Skewed T cell receptor V{alpha} repertoire among superantigen reactive murine T cells

Reactivity of murine T cells with viral or bacterial superantigensis clearly correlated with the expression of TCR V_ßdomains. Thus, T cells responding to the minor lymphocyte stimulatorylocus (Mls-1^a) or staphylococcal enterotoxin B (SEB) expresspredominantly TCR V_ß6 or V_ß8.2 respectively.We have investigated the involvement of the other major variableelement of the TCR, the V domain, in these superantigen responses.Using a panel of anti-TCR V mAbs, It is demonstrated that theTCR V repertoire among superantigen stimulated V_ß6⁺or V_ß8.2⁺ blasts (responding to Mls-1^a or SEB respectivelyin vitro) is altered in comparison with anti-CD3 stimulatedcells expressing the same V domains. Furthermore, the TCR Vrepertoire is strongly skewed in TCR V_ß8.2 transgenicmice that have undergone extensive peripheral clonal deletionafter SEB injection. These data imply that the V domain influencessuperantigen recognition by sthe TCR.     

Lambda5 is required for rearrangement of the Ig kappa light chain gene in pro-B cell lines.

Lambda5 associates with V(pre-B) to form the surrogate light (L) chain. The phenotype of lambda5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of lambda5 to lambda5-deficient pro-B cell lines which were established from bone marrow cells of lambda5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in lambda5 knockout mice. Restoration of lambda5 protein by retroviral-mediated gene transfer into established lambda5-deficient pro-B cell lines induced rearrangement of the Ig kappa L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored lambda5 in the cell line is coupled with V(pre-B) to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both lambda5 and V(pre-B), stimulates efficient rearrangement of the kappa L chain genes.     

Endogenous immunoglobulin expression in mu transgenic mice

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.     

Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice

The surrogate light chain (SL) is composed of two polypeptides, Vpre-B and lambda5. In large pre-BII cells the SL chain associates with Ig mu heavy chain (muH) to form the pre-B cell receptor (pre-BCR). In mice there are two Vpre-B genes which are 98% identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with lambda5. However, it is not known whether both gene products serve the same function in vivo. Here we have established mice that lack the Vpre-B1 gene (VpreB1(-/-)), but still express the Vpre-B2 gene, both as RNA and protein. In Vpre-B1(-/-) mice, the bone marrow cellularity and the percentage of B220+ cells is normal. However, among the B220+ cells, the percentage of pre-BI cells is increased, and the percentage of pre-BII and immature B cells is slightly decreased, suggesting that the lack of Vpre-B1 causes a partial block at the transition from pre-BI to pre-BII cells, i.e. into the pre-BCR stage. The number of cells that produce a functional pre-BCR is thus lower, but the cells that reach this stage are normal as they can be expanded by proliferation and then differentiate into more mature cells. The spleens of Vpre-B1 homozygous mutant mice show normal numbers of B and T lymphocytes. Moreover, the Ig loci are allelicly excluded and the homozygous mutant mice respond with normal levels of antigen-specific antibodies to T-dependent antigens. These results demonstrate that VpreB2 alone is capable of supporting B lymphocyte development in the bone marrow and can give rise to immuno-competent cells in the periphery.     

The level of expression of mu heavy chain modifies the composition of peripheral B cell subpopulations

The B cell receptor (BCR) has a decisive role in transducing signals required for the development of B cells and their survival in the periphery. However, the processes that initiate these signals remain unclear and concepts of constitutive and ligand-dependent signaling have been proposed. Using a mu-transgenic mouse model, we have analyzed the impact of high surface IgM expression on the composition of the splenic B cell population. kappa-deficient mice homozygous for the H3-mu transgene have B cells with a higher BCR surface density than H3 heterozygous mice. This higher BCR expression is associated with an increase in the percentage and the total number of splenic B cells. In addition, an important proportion of CD23(-)CD21(+) marginal zone (MZ) B cells can be observed in H3 homozygous mice. However, these modifications operate in the absence of impairment of the positive selection process of the H3-mu/lambda1 combination over the H3-mu/lambda2 + 3 ones. These results suggest that (i) a constitutive BCR signaling directly correlated with BCR surface density is responsible for the efficient B cell colonization of the periphery with an accumulation of B cells in the MZ and (ii) a ligand-dependent BCR signal is responsible for the clonotype composition of the mature B cell repertoire.     

The skewed heavy-chain repertoire in peritoneal B-1 cells is predetermined by the selection via pre-B cell receptor during B cell ontogeny in the fetal liver

As many as 5–15% of B-1 cells in the peritoneal cavityof adult mice produce antibodies reactive to phosphatidylcholine(PtC) and the vast majority of them express B cell receptors(BCRs) composed of V_H11-µH chains utilizing the J_H1 segmentand V9-L chains. This extremely skewed repertoire of PtC-reactiveB-1 cells is traditionally attributed to the expansion of particularclones in response to self or exogenous antigens. Here, we showthat the strong bias toward the J_H1 usage among V_H11-µHchains is already established prior to the BCR assembly, namelyat the transition from the large to the small pre-B cell stageduring B cell ontogeny in the fetal liver. Among V_H11-µHclones isolated from large pre-B cells where the J_H1 skewingwas not established yet, the J_H1 users showed the highest abilityto form pre-B cell receptor (pre-BCR) and to induce cellularproliferation and differentiation when expressed in fetal liverpro-B cells. Thus, the J_H1 users were positively selected andamplified at the pre-BCR checkpoint. When co-expressed withV9-L chains to form BCR, the J_H1 users almost exclusively conferredthe PtC reactivity on BCR even though other J_H users could alsoform BCR on the cell surface. Therefore, the pre-BCR-mediatedpositive selection of the J_H1 users among V_H11-µH chainsappears to be beneficial to the efficient generation of ‘innate-type’PtC-reactive B cells during the fetal B cell development, evenbefore the self-renewal or the antigen-driven clonal expansionof B-1 cells takes place in the peritoneal cavity.     

Expression of a fetal gamma delta T-cell receptor in adult mice triggers a non-MHC-linked form of selective depletion.

Day 14 fetal thymocytes and adult dendritic epidermal T cells (dEC) of all mouse strains express a characteristic non-polymorphic gamma delta T-cell receptor which is rarely found in the adult thymus or lymph nodes. We have made transgenic mice expressing this particular set of receptors on T cells in C3H and C57BL/6 mice. In adult mice of the latter strain, a dramatic depletion of transgene expressing T cells occurs and this effect is primarily mediated by thymic radiosensitive cells. The depletion is genetically dominant but not MHC-linked with major factor(s) mapping to chromosome 18. Taken together, our results show that strain-specific developmental changes in the thymic environment may play a role in shaping the gamma delta TCR repertoire.     

Monoclonal antibody against murine T cell receptor V alpha 14 cross-reacts with human CD3 epsilon and detects disulfide-linked dimeric form.

A monoclonal antibody against V14+ alpha-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/IgG3 protein containing murine TCR V alpha 14J alpha 281 in place of the VHDHJH of an IgG3. lambda 1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 alpha-chain. The anti-mouse V alpha 14 antibody precipitated TCR alpha beta molecules from Triton X-100-solubilized extracts of 125I-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3 epsilon molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3 epsilon, which is a novel family component of the CD3 complex and is associated closely with the CD3 zeta-zeta homodimer as well as TCR alpha beta or TCR gamma delta.     

Identification of CD19(-)B220(+)c-Kit(+)Flt3/Flk-2(+)cells as early B lymphoid precursors before pre-B-I cells in juvenile mouse bone marrow

The combined analysis of the expression of receptor tyrosine kinases c-Kit and Flt3/Flk-2 and of the human CD25 gene expressed as a transgene under the regulation of the mouse lambda5 promoter in the bone marrow of 1-week-old mice allows us to identify three stages of B lymphocyte development before the CD19(+)c-Kit(+) pre-B-I cells. Single-cell PCR analysis of the rearrangement status of the Ig heavy chain alleles allows us to order these early stages of B cell development as follows: (i) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(-), (ii) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(+) and (iii) B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(lo)lambda5(+) before B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(-)lambda5(+) pre-B-I cells. All these progenitors are clonable on stromal cells in the presence of IL-7 and can differentiate to CD19(+)c-Kit(-) B-lineage cells. A combination of stem cell factor, Flt3 ligand and IL-7 was also able to support the proliferation and differentiation of the progenitors in a suspension culture. Furthermore, the analyses indicate that the onset of D(H)J(H) rearrangements precedes the expression of the lambda5 gene. These progenitor populations were characteristic of juvenile mice and could not be detected in the bone marrow of adult mice. Hence the expression pattern, and probably the function, of the receptor tyrosine kinases in early B cell differentiation appears to be different in juvenile and adult mice.     

Maternal serum levels of interferon-gamma and interleukin-2 soluble receptor-alpha predict the outcome of early IVF pregnancies

BACKGROUND: Elevated maternal serum levels of interleukin-2 soluble receptor-alpha (IL-2 sRalpha), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been associated with pregnancy loss. The aim of our study was to evaluate the predictive value of these cytokines in the outcome of early IVF pregnancies. METHODS: One hundred and fifty-nine consecutive IVF patients who were subsequently diagnosed to have a biochemical pregnancy (n = 23), a first-trimester miscarriage (n = 19) or a normal term delivery (n = 117) were included in this study. Serum was collected from the initial pregnancy test, 11 days after a day 3 embryo transfer, and all samples were analysed for IL-2 sRalpha, TNF-alpha and IFN-gamma by commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IL-2 sRalpha levels were significantly higher in patients with an early pregnancy loss compared with patients with a normal term delivery (849.5 +/- 69.6 versus 693.5 +/- 31.2 pg/ml, P = 0.02), and a cut-off point of IL-2 sRalpha >1000 pg/ml predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sRalpha >or=1000 versus IL-2 sRalpha <1000 pg/ml; P = 0.02). IFN-gamma-positive patients had twice the risk for poor IVF pregnancy outcome compared with IFN-gamma-negative subjects (40.8 versus 20.0%, respectively; P < 0.02), including a significantly lower implantation rate (37.6 +/- 0.05 versus 50.0 +/- 0.03%, respectively; P = 0.02). There was no difference in pregnancy outcome based upon serum levels, or the ability to detect the presence of TNF-alpha. No differences in levels of these cytokines were found based on the aetiology of the patients' infertility. CONCLUSIONS: Elevated maternal serum levels of IL-2 sRalpha and IFN-gamma as early as 11 days after embryo transfer are associated with poor IVF pregnancy outcome.     

Monoclonal anti-{lambda}5 antibody FS1 identifies a 130 kDa protein associated with {lambda}5 and Vpre-B on the surface of early pre-B cell lines

mAbs specific for mouse 5 protein were prepared by fusion ofspleen cells from a hamster Immunized with recomblnant 5 proteinsynthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14.Here we report the characteristics of the antibodies producedby the FS1 hybridoma. FS1 antibody stains a variety of mousepre-B cell lines but not B cell lines or T cell lines. The stainingof the pre-B cell lines Awl and C-7 by phycoerythrin (PE)-con]ugatedFS1 (FS1 - PE) can be blocked by prelncubatlon of these cellswith unconjugated FS1 antibody or with affinity purified polyclonal5 specific Ig but not with normal hamster or mouse IgG or withaffinity purified polyclonal antl-Mb-1 Ig. From these experimentswe concluded that FS1 specifically recognizes 5 protein. Weused FS1 -PE to probe for surface (s) 5⁺ cells in normal BALB/cmouse bone marrow. Such cells were undetectable when total bonemarrow or FACS sorted subpopulatlons were analyzed. However,when B220^plus;, CD43⁺, s5⁺ bone marrow cells were cultured for4 days on the stromal cell line FLST2 in the presence of IL-7,s5 expression became apparent. Further expansion of these cellsin IL7 alone augmented the s5 expression to readily detectablelevels. This modulation may indicate that s5 expression on normalbone marrow cells in vivo is transient and that at any givenmoment only a small fraction of bone marrow cells expresseslow levels of 5 protein on the surface. Alternatively the bindingof our FS1 mAb to the s5 molecules on normal bone marrow cellsmay be blocked by other proteins binding to the 8X5 complexin vivo and directly ex vivo. Previous analysis of surface 5associated proteins on early mouse pre-B cell lines using apolyclonal anti-5 rabbit antlserum had suggested that s5 proteinwas associated with a high molecular weight protein. Analysisof 5 and Its associated proteins on early pre-B cell lines usingour FS1 mAb confirmed our previous finding and showed that theearly 5 receptor contains at least three proteins: 5, V_pre-B,and an as yet uncharacteiized protein with a molecular weightof 130,000 designated p130.     

High frequency of circulating gamma delta T cells with dominance of the v(delta)1 subset in a healthy population

TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V(delta)1(+) cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood gamma delta T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V(delta)1(+) cells, which is consequently the dominant subset. No age dependency of V(delta)1 frequencies was identified and the V(delta)1(+) cells in the African donors did not show preferential V(gamma) chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V(delta)1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8(+), CD38(+) and CD45RA(hi)CD45RO(-) cells within the V(delta)1(+) subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V(delta)1(+) cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V(delta)1(+) cells in the African study population. Our study shows that high frequencies of TCR gamma delta(+) cells with dominance of the V(delta)1(+) subset can occur at the population level in healthy people, raising questions about the physiological role of V(delta)1(+) T cells in the function and regulation of the immune system.     

Induction of human hsp60 expression in monocytic cell lines.

Both bacterial and mammalian heat shock proteins (HSP) are recognized by some T cells, and hsp60 recognition has been implicated in rheumatoid arthritis. We have developed a model to study the induction of hsp60 in human monocytic cell lines. An anti-mycobacterial hsp65 mAb (ML30), cross-reacting with human hsp60 was used to screen 21 human tumor cell lines in Western blot analysis. All T cell and B cell lymphomas constitutively expressed hsp60 protein at moderate to high levels, while little or no hsp60 protein was detected in two monocytic leukemia lines. Moderate to high levels of hsp60 mRNA and protein could be induced in the THP-I monocytic leukemia cell line by heat shock, retinoic acid, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha treatment, the highest levels obtained with a combination of IFN-gamma/TNF-alpha. This was also seen using two rabbit anti-hsp60 antisera directed against the N-terminal or C-terminal part of the human hsp60 protein. The determinants detected by the ML30 mAb or the two rabbit anti-hsp60 antisera were not cell surface expressed, as measured with immunofluorescence (FACS) analysis on control cultured or cytokine treated cell lines. This could be a useful model for studies related to the induction of hsp60 in human cells.     

A role for BP-3/BST-1 antigen in early T cell development

In the mouse thymus, pre-T cells are defined by their CD3^–CD4^–CD8^–triple-negative, CD44^10/– CD25⁺ phenotype. We made a ratmAb IF-7, that, among all Tcell subsets analyzed, reacted exclusivelywith pre-T cells. Molecular cloning revealed that the antigenrecognized by IF-7 was identical to BP-3/BST-1, a glycosyl-phosphatidylinositol-linked,CD38-related molecule previously described asa possible co-activationmolecule of pre-B cells. We found that IF-7 cross-linking enhancesthe proliferative response ofsorted pre-T cells to anti-CD3stimulation. In addition, IF-7 enhances and accelerates thedevelopment of fetal thymic organ culture (FTOC), although the lineage is unaffected by the treatment. In addition, sortedIF-7⁺ pre-T cells give preferentially rise to ß TCR⁺thymocytes in FTOC. Our observations strongly suggest that BP-3/BST-1is implicated in both early B and T cell growth and development,and is an early marker for the ß lineage.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Transient hypogonadotrophic hypogonadism in males with acute toxoplasmosis: suppressive effect of interleukin-1 beta on the secretion of GnRH

BACKGROUND: In September 2002, an outbreak of toxoplasmosis was noted in a male boarding high school on the Aegean coast of Turkey. We have focused our efforts to investigate the sex hormones in this population. METHODS: Blood samples were collected from 40 male patients, 17-18 years old, who also had positive titres of antibody to Toxoplasma gondii. Serum FSH, LH, free testosterone (FT), total testosterone (TT), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta) and macrophage-inflammatory protein-1alpha (MIP-1alpha) concentrations were measured in all patients and 20 control subjects. Initially, the patients were divided on the basis of the levels of sex hormones into the following groups: patients who had normal sex hormone levels (n = 31) as group A and patients with low sex hormone levels (n = 9) as group B. RESULTS: IL-1beta levels were found to be higher in group B patients than group A. The levels of IL-1beta correlated significantly in a negative manner with FSH, LH, FT and TT in all patients with acute toxoplasmosis (n = 40). CONCLUSIONS: Acute toxoplasma infection may cause temporary hypogonadotrophic gonadal insufficiency regardless of the course of the disease.     

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.     

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V{alpha}1 gene

Previous studies of the TCR chain gene have located promoterelements 5' to the start of the various V genes. The only fullycharacterized enhancer for the entire chain gene (V, J andC genes) has been located {small tilde}3 kb from the 3' endof C. We now report the existence of additional regulatory elementslocated in the introns of several murine V genes (V1, V3 andVB6.2.16). In the case of V1, this element appears to be a promoterwith bidirectional activity that is not T cell specific. Interestingly,upstream of the promoter in the antisense strand, an open readingframe has been found that codes for a small molecular weightprotein ({small tilde}60 amino acids) that contains a prollne-richregion and a tyrosine-isoleucine motif that has homology toIgß (the B29 gene product). A rabbit antiserum madeagainst this sequence has confirmed its existence by Westernblot and immunoprecipitation. Thus this V1 intronic promoterhas the potential not only to induce the formation of a truncatedV1 gene product, but also regulates the expression of a smallmolecular weight protein that may be involved in lymphocyteantigen receptor signaling. The activity of this promoter isregulated by changes in intracellular calcium. In the presenceof ionomycin the promoter is down-regulated in the sense directionand its activity is enhanced in the antisense direction. Thisresult suggests that this promoter can act differentially toproduce two very different gene products. The bidirectionalV1 promoter appears to be the first in the Ig superfamily toinduce potentially functional proteins in both directions.