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.     

Co-segregation of a gene encoding a deletion ligand for Tcrb-V3+ T cells with Mtv-3

A gene encoding the endogenous superantigen Mls^c, which deletes Tcrb-V3⁺ T cells in the NOD inbred mouse strain, was found to co-segregate with Mtv-3 on chromosome 11. This identifies a fourth gene encoding a deletion ligand for Tcrb-V3⁺ T cells and extends recently published observations in support of the hypothesis that a number of endogenous superantigens are the products of Mtv proviruses. Address correspondence and offprint requests to : K. Tomonari.     

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.     

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.     

Genetic resistance of CBA and a mice to transplanted lymphoid and hemopoietic cells of CBA.M523 mutants and their F1 hybrids

(CBA × M523)F₁, (A × M523)F₁ and M523 lymphocytes grafted into lethally irradiated CBA or A mice temporarily lose their capacity to respond to test antigens (SRBC, Vi-antigenS. typhi). Immunoresponsiveness of F₁ cells is affected to a lesser degree in lethally irradiated M523 mice. Depression of response is absent in the CBA F₁ combination, in the syngeneic combination and in CBA mice which have received transplanted cells from F₁ hybrids which do not share theM523 mutation. The number of hemopoietic (CBA × M523)F₁ colonies was also reduced in CBA mice. Resistance of CBA mice to lymphoid (CBA × M523)F₁ cells develops 18 days after birth. It can be reduced by additional recipient preirradiation or preinoculation with (CBA × M523)F₁ spleen cells. The abrogated resistance can be partially restored by CBA spleen cells. The activity of (CBA × M523)F₁ lymphocytes passaged through CBA spleen is restored in syngeneic F₁ secondary recipients but inhibited again in the CBA secondary recipients. These results are consistent with the suggestion that resistance of lethally irradiated CBA mice to hemopoietic and lymphoid (CBA × M523)F₁ cells is mediated by immunologically competent, radioresistant recipient cells rapidly reacting to transplantation antigens coded by the mutantH-2K ^ka allele. These cells temporarily suppress the functional activity of transplanted cells but do not eliminate them.     

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.     

Susceptibility to immunosuppression by ultraviolet B radiation in the mouse

Irradiation with ultraviolet B (UVB; 290–320 nm) initiates systemic immunosuppression of contact hypersensitivity (CHS). UV dose-responses for suppression of CHS to trinitrochlorobenzene were established in 18 strains of inbred mice. Three phenotypes with significantly different susceptibilities to UV suppression were identified. The phenotypes were: high (HI) susceptibility, 50% suppression with 0.7–2.3 kJ/m² UV (C57BL/6, C57BL/10, and C57L and NZB females); low (LO) susceptibility, 50% suppression with 9.6–12.3 kJ/m² UV (BALB/c, AKR, SJL and NZW), and intermediate (INT) susceptibility, 50% suppression with 4.7–6.9 kJ/m² UV (DBA/2, C57BR, C3H/HeJ, C3H/HeN, CBA/N and A/J). UV suppression was not correlated with skin pigmentation or with the magnitude of the CHS response in non-irradiated animals. Major histocompatibility complex (MHC) haplotype was not correlated with UV suppression in MHC congenic strains B10.D2/oSnJ, B10.D2/nSnJ, B10.BR/SgSnJ, and A.BY/SnJ. There were no sex differences in UV suppression in BALB/c, C57BL/6, or NZW animals. In the autoimmune NZB strain, however, male mice (LO) were seven times less sensitive to UV suppression than NZB female mice (HI). Both sexes of (NZB × NZW)F₁ and (NZW × NZB)F₁ mice were HI, supporting dominance of HI over LO. Thus there are genetic factors and interacting sex-limited factors determining susceptibility to UV suppression. These findings may be of relevance to UV-related diseases such as photosensitive lupus and skin cancer. Correspondence to: F. P. Noonan.     

Moused-amino-acid oxidase gene: Restriction fragment length polymorphism among mouse strains

Summary Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO⁺, ddY/DAO^–, ICR, NC, NZB and NZW) for the examination of the difference in the structure of thed-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingd-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO⁺ and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similard-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.     

Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Genetic control of T and B lymphocyte activation in nonobese diabetic mice.

Type 1 diabetes in nonobese diabetic (NOD) mice is characterized by the infiltration of T and B cells into pancreatic islets. T cells bearing the TCR Vbeta3 chain are disproportionately represented in the earliest stages of islet infiltration (insulitis) despite clonal deletion of most Vbeta3(+) immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen (SAg). In this report we showed that a high frequency of NOD Vbeta3(+) T cells that escape deletion are activated in vivo and that this phenotype is linked to the Mtv-3 locus. One potential mechanism of SAg presentation to peripheral T cells is by activated B cells. Consistent with this idea, we found that NOD mice harbor a significantly higher frequency of activated B cells than nondiabetes-prone strains. These activated NOD B cells expressed cell surface molecules consistent with APC function. At the molecular level, the IgH repertoire of activated B cells in NOD mice was equivalent to resting B cells, suggesting a polyclonal response in vivo. Genetic analysis of the activated B cell phenotype showed linkage to Idd1, the NOD MHC haplotype (H-2(g7)). Finally, Vbeta3(+) thymocyte deletion and peripheral T cell activation did not require B cells, suggesting that other APC populations are sufficient to generate both Mtv-3-linked phenotypes. These data provide insight into the genetic regulation of NOD autoreactive lymphocyte activation that may contribute to failure of peripheral tolerance and the pathogenesis of type I diabetes.     

A Cmv2 QTL on chromosome X affects MCMV resistance in New Zealand male mice

NK cell-mediated resistance to viruses is subject to genetic control in humans and mice. Here we used classical and quantitative genetic strategies to examine NK-mediated murine cytomegalovirus (MCMV) control in genealogically related New Zealand white (NZW) and black (NZB) mice. NZW mice display NK cell-dependent MCMV resistance while NZB NK cells fail to limit viral replication after infection. Unlike Ly49H⁺ NK resistance in C57BL/6 mice, NZW NK-mediated MCMV control was Ly49H-independent. Instead, MCMV resistance in NZW (Cmv2) involves multiple genetic factors. To establish the genetic basis of Cmv2 resistance, we further characterized a major chromosome X-linked resistance locus (DXMit216) responsible for innate MCMV control in NZW × NZB crosses. We found that the DXMit216 locus affects early MCMV control in New Zealand F₂ crosses and demonstrate that the NZB-derived DXMit216 allele enhances viral resistance in F₂ males. The evolutionary conservation of the DXMit216 region in mice and humans suggests that a Cmv2-related mechanism may affect human antiviral responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Xid-defective male (CBA/N x C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells

The reactivity of H-2^b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia.W39, a private specificity of I-A^b, on antigen-presenting cells. Cells of male (CBA/N x B6)F₁ mice carrying the mutation xid on the X chromosome lack Ia.W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N x B6)F₁ mice. We show here that spleen cells of male (CBA/N x B6)F₁ hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 x B10.BR)F₁ genotype, restricted to recognizing insulin in the context of F₁-unique I-A determinants. The epitope on the insulin molecule essential for stimulation was determined to depend on the glutamic acid residue in position 4 of the A chain of insulin. This contrasts with the H-2^b-restricted response of B6 mice to bovine insulin, which appears to be directed at the A chain loop determinant (amino acids A8 and A10). These data suggest that distinct I-A^b-encoded structures, the expression of which is regulated independently, may serve as components of restriction elements for H-2^b and (H-2^b x H-2^k)F₁ restricted T cells, which are specific for different epitopes of bovine insulin.     

T helper and T suppressor cells are restricted by the A and E molecules,respectively, in the F antigen system

The role of the A and E molecules as restriction elements was examined in the F antigen system. In the mouse the only responder haplotype known to date isk, and blocking studies with a monoclonal antibody show that in vitro T-cell proliferation is restricted by the A^k molecule. The (CBA × DBA/2) F₁ hybrid, which is a responder x nonresponder cross, is itself a nonresponder in terms of E-specific antibody production. Up to 10 days after priming, (CBA × DBA/2) F₁ T cells exhibited an E-specific proliferative response, but this diminished rapidly at later times. This diminution could be blocked with an E-specific monoclonal antibody, suggesting that suppression is restricted by the E molecule.     

Loss ofHindIII cleavage sites in thed-amino acid oxidase gene in some inbred strains of mice

Summary d-Amino acid oxidase cDNA was amplified by a polymerase chain reaction using RNA extracted from the mouse kidney. When digested withHindIII, the cDNAs of the BALB/c and ddY/DAO^– mice were cleaved into two fragments whereas the cDNA of the ddY/DAO⁺ mice was not. Sequencing revealed that nucleotide-471 of the cDNAs was G in the BALB/c and ddY/DAO^– mice whereas it was substituted for C in the ddY/DAO⁺ mice. This base substitution was the cause of the failure of the cleavage of the cDNA of the ddY/DAO⁺ mice.Examination of other strains of inbred mice showed thatd-amino-acid oxidase cDNAs of A/J, AKR, C57BL/6, CD-1, CF#1, ICR, DBA/2, NZB and NZW mice were cleaved withHindIII into two fragments whereas those of C3H/He, CBA/J and NC mice were not. Genomic DNAs extracted from the mice of these 15 strains were digested withHindIII and hybridized withd-amino-acid oxidase cDNA. A 18.2-kb fragment hybridized with the probe in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice whereas two fragments of 12 kb and 6.2 kb hybridized in the other mice. These results are consistent with those of the cDNAs, confirming the loss of theHindIII cleavage site in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice. The Southern hybridization revealed a loss of a differentHindIII cleavage site in the A/J, AKR, C57BL/6, DBA/2, ICR and NZB mice.The substitution at nucleotide-471 should cause a substitution of an amino acid residue. However, this substitution did not affect catalytic activity ofd-amino acid oxidase.     

B-Cell responses to male-specific antigen(s) in mice

It has been found that B-cell responses to male-specific antigen(s) can be clearly demonstrated by reversed plaque assays. Female mice injected with syngeneic male spleen cells showed significant increases (greater than 100 × in some strains) in the number of immunoglobulin-secreting cells in lymph nodes draining the injection site. There was a variation in B-cell responsiveness between strains and this correlated only partially with previously reported T-cell responsiveness to the H-Y antigen. C57BL (H-2 ^b ) mice were among the most responsive, while CBA (H-2 ^k ), (CBA × C57BL)F₁, and BALB/c (H-2 ^d ) were all much less responsive. These results apparently open up a new approach to the investigation of B-cell responses to male-specific antigen(s).     

The P2X<Subscript>7</Subscript>receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products

Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB × NZW)F₁ hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X₇, an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB × NZW)F₁ mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX ₇ gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB × NZW)F₁ lymphocytes to P2X₇ stimulation. NZB and NZW parental strains express the distinct P2X₇-L and P2X₇-P alleles of P2RX ₇, respectively, while lymphocytes from these and (NZB × NZW)F₁ mice differ markedly in their responses to P2X₇ receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X₇. We show that current mapping suggests that murine and human P2RX ₇ receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X₇ is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8+T Effector Memory Subset

CD26 is a T cell activation marker consisting in a type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. It has been described that DPPIV inhibition delays the onset of type 1 diabetes and reverses the disease in non-obese diabetic (NOD) mice. The aim of the present study was to assess the effect of MK626, a DPPIV inhibitor, in type 1 diabetes incidence and in T lymphocyte subsets at central and peripheral compartments. Pre-diabetic NOD mice were treated with MK626. Diabetes incidence, insulitis score, and phenotyping of T lymphocytes in the thymus, spleen and pancreatic lymph nodes were determined after 4 and 6 weeks of treatment, as well as alterations in the expression of genes encoding β-cell autoantigens in the islets. The effect of MK626 was also assessed in two in vitro assays to determine proliferative and immunosuppressive effects. Results show that MK626 treatment reduces type 1 diabetes incidence and after 6 weeks of treatment reduces insulitis. No differences were observed in the percentage of T lymphocyte subsets from central and peripheral compartments between treated and control mice. MK626 increased the expression of CD26 in CD8⁺ T effector memory (T_EM) from spleen and pancreatic lymph nodes and in CD8⁺ T cells from islet infiltration. CD8⁺T_EM cells showed an increased proliferation rate and cytokine secretion in the presence of MK626. Moreover, the combination of CD8⁺ T_EM cells and MK626 induces an immunosuppressive response. In conclusion, treatment with the DPPIV inhibitor MK626 prevents experimental type 1 diabetes in association to increase expression of CD26 in the CD8⁺ T_EM lymphocyte subset. In vitro assays suggest an immunoregulatory role of CD8⁺ T_EM cells that may be involved in the protection against autoimmunity to β pancreatic islets associated to DPPIV inhibitor treatment.     

Influence of the H-2 u haplotype on immune function in F1 hybrid mice

Recently we reported that antigen-primed T cells from (H-2 ^u × H-2 ^s)F₁ and (H-2 ^u × H-2 ^q)F₁ mice responded poorly in vitro to antigen in the context of antigen-presenting cells of the non-H-2 ^u parent. It was suggested that this effect might be due to unbalanced expression of parental antigens in the F₁ hybrid with the result that the non-H-2^u A antigens were greatly reduced or absent in these mice. If this were the case, non-H-2^u Ia-A cells might be expected to stimulate a mixed lymphocyte reaction (MLR) when cultured with Fl responder cells. When tested, (SJL × PL)F₁ responder cells reacted strongly to SJL stimulator cells. There was no significant reaction to PL stimulator cells. The use of major histocompatibility complex (MHC) congenic mice showed the stimulatory antigens to be associated with the MHC. The MLR could be blocked significantly by monoclonal A-specific antibody of the appropriate specificity. When a monoclonal antibody reactivewith a private epitope associated with A^s was used to probe for the presence of A^s on the surface of (SJL × PL)F₁ spleen cells, no antigen could be detected, indicating loss or alteration of this antigen. These findings suggest that an alteration of the expression of the parental A^s molecule may be responsible for this phenomenon.Abbreviations used in this paper APC antigen-presenting cells - BSS balanced salt solution - CTL cytotoxic T lymphocyte - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - T₂ suppressor T lymphocyte     

A new thymocyte membrane differentiation antigen for mouse and rat

A new thymocyte membrane-brain antigenic system is defined for the mouse and rat. Monoclonal antibody NIM-M129 recognizes a membrane antigen that has a similar size to Thy-1 (20–23 kd) but entirely different tissue distribution. It is absent from cortisone-resistant thymocytes, murine peripheral T cells, T-cell blasts, and T-cell lymphomas, or rat bone marrow. However, the determinant exhibits a quantitative difference in expression on thymocytes of Thy-1 ^a (low) and Thy-1 ^b (high) mice, with intermediate values for their F₁ progeny. Backcross and F₂ segregation tests between CBA and CBA-Thy-1^a congenic lines established firm genetic linkage between Thy-1 and the level of epitope expression on the thymocyte cell surface.