A critical role for the T cell receptor alpha-chain connecting peptide domain in positive selection of CD1-independent NKT cells.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.     

Characterization of Mouse Mammary Tumour Virus-Induced Migration of Lymphoid Cells into Lymph Nodes

We previously found that Mtv-2+ lymph nodes (LN) implanted into Mtv-2- mice underwent marked hyperplasia owing to the influx of lymphocytes. LN grafts infected with exogenous mouse mammary tumour viruses (MMTV), MMTV(FM) transmitted by FM mice and MMTV-2 produced by Mtv-2, also swelled in MMTV-free recipients. Mtv-3 and Mtv-7 also displayed this capability. Mtv-2-induced LN hyperplasia was earlier in onset and greater in extent when major histocompatibility complex (MHC) class II I-E was expressed than unexpressed. Mtv-3-induced LN hyperplasia was suppressed completely by Mtv-3 from a different mouse strain and partially by Mtv-6 slightly different from Mtv-3 in superantigen (SAg) Vbeta specificity. LN hyperplasia occurred bidirectionally in LN transplantation between mice carrying Mtv-2 and Mtv-3, which are different SAg Vbeta specificity. LN hyperplasia induced by MMTV-2 carrying SAg responsive to Vbeta14 alone and MMTV(FM) carrying SAg responsive to Vbeta14 and Vbeta8.2 was completely but partially suppressed by MMTV(FM) and MMTV-2, respectively. CD4+ T cells were essential for MMTV-induced LN hyperplasia. LN in situ also underwent significant hyperplasia when infected with MMTV. Thus, MMTV SAg may entice circulating lymphocytes into lymphoid organs and contribute to more efficient dissemination MMTV in vivo. Secondary lymphoid tissue chemokine (SLC) may not be directly involved in this event.     

T-cell receptor Valpha spectratype analysis of a CD4-mediated T-cell response against minor histocompatibility antigens involved in severe graft-versus-host disease.

Although CD4(+) T cells can have an important role in mediating lethal graft-versus-host disease (GVHD) directed to multiple minor histocompatibility antigens (miHA) after bone marrow transplantation, their precise characterization and effector function remains elusive. In this regard, T cell receptor (TCR) Vbeta spectratype analysis has been a powerful tool for identifying donor CD4(+) T cell populations expanding to host miHA after bone marrow transplantation in the major histocompatibility complex-matched C57BL/6 (B6) --> C.B10-H2(b) (BALB.B) model of lethal GVHD. Removal of all of the Vbeta(+) T cell families containing these responding cells from the donor inoculum has proven to be an effective means of preventing the development of GVHD. Previous studies have also found that of the 11 miHA-responsive B6 CD4(+) Vbeta(+) T cell families, transplantation of Vbeta2(+) and Vbeta11(+) T cells together into lethally irradiated BALB.B mice appeared to be primarily responsible for the severity of resultant GVHD. Further focusing on these critical CD4 responses, in this study we demonstrate that B6 CD4(+)Vbeta11(+) T cells alone can induce lethal GVHD in BALB.B recipients. In addition, immunohistochemical staining of host lingual and intestinal epithelial tissues supported the capacity of Vbeta11(+) T cells to infiltrate typical GVHD-associated target areas. To further characterize the specific CD4(+)Vbeta11(+) T cells involved in this anti-miHA response, TCR Valpha spectratype analysis was performed and indicated that 6 Valpha chains were used by this reactive population. These results provide further evidence that a restricted repertoire of T cell specificities, presumably recognizing a correspondingly low number of miHA, is sufficient for the induction of severe GVHD.     

Characterization of two infectious mouse mammary tumour viruses: superantigenicity and tumorigenicity

Mouse mammary tumour virus (MMTV) is a type B retrovirus that causes mammary tumours in susceptible mice. MMTV encodes a superantigen (SAg) that has the property of stimulating T-cell populations expressing a particular variable region of the T-cell receptor (TCR) beta chain (Vbeta) and needs to be presented in the context of major histocompatibility complex (MHC) class II molecules. Previously, we described two exogenous MMTV, MMTV BALB14, which encodes a superantigen that induces the deletion of Vbeta14+ Tcells, and MMTV BALB2, which encodes a SAg that induces the deletion of Vbeta2+ Tcells. We now describe their biological activity: the deletions involve both CD4+ and CD8+ populations, are progressive and can be detected in blood, lymph nodes and spleen. Such deletions reflect, at least in part, those occurring during intrathymic development. Both BALB2 and BALB14 viral variants are capable of inducing a strong increase of Vbeta-specific T cells in BALB/c mice (I-A+, I-E+). However, when injected into the footpad, their initial stimulatory capacity differs in that the presence of MHC I-E molecules is essential only for the stimulation of Vbeta2+ T cells. Both viral variants are able to induce deletion even in the absence of the I-E molecule in which case, however, deletion appears later and is less pronounced. Both exogenous MMTVs induce, at the end of a year, 30-35% of pregnancy-dependent mammary adenocarcinomas.     

Construction and binding analysis of recombinant single-chain TCR derived from tumor-infiltrating lymphocytes and a cytotoxic T lymphocyte clone directed against MAGE-1.

The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC. By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level. We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1. Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their Valpha and Vbeta gene usage. The TIL population showed a restriction in Valpha and Vbeta usage with only Valpha4 and Valpha9 and Vbeta2 and Vbeta7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only Valpha12 and Vbeta1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible Valpha-Vbeta combination (i.e. Valpha4-Vbeta2, Valpha9-Vbeta2, Valpha4-Vbeta7 and Valpha9-Vbeta7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, Valpha4-Vbeta2 scTCR demonstrated binding activity to HLA-A1(+) cells pulsed with MAGE-1 peptide. Results obtained from screening a panel of our scTCR constructs on HLA-A1(+) cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that Vbeta2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vbeta sequence is distinctly different from the others. These findings demonstrate that soluble TCR in single-chain format have binding activity. Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.     

Synergistic effect of KRN7000 with interleukin-15, -7, and -2 on the expansion of human V alpha 24+V beta 11+ T cells in vitro

KRN7000, (2S, 3S, 4R)-1-O-(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1, 3, 4-octadecanetriol, has been shown to prevent tumor metastasis to the liver through the activation of natural killer (NK) T cells in mice. In this study, the proliferation of human NK T cells, which express an invariant T cell antigen receptor (TCR) consisting of a Valpha24 chain and a Vbeta11 chain, was investigated using KRN7000, interleukin (IL)-15, IL-7, and IL-2 in vitro. KRN7000 stimulated the expansion of Valpha24(+)Vbeta11(+) T cells derived from peripheral blood mononuclear cells in a dose-dependent fashion, with some fluctuation between donors. IL-15, IL-7, and IL-2 synergistically stimulated the expansion of Valpha24(+)Vbeta11(+) T cells when combined with KRN7000. Intracellular expression of interferon (IFN)-gamma and IL-4 in Valpha24(+)Vbeta11(+) T cells expanded in the presence of KRN7000 was identified using flow cytometry. Valpha24(+)Vbeta11(+) T cells, expanded in the presence of KRN7000, contained granzyme (Gr) B-positive granules and perforin-positive granules. The addition of IL-15 to the culture containing KRN7000 increased GrB expression in Valpha24(+)Vbeta11(+) T cells while IL-7 and IL-2 failed to do it.In conclusion, the antitumor effect of KRN7000 may depend, in part, on granule-mediated cell killing through the activation of NK T cells and IL-15 may potentiate this effect.     

Role of the T cell receptor alpha chain in stabilizing TCR-superantigen-MHC class II complexes

Superantigens (SAGs) activate T cells by simultaneously binding the Vbeta domain of the TCR and MHC class II molecules on antigen-presenting cells. The preferential expression of certain Valpha regions among SAG-reactive T cells has suggested that the TCR alpha chain may modulate the level of activation through an interaction with MHC. We demonstrate that the TCR alpha chain is required for maximum stabilization of the TCR-SAG-MHC complex and that the alpha chain increases the half-life of the complex to match those of TCR-peptide/MHC complexes. The site on the TCR alpha chain responsible for these effects is CDR2. Thus, the overall stability of the TCR-SAG-MHC complex is determined by the combination of three distinct interactions: TCR-SAG, SAG-MHC, and MHC-TCR.     

Differences between two strains of myelin basic protein (MBP) TCR transgenic mice: implications for tolerance induction

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ T cells which preferentially use the Vbeta8.2 TCR in response to myelin basic protein (MBP). Two strains of Tg mice (Valpha2.3/Vbeta8.2 and Valpha4/Vbeta8.2) have T cell receptors that recognize the NAc1-11 immunodominant epitope of MBP. We previously reported that oral administration of MBP protects both Valpha2.3/Vbeta8.2 and Valpha4/Vbeta8.2 mice from EAE; however, tolerance induction differs between strains and is dependent on the timing of oral antigen. Here we analyze the peripheral and gut-associated lymphoid tissue (GALT) environments of the two strains of Tg mice. Tg cells in the Peyer's patch (PP) but not the spleen of Valpha2.3/Vbeta8.2 mice demonstrate increased CD69 and decreased CD45RB relative to Valpha4/Vbeta8.2 mice. High levels of Th1 and Th2 cytokines, proliferative activity and CC chemokines (MCP-1) are observed in the periphery and GALT of Valpha2.3/Vbeta8.2 Tg mice. In contrast, more non-Tg CD4+ cells are seen in the PP of Valpha4/Vbeta8.2 mice. These studies suggest that activated Tg T cells and fewer potential regulatory cells in the PP of Valpha2.3/Vbeta8.2 Tg mice may influence oral tolerance.     

Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML)

Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell lines specific to two dominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100) from an HLA-A0201+ PML survivor were generated for TCR cloning. Two distinct dominant TCR alpha chains (Valpha6 and Valpha12) and a unique TCR beta chain (Vbeta5.1) were cloned from the p36-specific cell line, while only one alpha (Valpha8.6) and one beta (Vbeta2) chains were dominant in the p100-specific line. Retroviral constructs encoding CIRs were created with the extracellular domains of TCR alpha and beta chains fused to the transmembrane and cytoplasmic portions of CD3zeta (ValphaCalphaCD3zeta or VbetaCbetaCD3zeta). Cellular expression and screening for binding specific peptide-HLA-A0201 tetramer confirmed the reactivity of the p100 TCRalphabeta and of one of the two pairs of p36 TCRalphabeta (Valpha12 and Vbeta5.1). Functional tests confirmed CIR-expressing T cells secreted cytokines and expressed potent cytotoxicity on contact with A0201+ B-lymphoblastoid line loaded with peptides and/or with HLA-A0201+ cells expressing native JCV VP1 protein. In conclusion, anti-JCV designer T cells were generated.     

Differences in the ligand specificity between CD1d-restricted T cells with limited and diverse T-cell receptor repertoire

The natural killer (NK) T-lymphocyte population consists of two subsets utilizing a diverse and restricted T-cell receptor (TCR) repertoire, respectively. Both populations have been shown to include autoreactive cells. NKT cells carrying restricted Valpha14(AV14S1)Jalpha281/Vbeta8.2(BV8S2A1 ) TCR have been shown to recognize alpha-galactosylceramide (alphaGalCer) presented in the context of murine CD1d. In this study we screened a set of murine CD1d-autoreactive T-cell hybridomas with diverse TCR for their reactivity with several glycosylated variants of ceramide, including alphaGalCer. These hybridomas showed a different pattern of reactivity to CD1d-expressing antigen-presenting cells (APC) and were not reactive with any of the tested variants of ceramide. A second set of hybridomas had been selected for expression of Valpha14 and Vbeta8.2 TCR chains. These cells responded to alphaGalCer presented on CD1d, but were only weakly reactive to syngeneic splenocytes or CD1d-transfected cells. Their fine specificity in the response to glycosylation variants of ceramide demonstrated a homogenous reactivity pattern, including reactivity to alpha-galactosylsphingosine, the variant of alphaGalCer with truncated fatty acyl chain. These findings underline the differences in ligand specificity between the two subsets of CD1d-restricted NKT cells, and demonstrate a similarity in reactivity among the hybridomas using the Valpha14-Jalpha281/Vbeta8.2 TCR.     

Potent expansion of human natural killer T cells using alpha-galactosylceramide (KRN7000)-loaded monocyte-derived dendritic cells, cultured in the presence of IL-7 and IL-15

Natural killer T (NKT) cells have an extremely restricted T-cell receptor repertoire, in man consisting of a Valpha24 chain preferentially paired with a Vbeta11 chain, and play crucial roles in various immune responses. Characterization of circulating Valpha24(+)Vbeta11(+)-T cells is hampered by their low frequencies. The alpha-galactosylceramide KRN7000 was reported to be presented by CD1d to NKT cells. Since dendritic cells (DC) are potent antigen presenting cells, and have been shown to express CD1d, we analyzed whether these cells could efficiently mediate expansion of Valpha24(+)Vbeta11(+)-T cells. During a 7-day co-culture of peripheral blood mononuclear cells and KRN7000-loaded mature monocyte derived DC (moDC) in the presence of interleukin-7 (IL-7) and IL-15, we observed up to 76-fold expansion of Valpha24(+)Vbeta11(+)-T cells. The expanded Valpha24(+)Vbeta11(+)-T cells expressed the cytotoxic molecule granzyme B, showed negligible expression of Fas ligand and could be induced to express high levels of interferon-gamma, while retaining the capacity to produce IL-4. B cells, expressing CD1d, could also present KRN7000, but Valpha24(+)Vbeta11(+)-T cell expansion was only observed in the presence of IL-7 and/or IL-15. Considering the low frequency of circulating Valpha24(+)Vbeta11(+)-T cells, the present method for expansion of Valpha24(+)Vbeta11(+)-T cells using KRN7000-loaded mature moDC will be of value for the further characterization of this unique T cell subset.     

Both B and gammadelta TCR(+) lymphocytes regulate alphabeta TCR(+) lymphocytes involved in superantigen specific responses

Endogenous superantigens (SAg) presented by MHC class II IA molecules induce slow-evolving negative selection of alpha beta T cells. The role of both B and gamma delta T cells on the regulation of these SAg-specific alpha beta T cell responses was addressed in IA(b+)IE(b-) C57BL/6 mice bearing genetically induced B cell and gamma delta T cell deficiencies. B lymphocytes were required in the negative selection of Vbeta5(+)/Vbeta12(+) CD4(+) T cells. In contrast, gamma delta T cells positively stimulated the utilization of the same SAg-responsive alpha beta T cell subsets. These differences started in mature CD4(+) thymocytes and extended to naive T cell pools for B cell negative selection, and up to memory T cells for gamma deltaT cell influences. The levels of SAg-responsive T cells did not vary between C57BL/6 and double deficient (B cell and gamma delta T cell-deficient) congenic mice, implying that both B and gamma delta T cells acted through independent mechanisms.     

Alleviation of renal disease and lymphadenopathy in MRL-Fasp(lrcg)/Fas(lprcg) (MR-lpr(cg)) mice neonatally infected with mouse mammary tumor virus encoding superantigen strongly reactive with TCR Vbeta8.2 element

Mouse mammary tumor virus transmitted by FM mice (FM-MMTV) encodes a superantigen (SAg) characterized by strong reactivity with TCR Vbeta8.2 element and broad spectrum of Vbeta reactivity. To investigate what effects the expression in vivo of FM-MMTV SAg exhibits on the course of the disease in a lupus-prone model, MRL/MpJ-Fas(lprcg)/Fas(lprcg) (MRL-lpr9cg) mice, neonatally FM-MMTV-infected MRL-lprcg(MMTV) and uninfected MRL-lpr(cg) mice were compared for various disease parameters. In MRL-lprcg(MMTV), survival was significantly prolonged, glomerulonephritis, proteinuria, and lymphadenopathy were clearly ameliorated, and the production of serum immunoglobulin G (IgG), complement-activating IgG2a, and cryogenic IgG3 autoantibodies, which are thought to be pathogenic to kidneys, and circulating immune complexes (IC), and glomerular IC deposition were significantly suppressed. FM-MMTV infection deleted Vbeta8.2+ cells by about 90% and Vbeta14+ cells less efficiently in all of the CD4+, CD8+, and B220+ CD4- CD8- or double-negative (DN) T-cell populations, and Vbeta8.1+ cells in the CD4+ population but not in the others. Similar deletion profiles of CD8+ and DN T cells support that DN T cells are derived from the CD8 lineage. The results imply that the specific regulation of the immune system with viral SAg has a potential for development of an attractive immunomodulatory therapy of autoimmune diseases.     

Chronic deletion, escape from deletion and activation of mouse mammary tumor virus superantigen-reactive T cells in C57BL/10 mice.

Though C57BL/10 mice express the mouse mammary tumor virus superantigens (sag) encoded by Mtv-8 and Mtv-9, it has been thought that these sag do not bind to the MHC class II molecule H2-Ab and consequently do not affect the T cell repertoire. However, we show that cells bearing TCR Vbeta chains specific for Mtv-8 and -9 sag are chronically deleted in C57BL/10 mice. Thymocytes and peripheral T cells escaping deletion by Mtv sag display a small reduction in the level of cell surface CD4. T cells escaping thymic deletion respond variably to endogenous Mtv sag with some, but not all, reactive populations appearing overrepresented in the activated/memory subset. The data suggest that in normal mice fine modulation of coreceptor expression levels may be a common way by which thymocytes escape elimination, that systems utilizing potentially Mtv sag-reactive TCR on a C57BL background may be inappropriate for the measurement of the affinity of TCR/MHC/peptide interactions required in thymic selection, and that detection of the activity of human sag may be aided by analysis of CD4 levels and activation markers on T cells in conjunction with studies of the frequency of cells bearing specific TCRVbeta chains.     

Characterization of Subpopulations of T-Cell Receptor Intermediate (TCRint) T Cells

CD1-autoreactive T cells of two types have been demonstrated among T cells expressing the T-cell receptor (TCR) alphabeta at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an alpha-chain with an invariant Valpha14-J281 rearrangement, and a beta-chain using Vbeta8. 2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1-reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1. 1- TCRint subsets from MHC class II-deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)-4 and interferon (IFN)-gamma after activation. Importantly, no IL-10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T-helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi-invariant Valpha14/Vbeta8.2-type TCR was lower in NK1.1+ cells from MHC class II-negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Valpha14-J281 rearrangement was also demonstrated among NK1.1- TCRint cells.     

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR alpha/beta-mediated recognition.

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8(+) CTL has been shown to lyse certain natural killer (NK)-susceptible target cells via still undefined mechanism(s). These CD8(+) T cells, hereafter referred to as NK-CTL, are frequently composed of cells expressing one single TCR Vbeta expansion (different in different individuals), display a memory phenotype and express HLA class I-specific inhibitory NK receptors. Here we show that cell populations or clones of NK-CTL isolated from three healthy donors homogeneously expressed Vbeta16, Vbeta9 and Vbeta3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vbeta16(+) cells of donor 1 displayed identical TCR Vbeta and Valpha rearrangements, thus suggesting a substantial monoclonality of the NK-CTL subset analyzed. NK-CTL lysed a number of NK-susceptible tumor target cells with the exception of those characterized by beta2-microglobulin (beta2m) deficiency. However, the latter targets became susceptible to lysis upon beta2m transfection. Using monoclonal antibodies specific for the relevant TCR Vbeta or beta2m we provide evidence suggesting that target cell lysis by NK-CTL is mediated by the TCR itself upon recognition of beta2m-associated proteins. The cellular distribution of the potential beta2m-associated proteins in susceptible target cells suggested, as a likely candidate for TCR-mediated recognition, the non-classical HLA-E molecule. The use, as target cells, of the murine TAP2-deficient RMA-S cells, either untransfected or transfected with HLA-E, and loaded with an appropriate HLA-E-binding peptide, provided the direct demonstration that HLA-E represents a ligand recognized by the TCR expressed by NK-CTL. This is the first evidence that human TCR alpha/beta can recognize HLA-E molecules, thus revealing a novel type of TCR-mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.     

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.