C57L Mice Research Articles

B cell lymphomas of C57L/J mice; the role of natural killer cells and T helper cells in lymphoma development and growth

The Hodgkin’s-like Type B neoplasms which arise spontaneously in aging C57L mice (25% incidence at 21 months of age) were first reported over 40 years ago, but since then relatively little has been published about these lymphomas. Based on previous studies in SJL mice, we investigated the phenotypic and functional properties of C57L-derived lymphomas in relation to Mtv29-encoded vSAg expression by the tumor cells, and their ability to stimulate TCR Vβ-restricted T cells. The cell surface phenotype of the C57L lymphomas indicates a B cell origin (sIg +, MHC II +). These B lymphoma cells also express co-stimulatory molecules [B7-1 (CD80) and HSA (CD24)], and stimulate marked proliferation of syngeneic CD4 + T cells. C57L B lymphoma cells exhibit Mtv-encoded mRNA by northern analysis, and also stimulate IL-2 production from Vβ16 + T cell hybrids, suggesting a role for Mtv 29 in this syngeneic T cell response. After transfer to syngeneic recipients, primary C57L lymphomas grow slowly, if at all. However, tumor growth is greatly accelerated by pretreatment of C57L recipients with anti-asialo GM1 antibody (but not anti-CD8 mAb), suggesting that NK cells play a major role in inhibiting lymphoma growth. If, in addition to anti-asialo GM1, the mice are also pretreated with anti-CD4 mAb, tumor growth is markedly inhibited, indicating that the lymphoma-responsive syngeneic CD4 + T cells promote tumor growth. Therefore, although the vSAg-induced response stimulated by vSAg29 expressing lymphoma cells in syngeneic TCR Vβ-restricted CD4 + T cells is an important etiologic factor in this type of B cell neoplasm both in C57L and in SJL mice, the final outcome of the spontaneous neoplastic process appears strongly influenced by endogenous NK activity in aging mice.

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: pathophysiology of biliary lipid secretion

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females. We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.

Sterol carrier protein 2 participates in hypersecretion of biliary cholesterol during gallstone formation in genetically gallstone-susceptible mice.

In inbred mice, susceptibility to cholesterol gallstone disease is conferred by Lith genes, which in part promote hypersecretion of cholesterol into bile in response to a high-fat/cholesterol/cholic acid (lithogenic) diet. Because cytosolic sterol carrier protein 2 (SCP2) is believed to participate in cellular cholesterol trafficking and is elevated in the liver cytosol of cholesterol gallstone patients, we defined the hepatic expression of SCP2 during cholesterol gallstone formation in gallstone-susceptible C57L and gallstone-resistant AKR mice fed the lithogenic diet. Steady-state cytosolic SCP2 levels in C57L, but not AKR mice increased as a function of time and were correlated positively with biliary cholesterol hypersecretion, cholesterol saturation indices of gall-bladder biles and the appearance of liquid and solid cholesterol crystals leading to gallstone formation. Steady-state mRNA levels increased co-ordinately, consistent with regulation of SCP2 expression at the transcriptional level. Our results suggest that overexpression of SCP2 contributes to biliary cholesterol hypersecretion and the pathogenesis of gallstones in genetically susceptible mice. Because of the different chromosomal localizations of the Lith and Scp2 genes, we postulate that Lith genes control SCP2 expression indirectly.

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile

Lith genes control susceptibility to cholesterol gallstone formation in inbred strains of mice on a lithogenic diet containing high fat, high cholesterol and 0.5% cholic acid. Our study defines the physical-chemical phenotypes of C57L, AKR, and (C57L x AKR) F1 mouse gallbladder biles during 56 days on the lithogenic diet. We found enhanced cholesterol supersaturation, accumulation of mucin gel, and larger gallbladders in all C57L and F1 mice, as well as more frequent gallstone formation in male C57L and F1 mice (80%) compared to females (40%) or AKR mice (15%). In male C57L and F1 mice, mucin gel accumulated at 3 days, followed by cholesterol supersaturation and phase separation of liquid crystals, solid monohydrate crystals, and, in 43% of mice, anhydrous cholesterol crystals; whereas, in females, phase separations were delayed 2 to 9 days, and anhydrous crystals did not form. In AKR mice, cholesterol supersaturation and phase separations were infrequent and delayed, and gender did not influence the phenotype. Taurocholate invariably replaced endogenous bile salts, especially tauro-beta-muricholate, with crystallization sequences matching taurocholate-containing model bile systems. We conclude: i) Lith genes determine biliary cholesterol supersaturation, mucin gel accumulation, gallbladder size, phase-separation, and prevalence of cholesterol gallstones. ii) Identical phenotypes in C57L and F1 mice indicate susceptibility to cholesterol gallstones is genetically dominant, favoring males 2:1. iii) Mucin gel accumulation, crystallization, and stone formation are rare in AKR mice. This definition of the physical chemistry of lithogenesis should aid in further elucidation of the Lith genes and the proteins they encode.

Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse V beta gene usage by T cells recognizing the core epitope encephalitogenic for T cell receptor V beta b and T cell receptor V beta a H-2b mice.

The nature of the autoimmune T cell response to myelin oligodendrocyte glycoprotein (MOG), recently recognized as a potential target antigen in multiple sclerosis (MS), has not yet been characterized, in contrast to the T cell reactivity to other potential target antigens in MS such as myelin basic protein and proteolipid protein. Here, we show that the encephalitogenicity of the recombinant Ig-like domain of human MOG is associated, in H-2 b mice, with an immunodominant T cell reactivity against a single region of MOG spanning amino acids 35-55, accounting for the previously reported strong encephalitogenic activity of pMOG 35-55. A single injection of pMOG 35-55 with or without administration of pertussis toxin was sufficient to induce severe clinical experimental autoimmune encephalomyelitis (EAE) in H-2 b mice. Encephalitogenic pMOG 35-55-specific T cell lines derived from C3H.SW (V beta b) mice were diverse in their TCR V beta gene usage (V beta 1, V beta 6, V beta 8 and V beta 15), although V beta 8.2 was most predominantly expressed (48%). However, V beta 8 + T cells may only be part of the encephalitogenic MOG-specific T cell repertoire in H-2 b mice, as demonstrated by the susceptibility of C57L (V beta a) mice to disease induced by pMOG 35-55. Encephalitogenic T cell lines from V beta a mice were also diverse in their TCR V beta gene usage (V beta 1, V beta 2, V beta 6, V beta 14 and V beta 16). Such a heterogeneous TCT V beta gene expression by pMOG 35-55/I-A b-reactive T cells from both V beta a and V beta b H-2 b mice suggested multiple epitopes within pMOG 35-55. Analysis of the pattern of reactivity by pMOG 35-55-reactive T cells to a set of truncated peptides was not commensurate with independent nested epitopes, but revealed a requirement for recognition of a core sequence, YRSPFSRVV (pMOG 40-48). However, optimal stimulation was obtained with longer peptides, with each additional amino acid flanking either the N or the C terminus differentially increasing the stimulatory capacity of pMOG 40-48. Nonetheless, pMOG 40-48 was the minimal encephalitogenic epitope for both V beta a and V beta b mice. Thus, the T cell reactivity against the immunodominant encephalitogenic region of MOG is characterized by a diverse V beta gene usage and a requirement for the same core epitope. This pattern of reactivity may favor epitope-directed, rather than TCR-targeted, approaches to immunospecific therapy for MOG-related autoimmune disease.

Characteristics of initial and reinduced experimental autoimmune encephalomyelitis.

The factors which influence expression of autoimmune disease in the central nervous system are still poorly understood. We determined the characteristics of experimental autoimmune encephalomyelitis (EAE) in twelve different inbred strains of mice using either mouse spinal cord homogenate or synthetic peptides as the encephalitogen. We also determined whether these strains were susceptible to reinduction of EAE at six weeks after the initial injection. The incidence, time of onset, severity, duration, and number of spontaneous relapses varied widely among the different strains. Duration of initial EAE correlated significantly with incidence of spontaneous relapses, and was greatest in C57L mice and in mice with a C57BL/10 background. Most strains of mice recovered from initial EAE, but recovery was unusual in A.SW and PERA mice. Incidence of reinduced EAE differed from incidence of initial EAE in some strains and did not correlate with incidence of spontaneous relapse. We conclude that the same factors control disease duration and incidence of spontaneous relapse, and that these factors are independent of the factors which control initial incidence. The factors controlling incidence of reinduced EAE are distinct from those controlling spontaneous relapse, and may also differ from those controlling initial incidence. Further investigation of the mechanisms effecting recovery from EAE and the genetic background underlying those mechanisms may help us understand human diseases such as multiple sclerosis.

Immunotherapy with IL-2-stimulated splenocytes reduces in vitro the level of Leishmania donovani infection in peritoneal macrophages

A study was done in vitro to determine if IL-2-stimulated lymphocytes (LAK cells) would activate infected macrophages to reduce their barden of Leishmania donovani. Macrophagedepleted splenocytes from normal or infected C57BL 6 (H-2 b; Lsh S) mice, stimulated in vitro by the IL-2-costaining supernatant of the MLA 144 cell line or by rIL-2, significantly reduced the number of syngeneic resting peritoneal macrophages infected by L. donovani; LAK cells from infected animals were significantly more effective in reducing the numbers of infected cells. Supernatants of MLA 144 stimulated aplees cells and rIL-2-stimulated splenocytes isolated in Millipore chambers also induced a significant reduction of the infection in vitro. Anti-Thy 1.2 eliminated the ability of the supernatant of MLA 144 to induce an activating function in C57BL 6 splenocytes; monoclonal anti-IL-2 abolished the ability of rIL-2 and of the MLA 144 supernatant to stimulate the splenocytes. Infected resting peritoneal macrophages of C57L (H-2 b; Lsh R) mice were more responsive to activation than those of the C57BL 6 animals, irrespective of the phenotype of the stimulating LAK cells. Lymphokine stimulation reverses the immunological anergy induced in T lymphocytes by Leishmania donovani; IL-2-stimulated LAK splenocytes are highly effective in reducing the intensity of experimental visceral leishmaniasis in vitro in peritoneal macrophages.

Localization of a novel chromosome 7 locus that suppresses development of N‐methyl‐N‐nitrosourea—induced murine thymic lymphomas

N-Methyl-N-nitrosourea (MNU) is a potent carcinogen that causes the development of murine thymic lymphomas. MNU-induced tumor incidence varies considerably among different inbred mouse strains. In particular, the AKR strain is highly susceptible, whereas the C57L strain is highly resistant to MNU-induced lymphoma formation. Crosses between AKR and C57L mice were established to investigate the genetic basis for the differential susceptibility of these inbred strains. A strong association between MNU-induced lymphoma development and coat color was observed in (AKR x C57)F2 and AKR x (AKR x C57)F1 progeny such that albino mice developed a higher tumor incidence than nonalbino animals. These data suggest that a locus on chromosome 7 influences tumor development. Analysis of four additional polymorphic loci (D7Rp2, Fes, Hbb, and Int-2) on chromosome 7 in AKR x (AKR x C57)F1 backcross mice revealed a significant linkage between high tumor incidence and homozygous inheritance of AKR alleles at the albino (tyrosinase) and Hbb loci. Thus, inheritance of at least one C57L allele at the albino or Hbb loci was associated with protection against MNU-induced lymphoma development. There was no association between tumor incidence and genotype at the D7Rp2, Fes, or Int-2 loci. Taken together, the data suggest that whereas C57L mice contain a dominant tumor suppressor gene on chromosome 7, in the AKR strain both alleles at this locus are defective resulting in enhanced susceptibility to MNU-induced lymphomagenesis.

Histoplasma capsulatumand Vβamice: cellular immune responses and susceptibility patterns

Certain strains of mice, designated V beta a, have a deletion of the gene segments encoding the beta chain of the T-cell receptor variable region. These mice do not express 40 to 50% of the T-cell receptor V beta chains. In this study, we examined the influence of this deletion on susceptibility to Histoplasma capsulatum. In addition, H. capsulatum-injected V beta a mice were tested for their capacity to generate T-cell dependent responses to H. capsulatum antigens. Susceptibility profiles of V beta a mice, SWR/J (H-2q), SJL/J (H-2s) and C57L-(H-2b), were compared to V beta b strains, C57BL/6 (H-2b) and DBA/l (H-2q), following intravenous (IV) injection of sublethal and lethal inocula of H. capsulatum yeast cells. One week after injection of 6 x 10(5) yeast cells, the spleens of SWR/J, SJL/J and C57L mice contained 5- to 7-fold fewer colony forming units (CFU) than spleens of C57BL/6 mice. Approximately 50% fewer CFU of H. capsulatum were recovered from the spleens of DBA/l mice compared to those from C57BL/6 animals. Subsequently, groups of mice were challenged IV with either 1.5 x 10(7) or 7.5 x 10(6) yeast cells and observed for 30 days. Survival of SWR/J,SJL/J, C57L and DBA/l mice was significantly prolonged compared to C57BL/6 mice. V beta a and DBA/l mice injected with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to an extract from the cell wall and cell membrane of yeast cells and to HIS-62, a purified antigen derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary hydronephrosis in C57L/MsNrs mice.

Inbred C57L/MsNrs mice (66 males and 72 females) maintained at the National Institute of Health, Japan, were examined grossly for hydronephrosis. Unlike the hydronephrotic strains of mice reported so far, C57L mice showed no sex-related difference in the incidence (62% in males and 75% in females; P > 0.05) and severity of hydronephrosis. The right kidney was more severely affected than the left kidney in both sexes. Age appeared to have no influence on the incidence.

Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection.

Lethally irradiated mice reject within 24 h certain marrow grafts, a phenomenon called either allogeneic or hybrid resistance. The cells responsible for this rejection (NK1+ CD3+ cells (TNK) express Ag of NK cells as well as the TCR-associated CD3 complex. This raises the question whether TCR participate in the function of these cells during graft rejection. By using flow cytometry it is shown that the majority of TNK cells expresses the TCR-alpha/beta chains and by using adoptive cell transfer assays evidence is presented that it is the TCR-alpha/beta expressing cells that cause rejection. To explore whether any particular TCR chains have to be expressed on these cells, C57L mice were assayed and found to be responders suggesting that the V beta chains deleted in these mice are not obligatory. However, introduction of a specific TCR V beta 5 chain into C57BL/6 mice as a transgene leads to inability to transfer resistance. TNK cells of V beta 5 transgenic mice express the introduced gene suggesting that it is the transgenic TCR that is responsible for the lack of function. In assessing T cell functions in V beta 5 transgenic mice it is shown that although these mice generate CTL specific for H-2d targets there is a deficiency to recognize H-2Dd, i.e., of determinants presumed to be recognized in the acute rejection mechanism. Thus TNK cells and CTL share the inability to recognize H-2Dd epitopes due to expression of the V beta 5 transgene. The notion that TCR on TNK cells play a role in the acute rejection process makes it necessary to postulate a receptor selection mechanism for these cells.

Role of T cell receptor V beta genes in Theiler's virus-induced demyelination of mice.

Intracerebral infection of certain strains of mice with Theiler's virus results in chronic immune-mediated demyelination in spinal cord. We used mouse mutants with deletion of the V beta class of TCR genes to examine the role of TCR genes in this demyelinating disease which is similar to multiple sclerosis. Quantitative analysis of spinal cord lesions demonstrated a markedly increased number and extent of demyelinated lesions in persistently infected RIII S/J mice which have a massive deletion of the TCR V beta-chain (V beta 5.2, V beta 8.3, V beta 5.1, V beta 8.2, V beta 5.3, V beta 8.1, V beta 13, V beta 12, V beta 11, V beta 9, V beta 6, V beta 15, V beta 17) compared with B10.RIII mice which are of identical MHC haplotype (H-2r) but have normal complement of V beta TCR genes. In contrast, infection of C57L (H-2b) or C57BR (H-2k) mice which have deletion of the V beta TCR genes (V beta 5.2, V beta 8.3, V beta 5.1, V beta 8.2, V beta 5.3, V beta 8.1, V beta 13, V beta 12, V beta 11, and V beta 9) resulted in few demyelinating lesions. Genetic segregation analysis of (B10.RIII x RIII S/J) x RIII S/J backcrossed mice and (B10.RIII x RIII S/J) F2 mice demonstrated correlation of increased susceptibility to demyelination with deletion of TCR V beta genes. The increase in number of demyelinating lesions correlated with increase in number of virus-Ag+ cells in spinal cord. These experiments provide strong evidence that the structural diversity at the TCR beta-complex can influence susceptibility to virus-induced demyelination.

Activation of murine T cells by staphylococcal enterotoxin E: Requirement of MHC class II molecules expressed on accessory cells and identification of Vβ sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by Staphylococcal enterotoxin E (SEE). L cells transfected with I-A b genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-A b markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4 + T cells and CD8 + T cells, and that larger parts of them bore Vβ11 (40–75%). mAb to Vβ11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; Vβ11 + T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/ 6 × BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and Vβ11 + T cells, deleted), and SJL and C57L mice (Vβ11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing Vβ11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4 + T cells and CD8 + T Cells.

Abrogation of resistance to Theiler's virus-induced demyelination in C57BL mice by total body irradiation

Theiler's murine encephalitis virus (TMEV) produces an unusual biphasic disease in susceptible mice characterized by poliomyelitis with early viral replication in neurons, followed by chronic demyelination with viral antigen expression in spinal cord white matter. In addition, infectious virus persists in the central nervous system (CNS) throughout the chronic pahse of disease. Previous studies have indicated an important role for major histocompatibility complex (MHC)-gene products in determining resistance/susceptibility to disease. In particular, certain calss I gene products of the D region of the H-2 gene complex render mice of the C57BL lineage resistant to induction of demyelination. Intracerebral infection of B10.S(D S) mice results in demyelination in the spinal cord while infection of C57BL/10(D b) or with gamma irradiation renders normally resistant B10.S(9R) and C57BL/10 mice susceptible to TMEV-induced demyelination and allowed for increased viral replication. In addition, the majority of irradiated C57BL/10 mice infected with virus showed extensive areas of CNS remyelination by oligodendrocytes beginning at 63 days post-infection. In contrast, immunosuppression of normally susceptible B10.S mice resulted in acute disease and high mortality accompanied by overwhelming destruction of neurons. The study supports the hypothesis that MHC-conferred resistance in C57L mice is associated MHC D region products and indicate an important active role for the immune system early in infection in limiting vital infection during disease induction in nonimmunosuppressed mice.

Analysis of V beta 17a expression in new mouse strains bearing the V beta a haplotype.

A set of new mouse strains were produced that carry the V beta a haplotype of the TCR-alpha/beta and any of a number of different H-2 haplotypes on backgrounds derived from related C57BL, C57L, and C57BR mice. Study of V beta 17a expression in these mice confirms the association between the presence of IE and the deletion of V beta 17a+ T cells. A second H-2 gene causing deletion of V beta 17a+ T cells was mapped in these mice to the K end of H-2k, and H-2 influences on the level of selection of CD4+ V beta 17a+ T cells were indicated.

Influence of complement C5 and V beta T cell receptor mutations on susceptibility to collagen-induced arthritis in mice.

SWR/J mice are resistant to collagen-induced arthritis (CIA) despite having a susceptible H-2q haplotype. We have earlier demonstrated the possible role of the V beta TCR mutation of SWR in the resistance to CIA. To investigate the influence of the C5 deficiency of SWR in this resistance, crosses were made between SWR and A/J (C5 deficient, TCRwild, H-2a), and between SWR and C3H.A (C5 sufficient, TCRwild, H-2a). Upon immunization with bovine type II collagen in adjuvant, there was a similar incidence and severity of arthritis in H-2q-bearing mice in the back-crosses A x (SWR x A) ad C3H.A x (SWR x C3H.A). The absence of hemolytic complement was confirmed in the arthritic A x (SWR x A) back-cross mice by standard SRBC hemolytic assays. In addition C57L (H-2b) mice, which were C5 sufficient but had a V beta TCR deletion mutation similar to SWR, could not complement for CIA susceptibility in H-2q-bearing C57L x (SWR x C57L) back-crosses and in (C57L x SWR)F2 hybrids. These studies show that complement C5 does not play a significant role in CIA susceptibility, and further implicate the V beta TCR mutation in the resistance to CIA in SWR mice.

Antibody-mediated activation of T cell clones as a method for screening hybridomas producing antibodies to the T cell receptor

In this study, supernatants (SN) of hybridomas established by fusing P3X63.Ag8.653 to spleen cells from C57L mice (Vß8 family of T cell receptor (TcR) gene negative) immunized with the H-Y specific Vß8 allotype positive helper T cell (HTL) clone OI6 were screened for the capacity to activate cloned T cells in the absence of interacting stimulator cells. In the first assay, SNs were mixed with Vß8 + H-Y specific CTL OH2 and 51Cr-labelled non-specific B lymphoma (L10.A). In this system, antibodies (Ab) which can bind to L10.A by Fc-Fc receptor interaction and recognize TcR can facilitate lysis of L10.A target cells by OH2 CTL. In the second assay, OI6 clone cells were cultured in microtiter well, previously coated with hybridoma supernatants (SN). In this assay, Ab recognizing OI6 TcR complex and bound to plastic plates can stimulate OI6 cells to proliferate in the absence of stimulator cells. Using these two screening methods, nine hybridomas were established. Analysis of these hybridoma SN using surface staining, inhibition of T cell function and immunoprecipitation of radiolabelled surface molecules followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) showed that five Ab were directed to the allotypic determinant (Vß8) of TcR and four Ab were specific to the clonotypic determinant of OI6 TcR. These results suggest that this Ab-mediated activation of T cell clone can be used for the screening of hybridomas secreting anti-TcR Ab and the immunogenicity of OI6 clonotypic determinants is similar to that of the Vß8 allotypic determinant even in strains which do not express the Vß8 TcR allotype.

Murine T-cell receptor mutants with deletions of beta-chain variable region genes.

Genomic Southern blots of DNA from eight strains of mice were examined for restriction fragment length polymorphisms in their loci encoding the variable region of the T-cell receptor beta chain (V beta), using 16 different V beta-specific probes. Mouse strains BALB/c, C57BL/6, C3H, and PL were identical, while strains SJL, C57BR, C57L, and SWR shared several polymorphisms with respect to the other four strains. In addition, SJL, C57L, C57BR, and SWR DNAs were missing 50% of the hybridizing bands visualized in BALB/c DNA. A cDNA library from concanavalin A-stimulated SJL spleen blasts was constructed and examined for V beta gene usage. Ten genes were found to account for all V beta-containing clones isolated, including three newly identified V beta genes. All 10 of these genes were found to be present in BALB/c mice. We conclude that SJL, C57L, C57BR, and SWR mice represent V beta deletion mutants of the BALB/c genotype.

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor.

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.

Ontogeny of B-lymphocyte function: XIV. Maturation of the capacity of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the ability of the B-cell population to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit anti-mouse immunoglobulin (RAMIg) was studied in LAF 1, C57BL/6, and C57L mice. As demonstrated by previous workers, the B-cell population from immature mice failed to re-express sIg after treatment with RAMIg. We have shown that the age at which the B-cell population acquires the capacity to re-express sIg is different in different strains and that the order in which the B-cell population of the different strains acquires the capacity to re-express sIg is different from the order in which their B-cell populations acquire the capacity to produce high-affinity antibodies. This suggests that these represent distinct differentiation events in the development of the B-cell population. In all of the strains studied the maturation of the capacity to re-express sIg occurred in two steps. After the first maturation step the B-cell population was able to re-express sIg after treatment with RAMIg for 1 hr but did not re-express sIg after treatment with RAMIg for 24 hr. After the second maturation step the B-cell population could re-express sIg even after 24 hr treatment with RAMIg. It has been suggested by previous workers that the inability of the immature B-cell population to re-express sIg could represent one of the mechanisms responsible for the development of B-cell self-tolerance. It is suggested here that the existence of a period during which cells become tolerant only upon prolonged exposure to antigen could protect the developing B cells from becoming unresponsive to transiently experienced foreign antigens but still permit them to become tolerant to self antigens which are continuously present.