoxicity required optimal cross-linking, as anti-p40 mAb could inhibit the lysis of Fcγ receptor (FcγR)-positive but not of FcγR-negative target cells. In addition, (Fab')₂ fragments of anti-p40 mAb failed to inhibit the lysis of FcγR-positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK-mediated cytolysis.     

Self-reactive T cell clones in a restricted population of interleukin-2 receptor β+ cells expressing intermediate levels of the T cell receptor in the liver and other immune organs

T cells expressing high levels of the T cell receptor (TCR^high) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCR^int) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCR^int cells constitutively express interleukin-2 receptor β-chain (IL-2R), two-color staining for CD3 (or TCR) and IL-2Rβ clearly distinguished IL-2RßT⁺ CD3^int (or TCR^int) cells from IL-2Rβ^?, CD3^high cells. CD3^int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti-Vβ mAb in conjunction with the endogenous superantigen Mls, the distribution of self-reactive clones among T cells generated in all of the above pathways was investigated in mice. Self-reactive T cell clones were confined to IL-2Rβ⁺, CD^int cells, in all of the organs tested. A significant proportion of self-reactive clones was never identified among CD3^high cells in the thymus and peripheral immune organs in either young (8 week old) or old (50 week old) mice. Possibly reflecting their self-reactivity, CD3^int cells, but neither NK cells nor CD3^high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti-CD3 mAb. These results raise the possibility that CD3^int cells seen in the liver and thymus might belong to a similar primordial lineage of T cells, and that self-reactive clones are not generated through the major intrathymic pathway, but only through extrathymic pathways and an alternative intrathymic pathway.     

CD45-mediated regulation of LFA1 function in human natural killer cells. Anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3^?CD16⁺ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3^?CD16⁺ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parralel the inhibition mediated (with the same tumor target cells) by the anti-LFAlα mAb TS.1.22 or by the anti-LFA1β mAb TS.1.18. However, different from the anti-LFAl mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3^?CD16⁺ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16⁺ NK cellsT205 orTA218 mAb were found to reduce by 50–70 % the intracellular Ca⁺⁺ ([Ca⁺⁺]i) mobilization induced by anti-LFAlα or anti-LFA1β mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca⁺⁺ mobilization induced by anti-CD 16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFAla mAb (as “triggering targets”). This effect of anti-LFAlα, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFAlα chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.     

Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus

A CD4⁺ T cell epitope of the influenza virus matrix protein corresponding to the C terminus (QAYQKRMGVQMQRFK) was inserted into the VP7 gene of bluetongue virus (BTV). The chimeric protein was expressed by a dual recombinant Autographa californica polyhedrosis virus (AcNPV), which encodes the two inner capsid proteins VP3 and VP7 of BTV. When Spodoptera frugiperda cells (Sf9 cells) were infected with this recombinant BTV, core-like particles (CLPs) were formed as demonstrated by electron microscopy. To study the immunogenicity of a foreign epitope deprived of its natural flanking sequences in vitro, purified CLPs expressing the T cell epitope were used to stimulate two different MHC class II-restricted CD4⁺ human T cell clones. One of these T cell clones, ALF 3.7 was specific for the inserted epitope, whereas the other T cell clone ALF 4.4 recognized shorter derivates of the given epitope. CLPs with the inserted epitope were presented as efficiently as purified influenza virus matrix protein to the clone ALF 3.7, whereas clone ALF 4.4 showed no proliferative response. Received: 17 February 1998     

Fine antigen specificity of human γδ T cell lines (Vγ9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium,Streptococcus sanguis

We have established human γδ T cell lines specific for Streptococcus sanguis (S. sanguis) KTH-1 present in normal oral cavity flora. The CD4^?CD8^? CD3⁺Vγ9⁺Vδ1^?CD45RO⁺ CD25⁺ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen-presenting cells without apparent evidence of HLA restriction. The proliferative response of the γδ T cell lines was completely blocked by anti-TcRγδ monoclonal antibody (mAb) and anti-HLA class I mAb (W6/32), whereas anti-HLA classical class Ia mAb (B-H9; anti-HLA-A,B,C), anti-HLA class II mAb (anti-DR, anti-DQ, and anti-DP) and anti-CD4 mAb did not have any inhibitory effects. Surprisingly, the γδ T cell lines showed the proliferative response against the original bacterial Ag KTH-1 exclusively, and exhibited no cross-reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH-3. Moreover, cytokine production of the γδ T cell lines was similar to the Th1 pattern [interferon-γ, tumor necrosis factor (TNF)-α and TNF-β]. They also produced interleukin-8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH-1 specific γδ T cell line and its subclones is rather limited. It is suggested that γδ T cells with canonical TcR could preferentially respond to KTH-1 Ag. Thus, in addition to a broad or cross-reactivity of γδ T cells against phylogenetically conserved stress/heat-shock protein, which is well characterized by others, some peripheral blood γδ T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.     

In vivo application of mAb directed against the γδ TCR does not deplete but generates “invisible” γδ T cells

mAb targeting the γδ TCR have been used for γδ T‐cell depletion with varying success. Although the depletion‐capacity of the anti‐γδ TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use γδ T‐cell depletion protocols involving the mAb clone UC7‐13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7‐13D5 antibodies does not deplete γδ T cells in vivo, but rather leads to TCR internalization and thereby generates “invisible” γδ T cells. We addressed this issue using anti‐γδ TCR mAb injections into WT mice as well as into reporter TCR delta locus‐histone 2B enhanced GFP knock‐in mice, in which γδ T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus‐histone 2B enhanced GFP mice provided here for the first time direct evidence that the “depleted” γδ T cells were actually still present. Our results show further that GL3 and UC7‐13D5 mAb are in part cross‐competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of γδ T cells through mAb. We conclude that γδ T‐cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of γδ T cells.     

T cell repertoire in patients with multiple myeloma and monoclonal gammopathy of undetermined significance: Clonal CD8+ T cell expansions are found preferentially in patients with a low tumor burden

The T cell receptor (TCR) variable (V) gene repertoire was analyzed in patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 17), multiple myeloma (MM) stage I (n = 16), MM stages II/III (n = 31) and agematched controls (n = 27) by immunofluorescence and flow cytometry using a panel of mouse monoclonal antibodies (mAb) (n = 10) against TCR Vα and Vβ gene products. T cell expansion was defined as a value ? thrice the normal median value for each respective TCR V mAb. Fifty-three percent of all patients displayed CD8⁺ expansion(s) as compared to 30% of age-matched controls (p = 0.001). Within the CD4 subset, 18% of the patients displayed T cell expansion(s) in comparison to 11% of the controls (not significant). Interestingly, the CD8⁺ expansion(s) were more frequently noted in patients with a low tumor burden (MGUS/MMI) (73%) as compared to those with advanced disease (MM II/III) (32% and control donors (30%) (p < 0.01). Likewise, multiple CD8⁺ expansions (two or more) were more common in MGUS/MM I patients than in MM II/III and controls (p = 0.01). The T cell expansions were stable over time in patients with a stable disease. A high degree of clonality of the expansions was detected by TCR CDR3 fragment length analysis, determination of Jβ gene usage and nucleotide sequencing. The frequent finding of oligoclonal CD8⁺ T cell expansions in patients with a low tumor mass, but not in patients with advanced disease justifies further work in order to identify the relevance of expanded CD8⁺ T cells. In one patient with T cell reactivity against the autologous myeloma idiotype, two expansions within the CD8 population (Vβ3 and Vβ5.2 respectively) displayed no reactivity against the idiotype. Instead, idiotype recognition was confined to a CD8 non-expanded Vβ22⁺ T cell population, with a highly restricted TCR usage (CDR3 fragment length analysis).     

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain,FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183^?EB6⁺ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4^? C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183⁺/EB6⁺ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and Fc?RI γ chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58⁺ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ-γ homodimers or in the form of ζ-γ heterodimers, while γ-γ homodimers were detectable in low amounts. However, p58⁺ clones displaying a unique association with γ-γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56^lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56^lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58-associated CD3 ζ, chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 ζ chains.     

Lymphocyte-activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4+ T lymphocytes

The activation requirements for antigen-dependent proliferation of CD4⁺ T cells are well documented, while the events leading to the inactivation phase are poorly understood. Here, we tested the hypothesis that the lymphocyte-activation gene 3 (LAG-3), a second major histocompatibility complex (MHC) class II ligand, plays a regulatory role in CD4⁺ T lymphocyte activation. CD4⁺ class II-restricted T cell clones were stimulated by their relevant antigen (hemagglutinin peptide or diphteria toxoid) and antigen-presenting cells with or without anti-LAG-3 monoclonal antibody (mAb). Kinetic studies were performed to monitor different activation parameters, including the measurement of thymidine incorporation, expression of activation antigens and cytokine secretion. Results showed that the time course from the initial time points up to the peak time point was not modified in the presence of anti-LAG-3 mAb. However, addition of these antibodies, either as whole IgG or as Fab fragments, led to increased thymidine incorporation values for late time points and, hence, to a shift in the decreasing proliferation curve. We also showed that expression of activation antigens, such as CD25, was higher in the presence of anti-LAG-3 mAb, and that cytokine concentrations, i.e. of interferon-γ or interleukin-4, were higher in the corresponding culture supernatants. In addition, we tested whether the effects of anti-LAG-3 mAb were limited to antigen-dependent. MHC class II-restricted responses. The proliferative responses of CD4⁺ T cell clones following stimulation with either interleukin-2, mitogens, a combination of anti-CD2 mAb, immobilized anti-CD3 or anti-T cell receptor mAb were not altered by anti-LAG-3 mAb. The allogeneic proliferative response of a CD8⁺ T cell clone was also not affected. Overall, the present analysis reveals a modulating effect of anti-LAG-3 mAb, mediated specifically on antigen-dependent, MHC class II-restricted responses of CD4⁺ T cell lines. These results support the view that LAG-3/MHC class II interaction down-regulates antigen-dependent stimulation of CD4⁺ T lymphocytes.     

Unusual selection and peripheral homeostasis for immunoregulatory CD4− CD8− T cells

Immunoregulatory CD4^? CD8^? (double‐negative; DN) T cells exhibit a unique antigen‐specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T‐cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2^k MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2^b MHC haplotype. Herein, we take advantage of this well‐defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC‐selecting haplotype. By defining DN T‐cell differentiation in 3A9 TCR transgenic CD47‐deficient mice as well as in mice bearing the NOD.H2^k genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.     

Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

Human double-negative (CD4-CD8-) T cells bearing alpha beta T cell receptor possess both helper and cytotoxic activities.

Expression of CD4 or CD8 on the cell surface is an important guide for discriminating the immunological functions of T cells. However, a minor T cell subset, which lacks both CD4 and CD8 molecules but bears the usual form of T cell receptor (TCR) alpha beta (CD4-CD8-TCR alpha beta+ T cells), has recently been found not only in mice but also in humans, and its role in immune response is now of considerable interest. In order to clarify the characteristics of this newly defined T cell subpopulation, we established five IL-2-dependent CD4-CD8-TCR alpha beta+ T cell clones from the peripheral blood of a healthy individual, and examined their various biological functions. It was found that all clones not only helped B cells in immunoglobulin production, but also exerted major histocompatibility complex-unrestricted cytotoxicity. Although their CD3/TCR complexes were functionally competent, the cytotoxicity seemed to be mediated via unknown molecules other than the CD3/TCR complex, as evidenced by the failure of CD3 MoAb to inhibit the cytotoxic activity. Our present findings showed that CD4-CD8-TCR alpha beta+ T cells possess potential bifunction, i.e. helper and cytotoxic activities. Their roles in the pathogenesis of immunodeficiency are discussed.