Positive and negative selection of Tcrb-V6+ T cells

Tcrb-V6⁺ T cells are deleted by an endogenous superantigen probably encoded by a mouse mammary tumor provirus (Mtv), Mtv-7, in association with major histocompatibility complex (MHC) class II molecules. In contrast, Tcrb-V6⁺CD4⁺ T cells are positively selected by MHC class II E molecules in Mtv-7 ^– mice. We have examined the levels of Tcrb-V6⁺CD4⁺ and Tcrb-V6⁺CD8⁺ T cells from six combinations of backcross mice. In this paper we show that: 1) Tcrb-V6⁺CD8⁺ T cells can be positively selected by MHC class I molecules; 2) MHC class II A molecules can also influence the levels of Tcrb-V6⁺CD4⁺ T cells; 3) Mtv-7 ^– NZW mice have a new Mtv, Mtv-44, which co-segregates with a gene encoding the partial deletion ligand for Tcrb-V6⁺ T cells; 4) the remaining Tcrb-V6⁺ T cells from mice with partial deletion of these T cells appear not to be anergized in the periphery. Address correspondence and offprint requests to: K. Tomonari.     

Tcrb-V3+ T-cell deletion and a mouse mammary tumor provirus,Mtv-27

Genes encoding superantigens which delete Tcrb-V3⁺ T cells co-segregate with mouse mammary tumor proviruses (Mtv), Mtv-1, Mtv-3, Mtv-6, Mtv-13, and Mtv-44. We have examined percentages of Tcrb-V3⁺ T cells and Mtv integrations in [(B10 × NZB)F₁ × B10.BR] mice, and show that Mtv-27 as well as Mtv-3 from NZB mice co-segregate with genes encoding deletion ligands for Tcrb-V3⁺ T cells without recombination. Adress correspondence and offprint requests to: K. Tomari.     

Tcrb-V3+3T-cell deletion and a new mouse mammary tumor provirus,Mtv-44

Genes encoding endogenous superantigens causing Tcrb-V3⁺ T-cell deletion co-segregate with mouse mammary tumor proviruses (Mtv),Mtv-3, Mtv-6, andMtv-13. In additionMtv-1 has been implicated in deletion of these T cells. We have examined levels of Tcrb-V3⁺ T cells andMtv integrations in the following off-spring and their parental strains, [(CBA-T6 × NZW)F₁ × CBA], [(CBA × C3H/He)F₁ × CBA], and [(B10.S (9R) × NOD]F₁ mice. We show that a newMtv (Mtv-44) from NZW mice andMtv-1 from C3H/He mice cosegregate with genes encoding ligands for partial deletion of Tcrb-V3^s+ T cells and that some NOD mice have an additionalMtv (Mtv-45) which is closely linked toMtv-3. Address correspondence and offprint requests to: K. Tomonari.     

Positive selection of Tcrb-V10b+ T cells

The Tcrb-V10b⁺ T cell population has been examined with a newly established antibody, KT10b, specific for Tcrb-V10b but not Tcrb-V10a. H-2E⁺ mice have higher levels of Tcrb-V10b⁺ T cells (4.3%–11.%) than H-2E^– mice (2.2%–4.9%). This difference appears to be determined by levels of Tcrb-V10b⁺ T cells in the CD4 population. F₁ mice between H-2E⁺ and H-2E^– mice dominantly express higher levels of Tcrb-V10b⁺ T cells. [NOD (E–) x (NOD x A (E⁺))F₁] backcross mice show positive selection of Tcrb-V10b⁺ CD4⁺ T cells by H-2E. On the other hand other backcross analyses reveal positive selection of Tcrb-V10b⁺ CD8⁺ T cells by certain major histocompatibility class I molecules. Involvement of non-H-2 antigens in these positive selections remain to be determined. Address correspondence and offprint requests to: K. Tomonari.     

The genetic basis of negative selection of Tcrb-Vll+ T cells

Non-H-2 genes responsible for negative selection of Tcrb-V 11⁺ T cells were examined using backcross mice of various strains with C58, which does not delete Tcrb-V 11⁺ T cells. Two independently segregating genes were found: one leading to partial deletion was closely linked toLy-2/Ly-3 on chromosome 6, and the second giving virtually complete deletion has not yet been mapped. The A strain had only the former, whereas BALB/c, BALBK, B10.BR, CBA-T6, C3H/He, and DBA/2 expressed both of these genes. Although a gene(s) of the NIH strain led only to partial deletion, the chromosomal localization of the gene(s) has not yet been determined: no informative polymorphic molecules are expressed from genes on chromosome 6 of this strain.     

Neuritogenic Lewis rat T cells use Tcrb chains that include a new Tcrb-V8 family member

The P2 protein obtained from Schwann cells induces a population of T cells which, upon adoptive transfer, causes the disease experimental allergic neuritis (EAN), an animal model for Guillain-Barre syndrome. In this report, a truncated peptide, FR22, derived from a previously reported neuritogenic T-cell determinant, was used to generate from Lewis rats T cells that were shown to cause EAN. Since our previous studies showed that Tcrb-V8 was used by a majority of T-cell hybridomas specific for the neuritogenic peptide P26, which contains the FR22 sequence, we sequenced the Tcrb-V8 ⁺ mRNA from FR22-specific T-cell lines, and compared the sequences obtained with those obtained from similarly generated myelin basic protein (MBP) 68–88-specific Lewis rat T-cell lines. We found that in the EAN lines, several members of the Tcrb-V8 family were used, including a new family member, Tcrb-V8E. This was more diverse than the MBP-68–88-specific response in which only a single Tcrb-V8 family member was used. Also, in the EAN lines, the beta chain sequences did not show the same conserved junctional regions seen in the MBP lines. Thus, T-cell receptor beta chain usage in the response to this dominant neuritogenic peptide appears to be less restricted than the response to the dominant encephalitogenic determinant of MBP both in V region usage and in CDR3 usage.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databse and have been assigned the accession numbers UO6100 (Tcrb-V8A), UO6101 (Tcrb-V8B), UO6102 (Tcrb-V8C), UO6103 (Tcrb-V8D), and UO6104 (Tcrb-V8E)     

FVB/N (H2 q ) mouse is resistant to arthritis induction and exhibits a genomic deletion of T-cell receptor V beta gene segments

 Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis (CIA) in mice is an autoimmune disease model of rheumatoid arthritis. Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). CD4⁺ T cells that express the T-cell receptor (TCR) Tcra-V11.1 and/or Tcrb-V8.2 play a key role in the pathogenesis of arthritis in the DBA/1 mouse (H2 ^q ). We identified an inbred mouse strain, FVB/NJ (H2 ^q ), that is resistant to arthritis induction and exhibits a genomic deletion of certain Tcrb-V gene segments. We report a novel polymerase chain reaction-based method for the rapid identification of new mouse strains that exhibit germline Tcrb-V gene deletions. We mapped for the first time both the 5′ and 3′ breakpoints of the Tcrb-V deletion in the FVB/NJ, SWR, SJL, C57L, and C57BR strains to within 1.1 kilobases. Since there is an association between a particular Tcra-V allele (Tcra-V11.1 ^d ) and arthritis susceptibility in H2 ^q mouse strains, we examined the allelic polymorphisms of the Tcra-V11 gene subfamily members between the arthritis-susceptible DBA/1 mouse and the arthritis-resistant FVB/NJ mouse strain. The amino acid sequences of the Tcra-V11.1 alleles differ at two positions (codons 18 and 68). Therefore, the resistance of FVB/NJ mouse to arthritis induction may be due in part to Tcra-V11.1 coding sequence polymorphism and Tcrb-V8.2 gene segment deletion, as we have recently demonstrated in the case of SWR mouse strain. Received: 12 January 1999 / Revised: 17 March 1999     

Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L,and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c⁺ DCs and CD4⁺/CD8a⁺ T cells into tumors. Depletion of CD4⁺ or CD8a⁺ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.     

Polymorphonuclear neutrophils enhance suppressive activities of anti-CD3-induced CD4 suppressor T cells

We investigated the effects of polymorphonuclear neutrophils (PMN) on the suppressive activities of CD4⁺ suppressor T cells induced by immobilized mAb to the CD3 molecular complex in order to explore the role of PMN in the regulation of humoral immune responses. CD4⁺ T cells that had been treated with mitomycin C induced the IgM production from highly purified B cells in cultures stimulated with immobilized anti-CD3. Addition of CD4⁺ T cells that had not been treated with mitomycin C (control T4 cells) suppressed the IgM production induced by immobilized anti-CD3-stimulated T4 mito. PMN enhanced the degree of suppression of the IgM production by anti-CD3-stimulated control T4 cells. The capacity of PMN to enhance the suppressive activity of anti-CD3-stimulated control T4 cells was restored when PMN were fixed with paraformaldehyde (PFA), suggesting that direct interactions between PMN and CD4⁺ T cells, but not soluble factors secreted by PMN, were involved in the enhancement of suppression. Fresh PMN as well as PFA-fixed PMN enhanced the endogenous IL-2 production by immobilized anti-CD3-stimulated CD4⁺ T cells. Moreover, neither fresh PMN nor PFA-fixed PMN significantly augmented the suppressive activity of anti-CD3-stimulated control T4 cells in the presence of exogenous IL-2. These results indicate that PMN enhance the suppressive activity of anti-CD3-stimulated control T4 cells through direct interactions between PMN and CD4⁺ T cells. The enhancement of the suppressive activity of CD4⁺ suppressor T cells by PMN is accounted for by the enhancement of the endogenous IL-2 production by anti-CD3-stimulated CD4⁺ T cells. Thus, the data demonstrate that PMN influence the magnitude of humoral immune responses by regulating the production of IL-2 through direct interactions with T cells.     

Genetic analysis of the Mls system. Formal Mls typing of the commonly used inbred strains

In order to elucidate the biological role of minor lymphocyte stimulating (Mls) gene products, we have been investigating the fundamental immunogenetic characteristics of the Mls system. In this report, describe the distribution of stimulatory Mls products, Mls^a and Mls^c, in a panel of laboratory inbred strains based on the response pattern of H-2-compatible naive T-cell populations as well as monospecific Mls^a- or Mls^c-reactive T-cell clones. In addition, the expression of four different T-cell receptor (Tcr) b-V segment Tcrb-V3, –V6, –V8.1, and –V9, which were recently reported to be associated with T-cell recognition of Mls gene products in these strains, was examined. The results indicate that the majority of commonly used laboratory strains including those originally typed as Mls ^aare also expressing Mls^c determinants and that very few independent inbred strains are non-Mls ^c. Moreover, the pattern of Tcrb-V expression in spleen as well as in thymus suggests that the association between Mls expression and clonal deletion of self Mls-reactive T cells appears to be the general rule in inbred strains. Based on these results, implications for the nondetectable Mls-like gene products in other species besides the mouse are discussed.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M67511 for V6.1A and M67512 for V6.1B.     

Participation of NK1.1+ T Cells in the Rejection of lpr αβT Cells When Bone Marrow Cells of Ipr Mice Are Transplanted into B6 Mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3^? IL-2Rβ⁺ NK cells and IL-2Rβ CD3^int cells (i.e., CD3^int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1⁺ and NK1.1^?, among the IL-2Rβ⁺ CD3^int cells. However, the NK1.1⁺ subset (i.e., NK1.1⁺ T cells) was much more radioresistant, and the majority of CD3^int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM₁ antibody to eliminate NK cells alone or both NK cells and NK1.1⁺ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1⁺ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4^?8^? cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.     

Sequential Involvement of NK Cells and CD8+ T Cells in Granuloma Formation of Rhodococcus aurantiacus-Infected Mice

We investigated the effect of in vivo administration of antibodies against T-cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN-γ) production and granuloma formation in Rhodococcus aurantiacus-infected mice. High titers of endogenous IFN-γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN-γ production was reduced by in vivo administration of anti-NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1⁺ cells. Endogenous IFN-γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN-γ at 1 and 3 weeks was reduced by in vivo administration of anti-CD8 MAb, but not by anti-CD4 MAb or anti-NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti-IFN-γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8⁺ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN-γ is attributable to NK cells in the early phase of the infection and CD8⁺ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8⁺ T cells through the secretion of endogenous IFN-γ.     

Influence of phage T3 and T7 gene functions on a type III(EcoP1) DNA restriction-modification system in vivo

Summary The ocr ⁺ gene function (gp 0.3) of bacteriophages T3 and T7 not only counteracts type I (EcoB, EcoK) but also type III restriction endonucleases (EcoP1). Despite the presence of recognition sites, phage DNA as well as simultaneously introduced plasmid DNA are protected by ocr ⁺ expression against both the endonucleolytic and the methylating activities of the EcoP1 enzyme. Nevertheless, the EcoP1 protein causes the exclusion of T3 and T7 in P1-lysogenic cells, apparently by exerting a repressor-like effect on phage gene expression. T3 which induces an S-adenosylmethionine hydrolase is less susceptible to the repressor effect of the SAM-stimulated EcoP1 enzyme. The abundance of EcoP1 recognition sites in the T7 genome is explained by their near identity with the T7 DNA primase recognition site.Abbreviations d.p.m. decompositions per min - EcoB, EcoK, EcoP1, EcoP15, EcoRII, EcoR124, HinfIII restriction endonucleases coded by Escherichia coli strains B or K, E. coli plasmids P1, P15, RII or R124, and Haemophilus influenzae Rf 232, resp. - e.o.p. efficiency of plating - gp gene product (in the sense of protein) - m.o.i. multiplicity of infection (phage/cell) - ocr ⁺ gene function which overcomes classical restriction - p.f.u. plaque-forming units - SAM S-adenosylmethionine - sam ⁺ gene function with S-adenosylmethionine-cleaving enzyme (SAMase) activity - UV ultraviolet light Dedicated to Professor Konstantin Spies on the occasion of his sixtieth birthday     

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

Antigen-specific CD4⁺ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4⁺ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4⁺ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4⁺ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4⁺ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4⁺ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4⁺ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.     

Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4⁺CD8⁺ double positive stage, were both upregulated in CD4⁺ or CD8⁺ single positive thymocytes, and both continued to be co-expressed in na?ve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8⁺ T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.