Shared amino acid sequences in the NDβN and Nα regions of the T cell receptors of tumor-infiltrating lymphocytes within malignant glioma

The purpose of this study was to assess the V-(D)-J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma-specific antigen contact in αβ TCR-mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor-infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1⁺ clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1⁺ clones from the PBL (p < 0.001). A common VDJ sequence, FCASS (Vβ13.1)-YRLPWGTSDS (NDβN)-GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1⁺ PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V-J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7-bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p < 0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage-specific T cells occurred in vivo at the tumor site and that these T cells may be recognizing glioma-specific antigens.     

Interleukin-12 increases interleukin-4 production by established human ThO and Th2-like T cell clones

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-γ-producing T cells, increases IFN-γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells. We found that IL-12 (i) enhances, as previously reported, IFN-γ production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-γ and also of IL-4 by established IL-4-producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.     

Bacille Calmette Guérin and interleukin-12 down-modulate interleukin-4-producing CD4+ NK1+ T lymphocytes

Early production of interleukin-12 (IL-12) by macrophages and of IL-4 from CD4⁺ NK1⁺ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon-γ-secreting T helper 1 (Th1) cells against intracellular pathogens and of IL-4-producing Th2 cells against helminths. Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL-4 or IL-12, respectively. Moreover, it is likely that IL-4 dominates over IL-12, suggesting that Th2 cell development is preferred in the presence of both cytokines. Mycobacterium bovis Bacille Calmette Guérin (BCG) and IL-12 are potent inducers of Th1 responses. Here we show that BCG and IL-12 down-modulate IL-4-producing CD4⁺ NK1⁺ TCRα/β^intermediate liver lymphocytes. Our data provide further insights into the mechanisms by which BCG and IL-12 may promote unrestricted development of Th1 responses in vivo: BCG and IL-12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate

Type 2 cytokines are thought to have a protective role in psoriasis vulgaris by dampening the activity of T helper 1 (Th1) lymphocytes. The aim of the present study was to determine the effect of monomethylfumarate (MMF), the most active metabolite of the new anti-psoriatic drug Fumaderm®, on the production of cytokines and the development of Th subsets. MMF was found to enhance interleukin (IL)-4 and IL-5 production by CD2/CD8 monoclonal antibody-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Maximal effects of MMF were found at a concentration of 200 μM and resulted in tenfold enhanced levels of IL-4 and IL-5 production. MMF did not affect the levels of IL-2 production, interferon (IFN)-γ production or proliferative T cell responses in these cultures. Similar effects of MMF were observed in cultures of purified peripheral blood T cells indicating that this compound can act directly on T cells. MMF did not influence cytokine production by purified CD4⁺CD45RA⁺ (unprimed) T cells, but greatly enhanced IL-4 and IL-5 production without affecting IFN-γ production by purified CD4⁺CD45R0⁺ (primed) T cells. Furthermore, MMF also augmented IL-4 and IL-5 production in established Th1/Th0 clones that were stimulated with CD2/CD28 monoclonal antibody. Finally, when PBMC were challenged with Mycobacterium tuberculosis that typically induces Th1 recall responses with strong IFN-γ secretion, MMF again appeared to induce high levels of IL-4 and IL-5 secretion while IFN-γ production was unaffected. These results may be relevant for the development of therapeutic regimens designed to correct inappropriate Th1 subset development in immunopathologic conditions.     

Broadly reactive human CD4+ T cells against Enterobacteriaceae are found in the naïve repertoire and are clonally expanded in the memory repertoire

Enterobacteriaceae are a large family of Gram-negative bacteria that includes both commensals and opportunistic pathogens. The latter can cause severe nosocomial infections, with outbreaks of multi-antibiotics resistant strains, thus being a major public health threat. In this study, we report that Enterobacteriaceae-reactive memory Th cells were highly enriched in a CCR6⁺CXCR3⁺ Th1*/17 cell subset and produced IFN-γ, IL-17A, and IL-22. This T cell subset was severely reduced in septic patients with K. pneumoniae bloodstream infection who also selectively lacked circulating K. pneumonie-reactive T cells. By combining heterologous antigenic stimulation, single cell cloning and TCR Vβ sequencing, we demonstrate that a large fraction of memory Th cell clones was broadly cross-reactive to several Enterobacteriaceae species. These cross-reactive Th cell clones were expanded in vivo and a large fraction of them recognized the conserved outer membrane protein A antigen. Interestingly, Enterobacteriaceae broadly cross-reactive T cells were also prominent among in vitro primed naïve T cells. Collectively, these data point to the existence of immunodominant T cell epitopes shared among different Enterobacteriaceae species and targeted by cross-reactive T cells that are readily found in the pre-immune repertoire and are clonally expanded in the memory repertoire.     

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor β (IL-2Rβ CD122) and the polymorphic antigen NK1.1. These “NK-like” T cells are distinguished by their highly skewed Vα and Vβ repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1⁺ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2Rβ⁺ T cells with a skewed Vβ repertoire characteristic of “NK-like” T cells. Furthermore lack of NK1.1 expression on IL-2Rβ⁺ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2Rβ⁺ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that “NK-like” T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.     

Influence of both TCR repertoire and severity of the atopic status on the cytokine secretion profile of Parietaria officinalis -specific T cells

Peripheral blood mononuclear cells (PBMC) from both nonatopic and Parietaria officinalis -sensitive donors proliferated in response to the allergen Par o 1 and developed into Par o 1-specific T cell lines and clones, which also showed reactivity for Par o 1-derived peptides. Virtually all Par o 1-specific T cell lines and large numbers of Par o 1-specific T cell clones proliferated in response to two Par o 1 nonapeptides (p92 and p96), which probably contain immunodominant epitopes of the Par o 1 allergen. Both p92- and p96-specific T cell clones showed the ability to produce IFN-γ, but p92-specific T cell clones produced significantly lower amounts of IL-4 and IL-5 than p96-specific T cell clones, indicating that distinct epitopes, able to elicit functionally different T helper cell responses, may coexist in Par o 1. How ever, p92-specific T cell clones derived from atopic subjects with high IgE serum levels (high IgE producers) secreted significantly higher amounts of IL-4 and IL-5 than corresponding T cell clones generated from non atopic subjects or patients with low IgE serum levels (low IgE producers), whereas p96-specific T cell clones secreted high IL-4 and IL-5 concentrations irrespective of whether they derived from high or low IgE producers. The addition of IL- 4 and anti-IL-12 mAb to bulk culture significantly up-regulated the development of p92-specific T cells into IL-4-producing cells, whereas the addition of IL-12 and anti-IL-4 mAb shifted the differentiation of p96-specific T cells towards IFN-γ-producing cells. Taken together, these results suggest that the cytokine profile of allergen-specific T cells is influenced by both the T cell receptor repertoire and the severity of atopic status and can be modulated, at least in vitro, by stimulation with the specific peptide in the presence, or after removal, of appropriate cytokines.     

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine-producing cells, the production of type 1 [interleukin-2 (IL-2) and interferon (IFN)-γ], type 2 (IL-4, IL-5, IL-10), inflammatory [tumor necrosis factor (TNF)-α, IL-1α, IL-6] and regulatory (transforming growth factor-β) cytokines were examined. With the exception of IL-4 and IL-5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL-4 and IL-5 were not detected until later in infection in the resistant mice (>34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL-2 and IFN-γ and an earlier appearance of TNF-producing cells, in comparison to resistant mice. Cells producing IL-2 or IL-10 were transient in their appearance in the spleen while cells producing IL-1, IL-4, IL-5, IL-6, IFN-γ, TNF, or TGF-β were first detectable in either the acute or post-acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN-γ, TNF-α and TGF-β were particularly numerous even very late in the infection. Double-staining techniques were used to show that the vast majority of the IFN-γ-producing cells in the spleen were CD4^?, CD8^? α/β TCR⁺ T cells. This study confirms the transience of IL-2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late-acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine-producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte–M2 macrophage pathway

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3⁺ CD8⁺ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1β and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3⁺ CD8⁺ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1β and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.     

Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: Selective in vivo expansion of T cells bearing defined receptors

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2⁺ and Vβ5.6⁺ clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2⁺ and Vβ5.6⁺ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.     

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4⁺ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2⁺, CD4⁺, CD8^?, T cell receptor (TcR) αβ⁺ and produced interleukin-2 in response to CII peptides presented by I-A^q molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical Vα and Jα (VαBMB, Jα20) gene segments and two distinct Vβ (Vβ1 and Vβ4) associated with the Jβ2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both α and β chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4⁺ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.