Susceptibility and resistance to Leishmania amazonensis in H-2q syngeneic high and low antibody responder mice (Biozzi mice).

H-2 syngeneic H and L (Biozzi) mice provide a model to study Leishmania infections in which polar resistant and susceptible phenotypes are independent from H-2 differences. High-Ab-responder (H) and low-Ab-responder (L) mice syngeneic at the H-2 locus (H-2q) were, respectively, susceptible and highly resistant to Leishmania amazonensis infection. L-mice resistance was associated with high IFN-gamma and transient IL-4 production by lymph node (LN) cells, in contrast with sustained IL-4 and decreasing IFN-gamma production by susceptible H mice. IL-12 production could be detected only in LN from resistant mice. The cytokine production pattern was consistent with preferential progression to a Th1-type response in resistant L-mice, and to a Th2-type response in susceptible H-mice. We also investigated whether this shift towards Th1- or Th2-type cytokine responses was dependent upon H or L antigen presenting cells' (APC) intrinsic ability to preferentially stimulate either T-cell subset. To this end, LN-derived T-cell lines were grown from 12-day infected mice, when both strains produced IFN-gamma and IL-4. L-derived T-cell lines developed a Th2 cytokine pattern whereas H-derived T-cell lines produced IFN-gamma, IL-4 and IL-10 whatever the APC origin (H or L) used for their derivation. This work constitutes the first characterization of cellular immune responses to the intracellular parasite, L. amazonensis in H-2 syngeneic mice, an infection model in which polar resistant and susceptible phenotypes are determined by non-MHC genes.

Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8.

Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness.

Functional differences in the specific B-cell compartment in high or low antibody responder mice.

The role of antigen-presenting cells (APC) in quantitative antibody synthesis regulation was studied in mice genetically selected for high (HI) or low (LI) antibody response. Irradiated spleen cells and enriched specific B cells from HI and LI mice co-isogenic at H-2s locus, were compared for their capacity to present chicken ovalbumin (OVA) to specific T-cell hybridomas. Minor differences were observed between HI and LI mice when three distinct hybridomas were stimulated in the presence of OVA and splenic macrophages APC. These differences were totally abolished when APC were pulsed with OVAxAb complexes. Looking at the B-cell compartment, hybridoma IL-2 responses were similar when TNP primed B cells were pulsed with OVA. However, when OVA was targeted on TNP-specific enriched B cells by pulsing with TNP-OVA, the IL-2 production by the T-cell hybridomas was stronger in the presence of HI B cells than in the presence of LI B cells. These results strongly suggest that an efficient Ag handling/processing by specific B cells is a major component of the high Ab responder status in Biozzi mice.

T-cell compartment involvement in two high antibody responder lines of mice (HI and HII Biozzi mice) respectively susceptible and resistant to collagen-induced arthritis.

The T-cell compartment was investigated in two high antibody responder lines of mice respectively susceptible (HI) and resistant (HII) to chicken collagen (CII)-induced arthritis (CIA). Previous data had shown that both lines were high anti-CII Ab producers, without any TCR V-beta gene defect or membrane expression impairment. The present studies demonstrate that anti-CII proliferation is much lower in HII than in HI. These results are confirmed by the limiting dilution analysis of anti-CII T-precursor frequencies (1/991 in HI and 1/12175 in HII). The percentage of CD8+ T cells is constitutively higher in HII mice, this difference increasing after CII immunization. This finding suggests a suppressive effect accounting for resistance to CIA. However, no restoration of specific response was achieved by in-vivo or in-vitro depletion of CD8+ T cells. T clones specific for Chicken CII could be obtained only from primed HI mice. Four of five clones with CD8+ phenotype proliferated in vitro to native and denatured CII and showed cytotoxic function in an anti-CD3 redirected assay. The CD4+ clone was shown to proliferate on both HI and HII-pulsed APC, which rules out a major CII processing/presentation defect in HII.

Migration stimulation factor (MStF), from murine B cells, constitutively produced by a T-B hybridoma.

Hybridomas were established between murine spleen B cells and the thymoma cell line BW5147, to purify the migration stimulation factor (MStF), a molecule likely involved in immunosuppression. The parental B cells were from Lo/PHA mice previously shown to produce high levels of MStF after immunization by appropriate (tolerogenic) doses of ovalbumin. Among the positive clones, B9 was selected, since it produced high levels of MStF constitutively, and no immunoglobulin. This clone was shown to contain the genome of the B-cell fusion partner, since one of its L chain genes had undergone a VK-JK rearrangement. Isolation of MStF by size-exclusion chromatography showed 2 major peaks of activity, one of which eluted in a 20-kDa, almost protein-free fraction. This elution is unlikely to correspond to the true molecular mass, since MStF was found not to be a protein. Indeed, MStF was TCA-soluble, thermoresistant, highly hydrophobic and protease-resistant, but activity was abolished by neuraminidase digestion. The possibility of its being a small molecule transported by a protein carrier was also ruled out. These results suggest that MStF is a complex molecule containing both sialic residues and a lipid moiety. Experiments are planned to further investigate the chemical structure of this unusual B-cell factor.

Genetics of nonspecific immunity: I. Bidirectional selective breeding of lines of mice endowed with maximal or minimal inflammatory responsiveness.

The genetic regulation of acute inflammatory reaction (AIR) was studied by the method of bidirectional selective breeding, used to produce a line of mice giving the maximal and a line of mice giving the minimal inflammatory reaction (AIR max and AIR min, respectively). The AIR was triggered by subcutaneous injection of a neutral substrate (suspension of polyacrylamide microbeads), and measured by the leukocyte and serum protein accumulation in the exudate. The two parameters are positively correlated and present a normal frequency distribution. The highly genetically heterogeneous foundation population was produced by the equipoised intercrossing of eight inbred strains of mice, and selective breeding carried out by assortative matings of extreme phenotypes. The response to selection in 11 consecutive generations was highly asymmetrical: a marked AIR increase in the AIR max and no change in the AIR min line occurred. The mean value of realized heritability in the AIR max line was 0.26 and 0.18 for cell and protein concentrations, respectively. The response to selection must have resulted from the interaction of seven to nine independent gene loci endowed with additive effects. The lack of response to selection of the AIR min line is discussed. The large inter-line difference opens new possibilities for studying the biochemistry and molecular genetics of inflammation, and also for investigating the beneficial or detrimental effect of inflammatory responses.

Differential expression of migration inhibitory and migration stimulatory factors in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin. 1. Migration stimulatory factor(s) from T and B cells of immune spleen.

Expression of the lymphokine migration inhibition factor in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) response of their lymph node cells to phytohemagglutinin was found to be modulated by concomitant expression of migration stimulation factor(s) [MStF(s)]. The expression of both lymphokines was dependent on genetic character and the immunizing dose of antigen. In mice immunized 5 days earlier with 50 micrograms ovalbumin in Freund's complete adjuvant (Ova in FCA immune), migration inhibition factor, assessed with a sensitive photoelectric method, was well expressed by male spleen or lymph node 24-hour culture supernatants of Lo/PHA but Hi/PHA, especially female, expressed marked MStF(s) instead. Immunization with 500 micrograms Ova in FCA markedly enhanced expression of MStF(s) in Lo/PHA but inhibited it in Hi/PHA. MStF(s) of Ova in FCA immune spleens of the two lines were found to derive from both T and B cells, B cell activity being greater. Lo/PHA were by far better expressors of both T- and B-cell-derived MStF(s) as compared to Hi/PHA (p less than 0.01). Spleen cells of mice immunized with FCA alone also expressed MStF(s) but to lesser extent than Ova in FCA immune spleens, expression by Lo/PHA B cells being significantly higher than in Hi/PHA (p less than 0.05). The MStF(s) of Ova in FCA immune spleens was found to be non-immunoglobulin in nature.

Differential expression of migration inhibitory and migration stimulatory factors in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin. 2. Effects of mitogenic or allogeneic stimulation.

Expression of migration inhibition factor (MIF) following in vitro stimulation with phytohemagglutinin (PHA) or allogeneic cells was explored in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) response of their lymphoid cells to PHA. Hi/PHA mice also have greater cell-mediated immune responses in mixed lymphocyte culture and graft-versus-host reactions, the poorer cell-mediated immune response of Lo/PHA being accompanied by a higher frequency of malignant tumours. Expression of MIF in PHA-pulsed spleen cell supernatants measured by a sensitive photoelectric method was found to be modulated by the concomitant presence of migration stimulation factor (MStF) derived from T cells. Both lymphokines were better expressed in Lo/PHA, as compared to Hi/PHA, under appropriate experimental conditions. Use of a low proliferative dose of mitogen (5 micrograms/ml PHA, 2-hour pulse) followed by culture in serum-free medium led Lo/PHA to express the highest titres of MIF, whereas a proliferative dose of PHA (50 micrograms/ml, 2-hour pulse) caused abrogation or occultation of expression of MIF and elective expression of MStF in this line. Hi/PHA mice expressed MIF equally at both mitogen doses, with transient expression of MStF followed by MIF after 50 micrograms/ml PHA, the kinetics of expression of the two lymphokines being different. Expression of MStF by spleen cells was an early event after PHA stimulation. In contrast to mitogenic stimulation, allogeneic stimulation in one-way mixed lymphocyte culture led to similar expression of MIF by both lines of mice. The implications of these findings are discussed.

Genetics of acute inflammation: inflammatory reactions in inbred lines of mice and in their interline crosses.

Acute inflammation is induced by the subcutaneous injection of swollen polyacrylamide microbeads, its intensity measured by the cell and protein concentration of the local exudates. A large and continuous range of responses is obtained in different inbred strains of mice, which suggests a polygenic control of the inflammatory response. The variable levels of the global dominance observed in F1 hybrids issued from several parental combinations indicated that the pattern of alleles controlling high or low response was different in each parental strain. Balanced intercrossing of the 8 inbred strains studied has provided a genetically heterogeneous F3 population, presenting a high variability of responses. The value of the genetic part of F3 phenotypic variance, the spread of the interstrain differences, as well as the polygenic nature of the regulation of inflammatory responses pointed out the possibility to perform a bidirectional genetic selection by using the F3 mice as the foundation population, and response to microbeads as the selective phenotypic character.

Collagen-induced arthritis in Biozzi mice. Joint involvement is not correlated with collagen II IgG2a autoantibodies nor restricted to only H-2q and H-2r.

High (H) and low (L) immune responder "Biozzi" mice, obtained by four different selections, were investigated for their ability to develop collagen-induced arthritis. Both LI and LII lines--characterized by their low antibody responses to a wide variety of Ag--developed arthritis though they do not bear the susceptible H-2q and H-2r haplotypes. Out of the two lines (HI and HII) selected for their high antibody responses and bearing H-2q, only one (HI) developed arthritis. Both the lines with amplified high or low antibody responses (HG and LG), and the lines differing in the levels of cell-mediated immunity (Hpha and Lpha), failed to develop arthritis. Collagen II autoantibodies were found in all the lines: the responses being high (HI and HG), low (LI, LII and LG), or intermediate (HII, Hpha and Lpha). The level of IgG2a autoantibodies, presumed to be the most pathogenic, was low in two (HI and LII) of the three arthritic lines, and was high in the unaffected HG line. These results show that this arthritis is not solely restricted to H-2q and H-2r haplotypes, and argue against a correlation between collagen autoantibody levels and disease incidence.

Impact of genetically regulated T cell proliferation on acquired resistance to Listeria monocytogenes.

Two lines of mice genetically selected for high and low in vitro responses to PHA were used to evaluate the impact of T cell polyclonal expansion on acquired resistance to Listeria monocytogenes. The selective breeding induced two major consequences in low responder mice: (1) a reduction of the number of L3T4+ cells and (2) a restriction of T cell expansion upon PHA stimulation, predominantly affecting the Lyt-2+ subset, and associated with an abridgment of IL-2 production. In vivo PHA stimulation induced anti-Listeria protection in high responder mice, but was much less effective in low responder mice. Flow cytometer analysis revealed that T cell proliferation was also reduced in low responder mice during the course of Listeria infection, implying both L3T4+ and Lyt-2+ subsets. This defect did not apparently influence the kinetics of bacterial elimination in host tissues, which was similar in both lines during primary Listeria infection. In contrast, the expression of delayed-type hypersensitivity to Listeria antigens and the level of immunologic memory were significantly reduced in low responder mice. In vivo selective T cell depletion by anti-L3T4 or anti-Lyt-2 mAb allowed us to demonstrate the predominant role of Lyt-2+ cells in protection and that of L3T4+ cells in the expression of delayed-type hypersensitivity.

Genetic regulation of the specific and non-specific component of immunity.

Bi-directional selective breeding for antibody (Ab) responsiveness to heterologous erythrocytes (Selection I) produced a high (H) and a low (L) responder line of mice which were also remarkably separated for Ab responses to many unrelated natural antigens (Ags) such as heterologous proteins, viruses, bacteria, parasites and haptens carried by these immunogens. The character "quantitative Ab responsiveness" is controlled by several independently segregating loci (polygenic regulation). The major genetic modification is produced at the level of macrophage activities. The Ag is slowly catabolized and persists for a long time on the macrophage membrane of the H line, whereas it is rapidly destroyed in L line macrophages. The bactericidal and bacteriostatic activity of the macrophage is also strong in the L line and weak in the H line. The opposite genetic regulation of Ab responsiveness and macrophage activity is a fundamental phenomenon for understanding natural and vaccination-induced anti-infectious immunity. The L line is more resistant than the H line against the infections due to intracellular microorganisms (Salmonellae, Yersinia, Mycobacteria, Brucellae, Leishmania) where the macrophage plays the dominant defensive barrier. The H line is more resistant than the L line to the extracellular microorganisms which are efficiently counteracted by a strong antibody response (Pneumococcus, Klebsiella, Plasmodia, Trypanosoma). The intensity of T cell-mediated immunity as measured by delayed type hypersensitivity, which is independent of the genetic regulation of antibody responsiveness, is correlated with the degree of nonspecific inflammation produced at the site of the reaction by the Ag injection in non-sensitized mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic difference in the proliferative response to T mitogens between Hi/PHA and Lo/PHA lymphocytes is independent of accessory cell function.

The role of the macrophage as accessory cell in the proliferative response of lymphocytes to phytohemagglutinin (PHA) was studied in two lines of mice genetically selected for high and low responsiveness to T mitogens. Adherent cell depletion of lymph node cells abrogated the low (Lo)/PHA response, but only partially inhibited the high (Hi)/PHA response. Addition of peritoneal cells provided either by Hi/PHA or by Lo/PHA mice equally restored Hi/PHA responsiveness but had only a slight reconstituting effect on the inhibited Lo/PHA response. Equivalent enhancement or suppression of proliferation of untreated lymph node cells was obtained by the addition of increasing percentages of each of the two peritoneal cell populations. However, the maximum level of the Lo/PHA response never reached that of Hi/PHA cells. These data indicate that the bidirectional selective breeding has not modified the potentialities of the macrophages as accessory cells but has resulted in an impaired response of Lo/PHA lymphocytes to the signals delivered either by accessory cells or by T mitogens.

Effects of T mitogens on in vivo antibody production to a T-dependent antigen in lines of mice genetically selected for high or low in vitro responsiveness to PHA.

The influence of genes which regulate the in vitro T-cell proliferative response to T mitogen upon in vivo antibody production to a T-dependent antigen was studied in two lines of mice genetically selected for a high or a low in vitro lymphocyte response to phytohaemagglutinin (PHA). Kinetics of agglutinin production to increasing doses of sheep erythrocytes was similar in the two lines, except for the titres of mercaptoethanol-resistant antibodies, which were slightly lower in the low-responder line. Treatment with mitogen prior to immunization modified the antibody response in the two lines differently. This finding would indicate that the genes which regulate in vitro stimulation of T cells by PHA also control in vivo activation of T-cell subsets involved in immunoresponsiveness.

In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

The kinetics of viability of lymph node and spleen cells of mice genetically selected for "high" or "low" in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.

[Genetic regulation of T-lymphocyte responsiveness to PHA is independent of culture conditions (author's transl)]. / La régulation génétique de la réponse des lymphocytes T a la PHA est indépendante des conditions de culture.

A maximal interline separation has been obtained after 10 consecutive generations of selective breeding for the character "quantitative in vitro response of lymph node lymphocytes to the mitogenic effect of phytohaemagglutinin". At the selection limit the difference between high and low responder lines was about 20-fold. A similar interline separation has been demonstrated for the T-mitogen effect of concanavalin A. The identical response to PPD (purified protein derivative of tuberculin), a B mitogen, proved that the genetic selection has only modified the potentialities of T lymphocytes. During the selective breeding, responsiveness to PHA stimulation has been always measured under identical culture conditions. To demonstrate that the interline difference in responsiveness was due essentially to genetic factors independent of environmental effects, a systematic study of various culture conditions has been undertaken. The optimal stimulation was found after two days of culture for high line cells and after three days for low line cells. The difference between maximal responses was only slightly lower than that obtained after a two-day culture as used for the selection test. Increase in cell concentrations produced higher thymidine incorporation. In the two lines, a linear correlation was established between the cell concentration and the response produced. The maximal response given by the highest number of low line lymphocytes was equivalent to that given by a number, 11-fold smaller, of high line cells. Within certain limits, changes in the amount of tritiated thymidine added to the culture did not affect the interline separation. With a thymidine of high specific activity, a sub-evaluation of uptake by high line cells decreased the interline difference. Results in mixed culture of lymph node cells from high and low lines indicated that the low response was not due to the release of inhibiting factors or to the presence of suppressive cells in low responder mice. In conclusion, separation of these two lines was due to genetic factors acting independently of the cell culture conditions.