Mapping lipopolysaccharide response loci in mice using recombinant inbred and congenic strains

Lipopolysaccharide (LPS) induces proliferation of splenic B-cells, and this response was found to be significantly lower in A/J than in C57BL/6J (B6) mice. Several strains and substrains mirrored the high and low responses of B6 and A/J. Assessment of 26 AXB/BXA recombinant inbred (RI) mouse strains identified 23 strains with a low (A/J-like), high (B6-like), or intermediate response. The three remaining RI strains exhibited a novel hyperresponsive phenotype significantly different from that of either founder strain. RI analysis identified four suggestive loci contributing to the LPS response, two of which were confirmed by analysis of congenic strains containing the donor genomic segment from a high- or low-responder strain on the opposite background. The combination of A/J and B6 alleles fixed to homozygosity at the four suggestive loci would occur in only 1 of 256 intercross progeny, but occurred several times among the RI strains.     

Synergistic interaction between anti-CD3 and IL-2 demonstrated by proliferative response, interferon production, and non-MHC-restricted killing

Anti-CD3 monoclonal antibody acts on normal peripheral blood mononuclear cells to induce T cell proliferation, interferon-gamma production, and non-MHC-restricted cytotoxicity against both NK (CD16+)-sensitive and -resistant target cells. Moreover, anti-CD3 and interleukin 2 (IL-2) act synergistically to give greater proliferative, interferon-gamma (IFN-gamma), and natural cytotoxicity responses than those expected by the simple addition of the individual responses to each stimulus acting alone. This synergistic response is macrophage independent, greatest at low concentrations of anti-CD3, inhibited by anti-IL2 receptor, and depends upon the induction of IL-2 receptors by CD3 activation which are then available to respond to exogenously added IL-2. Natural cytotoxicity induced by anti-CD3 and IL-2 correlates with IFN-gamma production, is inhibited by anti-IFN-gamma, and is still present after depletion of CD16-positive cells by specific monoclonal antibody and complement. The use of anti-CD3 in concert with IL-2 may be worthy of examination in a clinical setting, presumably because CD3/IL-2-generated LAK effector cells could be followed by in vivo administration of potentially lower and less toxic quantities of IL-2 than have been used in the past.     

Genetic composition of the recombinant congenic strains

For the study of biological phenomena influenced by multiple genes in mice, the Recombinant Congenic Strains (RCS) have been developed. An RCS series comprises approximately 20 homozygous strains, each of which contains on average 87.5% genes of a common background strain and 12.5% of a common donor strain. In an RCS series, non-linked genes involved in the control of a multigenic trait become distributed into different recombinant congenic strains. In this way a multigenic trait is transformed into a series of single gene traits in which each gene can be studied individually. For the ability to use the strength of the recombinant congenic strains system to its full extent, a thorough genetic characterization is indispensable. We have typed the CcS/Dem and OcB/Dem series for 611 and 550 markers, respectively. This results in a genetic characterization sufficient to detect most donor strain genes. In addition, we report the genetic characterization of the HcB/Dem and HcB(N4)/Dem series. Strains of the latter series contain on average 6.25% of the donor strain genome. Both series have been typed for 130 markers. All the typing data have been deposited in the Mouse Genome Database at The Jackson Laboratory. Received: 11 July 1995 / Accepted: 18 August 1995     

Segregation of genes from donor strain during the production of recombinant congenic strains

Recombinant congenic strains (RCS) constitute a set of inbred strains which are designed to dissect the genetic control of multigenic traits, such as tumour susceptibility or disease resistance. Each RCS contains a small fraction of the genome of a common donor strain, while the majority of genes stem from a common background strain. We tested at two stages of the inbreeding process in 20 RCS, derived from BALB/cHeA and STS/A, to see whether alleles from the STS/A donor strain are distributed over the RCS in a ratio as would theoretically be expected. Four marker genes (Pep-3; Pgm-1; Gpi-1 and Es-3) located at 4 different chromosomes were selected and the allelic distribution was tested after 3-4 and after 12 generations of inbreeding. The data obtained do not significantly deviate from the expected pattern, thus supporting the validity of the concept of RCS.     

Progression mechanisms in colon cancer: soluble interleukin-2 (IL-2) receptor,IL-2 plus anti-CD3 proliferative response and tumour stage correlations

Soluble interleukin-2 receptor (sIL-2R) levels have been found to be elevated in several clinical conditions, including disseminated solid neoplasms, whereas they are generally within the normal range in patients with locally limited neoplastic disease. The aim of the present study was to examine this in our colon cancer patients, and to assess if this situation can affect the in vitro activation of peripheral blood mononuclear cells (PBMC) examining the proliferative response to IL-2 and anti-CD3 monoclonal antibody, the IL-2 serum levels and the PBMC phenotype. The results show that sIL-2R levels were significantly correlated with the stage of the disease, showing an increase from stage I to stage IV; moreover, it is worth noting that the proliferative response to IL-2 plus anti-CD3 is significantly higher than to IL-2 alone in stage IV, without significant alteration in the numerical presence of T and natural killer cells. So it seems that in the peripheral blood of patients, connected with the disease progression, are present cellular populations showing a different response to activation, and that T cells acquire a better response condition than NK. Thus, since the T cellular population includes the tumour-specific cytotoxic precursor cells, this should be helpful for its tumour regressive activity, but it is conceivable that this population cannot perform its functions, owing to a deficiency in responsiveness of the specific ThCD4⁺ subpopulation.     

Genetic analysis of the non-H-2-linked Ir genes controlling the cytotoxic T-cell response to H-Y in H-2 d mice

The T-cell mediated immune responses to the male specific minor histocompatibility antigen H-Y in mice have been studied extensively as a model for immune responses to other weak antigens like tumor antigens or autoantigens. In a recent analysis of the strain distribution of the cytotoxic T-cell (Tc-cell) responsiveness to H-Y, it has been found that genes both within and outside the H-2 complex exert an interactive control. Whereas the H-2 ^b strains all are high responders, independent of their non-H-2 background, other H-2 haplotypes (d, k, and s) only allow for a response if they are combined with certain non-H-2 genes. The H-2-linked immune response genes (Ir-genes) have been previously mapped to the I and K or D region of the H-2 complex, but the mapping of the non-H-2 genes has not yet been established. In this study evidence is presented, using recombinant inbred strains and immunoglobulin heavy chain (Igh) congenic strains of mice, to show that there is more than one non-H-2 Ir-gene involved, that the main controlling genes are not linked to the Igh complex, and that at least one non-H-2 Ir-gene is linked to the H-3 region on chromosome 2. This region includes genes for beta-2-microglobulin (2m), the Ly-mllalloantigen a polymorphic cell surface glycoprotein (Pgp-1), a B-cell specific antigen Ly-4, a transplantation antigen H-3, and genes (Ir-2) controlling the immune response to Ea-1 and H-13.     

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

Alloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2^pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2^b), BALB/c (H2^d), CBA (H2^k), and DBA/1 (H2^q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.     

Growth factor production and requirements during the proliferative response of human T lymphocytes to anti-CD3 monoclonal antibody

Anti-CD3 was administered with three different accessory stimuli to purified populations of human T cells. Sepharose conjugated anti-CD3, monocytes, and PMA each could induce the p55 component of the IL-2R as well as responsiveness to exogenous IL-2. Sepharose anti-CD3 did not induce IL-2, although the levels of IL-2 protein and mRNA were 10 to 30 times higher with PMA than with monocytes. Despite these differences in IL-2 production, the amount of DNA synthesis and the number of lymphoblasts were comparable when monocytes or PMA were used as the accessory stimulus, and the responses were equally sensitive to inhibition by an anti-IL-2R antibody. To pursue the functional relevance of the "supraoptimal" levels of IL-2 that are induced by PMA, anti-CD3-induced lymphoblasts were isolated free of monocytes and challenged with lymphokines. It could be shown that 1) the small amounts of IL-2 in the monocyte-T cell conditioned medium would drive DNA synthesis, but that 2) higher levels of IL-2 (20 to 100 U/ml) were needed to induce IFN-gamma, as well as the mRNA for IL-4 and the p55 IL-2R. We suggest that the capacity to produce high levels of IL-2, as seen with PMA, is required under physiologic conditions for two reasons: to up-regulate the IL-2R when small amounts of Ag rather than large amounts of anti-CD3 are ligands for the T cell, or to induce the release of lymphokines like IL-4 and IFN-gamma from sensitized lymphoblasts.     

Non-H-2 and H-2-Linked immune response genes control the cytotoxic T-cell response to H-Y

The immunoregulation of cytotoxic T-cell responses to the male-specific antigen H-Y in mice has been found to be genetically controlled by genes of the major histocompatibility complex (H-2). Responsiveness was mainly confined to H-2 ^b strains, but it has also been found in recombinant strains, F₁ hybrids, and chimeras that carry at least part of the H-2 ^b haplotype. By using a different immunization procedure it has been shown recently that an H-2 ^k mouse strain (CBA) is also able to mount an equivalent H-Y-specific response. We investigate here, by applying this immunization technique, the responsiveness of other H-2 ^k strains and of strains of other independent H-2 haplotypes. Both responders and nonresponders are found in three haplotypes: k, s, and d. The strain distribution pattern of responsiveness shows a combined influence of non-H-2 and H-2 genes. In certain strains there is a high variability in responsiveness between genetically indentical individual animals. We discuss a model of immune response (Ir) gene function which could account for these observations.     

Single and multiple congenic strains for hydrocephalus in the H-Tx rat

The H-Tx rat has fetal-onset hydrocephalus with a complex mode of inheritance. Previously, quantitative trait locus mapping using a backcross with Fischer F344 rats demonstrated genetic loci significantly linked to hydrocephalus on Chromosomes 10, 11, and 17. Hydrocephalus was preferentially associated with heterozygous alleles on Chrs 10 and 11 and with homozygous alleles on Chr 17. This study aimed to determine the phenotypic contribution of each locus by constructing single and multiple congenic strains. Single congenic rats were constructed using Fischer F344 as the recipient strain and a marker-assisted protocol. The homozygous strains were maintained for eight generations and the brains examined for dilated ventricles indicative for hydrocephalus. No congenic rats had severe (overt) hydrocephalus. A few pups and a significant number of adults had mild disease. The incidence was significantly higher in the C10 and C17 congenic strains than in the nonhydrocephalic F344 strain. Breeding to F344 to make F.H-Tx C10 or C11 rats heterozygous for the hydrocephalus locus failed to produce progeny with severe disease. Both bicongenic and tricongenic rats of different genotype combinations were constructed by crossing congenic rats. None had severe disease but the frequency of mild hydrocephalus in adults was similar to congenic rats and significantly higher than in the F344 strain. Rats with severe hydrocephalus were recovered in low numbers when single congenic or bicongenic rats were crossed with the parental H-Tx strain. It is concluded that the genetic and epigenetic factors contributing to severe hydrocephalus in the H-Tx strain are more complex than originally anticipated.     

IL-2 suppression of IL-12p70 by a recombinant HSV-1 expressing IL-2 induces T-cell auto-reactivity and CNS demyelination

To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2rα) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8(+) and CD4(+) T cells contribute to HSV-IL-2-induced CNS demyelination with CD8(+) T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS.     

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.     

Use of recombinant inbred strains to map genes of aging

Recombinant inbred strains have been used in a number of organisms for segregation and linkage analysis of quantitative traits. One major advantage of the recombinant inbred (RI) methodology is that the genetic identity of individuals within a strain permits replicate measures of the same recombinant genotype. Such replicability is important for traits such as aging inDrosophila, where phenotypic expression is highly influenced by different environmental conditions. RI strain methodology has an added advantage for DNA marker-based linkage analysis of traits measured over the lifespan of the organism. The DNA can be extracted from individuals of the same genotype as those measured in a longevity study. In this paper an argument is presented for the use of a set of recombinant inbred strains to map the quantitative trait loci involved in the aging process inDrosophila. A unique use of a set of stable, transposable moleular markers to trace the quantitative trait loci involved is suggested.     

Genetics of susceptibility to 6-aminonicotinamide-induced cleft palate in the mouse: studies in congenic and recombinant inbred strains

In a search for genetic differences in susceptibility to cleft palate, congenic and recombinant inbred strains of mice were treated with 6-aminonicotinamide or control injections. Of six loci tested, only the chromosome segment marked by N-acetyl transferase was found to affect susceptibility to 6-aminonicotinamide-induced cleft palate. This chromosome segment is known to affect glucocorticoid-induced cleft palate and phenytoin-induced cleft lip with or without cleft palate in these strains of mice.     

Freshly isolated, murine neonatal T cells produce IL-4 in response to anti-CD3 stimulation.

In previous studies of chimeric animals, we found that fetal intrathymic T cell precursors give rise to phenotypically abnormal peripheral T cell populations. Because most peripheral T lymphocytes in newborn mice are the progeny of fetal T cell precursors, this result led to the hypothesis that neonatal and adult T cells differ in their functional capacities. To investigate this issue, the responses of neonatal and adult T cells to anti-CD3 antibody and TCR-independent stimulation were compared. When stimulated with soluble anti-CD3 antibody in the presence of adult accessory cells, neonatal T cell proliferation was markedly decreased compared with that of adult T cells. This reduction in proliferation was associated with both quantitative and qualitative differences in lymphokine production. At 48 h of stimulation with anti-CD3 antibody, neonatal T cells produced at least 10-fold less IL-2 than adult T cells. This apparently accounted for their reduced proliferation because the addition of exogenous IL-2 restored their proliferation to the levels achieved by adult T cells. In striking contrast to adult T cells, neonatal T cells secreted large amounts of IL-4 upon primary stimulation in vitro. The differences between neonatal and adult T cells in proliferation and lymphokine production were shown to be specific for CD3-mediated stimulation. In the presence of phorbol ester and calcium ionophore, neonatal and adult T cells showed equivalent proliferation and IL-2 production. Under these conditions, IL-4 production by neonatal or adult T cells was essentially undetectable. Thus, in response to TCR-independent stimulation, freshly isolated neonatal and adult T cells show similar functional responses. However, when stimulation occurs via the CD3 components of the TCR, the responses of neonatal T cells resemble those of primed T cells from adult animals.     

The role of Ia-bearing cells in T-cell proliferative response to concanavalin A

Ia-bearing cells are required for the concanavalin A (Con A)-induced T-lymphocyte proliferative response. We attempted to determine how and when the Ia-bearing cells are required in this response. As Ia-bearing cells, mitomycin C-treated nylon wool- and plastic dish-adherent cells (AACmc) were used. AACmc didn't lose their restoring capacity on treatment with anti-Thy-1, anti-Ig, but they did with anti-Ia. Using a Marbrook chamber system, it was shown that cellular contact between T cells and Ia-bearing cells is necessary for restoring the response. Results with AACmc addition after various times from Con A addition, or Ia-bearing cell elimination after various times from Con A addition indicate that the Ia-bearing cells are required for about 12 hr from the start of incubation with Con A. And it was shown that Con A-pretreated AACmc could stimulate Ia-depleted T cells, and allogenic AACmc could replace syngenic ones.