Nonresponder Mouse Strains Research Articles

Neutralization of IL-4 reverses the nonresponsiveness of CD4 + T cells to regulatory T-cell induction in non-responder mouse strains

It is well established that naive cells can be converted by TGF-β into CD4 +CD25 + regulatory T (Treg) cells with therapeutic potentials. Likewise, it is shown that all-trans retinoic acid (ATRA) can greatly enhance TGF-β-induced Treg conversion, a phenomenon which has mainly been studied in C57BL/6 mice. Here we show that, although purified naive cells are highly susceptible to Treg generation, total CD4 + T-cell populations from different mouse strains display significantly different sensitivities to TGF-β/ATRA-induced Treg conversion. The resistance of “non-responder” strains is associated with an enhanced production of IL-4 by memory T cells as well as an increased sensitivity of naive T cells to the action of IL-4. Importantly, neutralization of IL-4 overcomes the differences, thereby enabling TGF-β/ATRA to generate large numbers of functional Treg cells from total CD4 + T cells in a consistent manner across different mouse strains. Likewise, blockade of IL-4 significantly enhances TGF-β/ATRA-induced Treg conversion from human naive T cells in the presence of memory cells. These results show that the inherent resistance of “non-responder” mouse strains to Treg conversion induced by TGF-β and ATRA can be reverted by neutralization of IL-4 and that inhibiting the action of IL-4 is beneficial or even necessary for consistent inducible Treg generation.

Processing and Presentation of Variant Surface Glycoprotein Molecules to T Cells in African Trypanosomiasis

Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.

Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and “naturally activated” large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among “natural” Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.

Role of non-RT1 genes in the response of rat lymphocytes to Mycoplasma arthritidis T cell mitogen, concanavalin A and phytohemagglutinin.

A comparison of splenic cells from various inbred rat strains indicated that DA, Lewis, Buffalo, August, Wistar Furth, and (LEW X BN)F1 all responded well to the Mycoplasma arthritidis T cell mitogen, phytohemagglutinin and concanavalin A, but cells from BN and MAXX rats were very weakly or nonresponsive. Cells from congenic strains expressing nonresponder background genes, and responder haplotypes at RT1 (BN.1L(LEW), RT1; BN.1A(DA), RT1av1) failed to respond significantly to the mitogens. Rats expressing responder background genes but the nonresponder haplotype at RT1 at RT1 (WF.1N-(MAXX), RT1n) exhibited high responses to all mitogens. The controlling role of non-RT1 genes was confirmed by testing tissue-typed (DA X BN)F2 progeny and (DA X BN)F1 X DA and (DA X BN)F1 X BN progeny. No association was seen between the expression of a/a, a/n, or n/n at RT1 and the degree of response to the mitogens. In contrast, as the proportion of DA non-RT1 genes increased, so did the degree of mitogenic responsiveness. Similar results were obtained by using a partially purified preparation of the mycoplasma T cell mitogen. The results indicated that in the (DA X BN)F1 hybrids, responsiveness to all mitogens was recessive: this contrasts with the (LEW X BN)F1 hybrids in which responsiveness was dominant. Finally, we showed that both responder and nonresponder splenic cells were capable of binding the M. arthritidis mitogen. The data contrast with those obtained with nonresponder mouse strains the cells of which failed to bind mitogen due to the absence of the E alpha chain of the I-E-coded molecule.

Ir gene control of immune responses to insulins. II. Phenotypic differences in T cell activity among nonresponder strains of mice.

Immune responses by mice to heterologous insulins are controlled by H-2 linked Ir genes. In studies to determine the mechanisms responsible for nonresponsiveness, we found that although pork insulin failed to stimulate antibody or proliferative responses in H-2b mice, it did prime T cells that can express helper activity in adoptive recipient mice. This helper activity was insulin-specific in both elicitation and expression. In studies presented in this paper, we have extended this analysis to the response patterns of helper T cells stimulated by sheep, horse, and rat insulins in mice bearing different H-2 haplotypes. The results demonstrate that nonresponder forms of insulin, including rat insulin, prime T cells in H-2b and H-2d, but not H-2k, mice. These results suggest that regulation of nonresponsiveness to insulin appears to be through different pathways in mice bearing different H-2 haplotypes.

Lithium chloride induces partial responsiveness to LPS in nonresponder B cells.

The lipopolysaccharide (LPS)-nonresponder mouse strains C3H/HeJ, C57BL/10ScCR and C57BL/10ScN do not respond to LPS acting as a polyclonal B-cell activator, a mitogen, or an adjuvant. The genetic basis for the defective LPS response has been extensive studied in C3H/HeJ and C57BL/10ScCR mice, in which it was demonstrated that a single gene locus on chromosome 4 was responsible for LPS unresponsiveness. Lithium chloride, a potent inhibitor of adenylate cyclase, not only improved lymphocyte activity in a patient with adenosine deaminase deficiency but also enhanced the phytohaemagglutinin (PHA)-induced responses of normal human lymphocytes. Therefore, we investigated whether LiCl could restore LPS responsiveness in spleen cells of C3H/HeJ mice. We show here that LPS, in the presence of LiCl, induced polyclonal IgM and IgG antibody formation and DNA synthesis in C3H/HeJ mouse spleen cells in vitro. Moreover, LiCl (10 mM), which by itself is non-mitogenic, increased RNA synthesis in spleen cells from both LPS-nonresponder and high responders strains; in contrast, LPS failed to increase RNA synthesis in cells from such LPS-nonresponder strains as C3H/HeJ and B10ScCr mice.

The role of interleukin 1 in acute phase serum amyloid A (SAA) and serum amyloid P (SAP) biosynthesis.

The acute phase SAA and SAP profiles have been compared for localized and endotoxin induced inflammation in LPS responder and nonresponder strains of mice. The SAP profile can reflect a delay with respect to the start of the increase. Its maximum is on the order of ten times the nonacute phase concentration and elevated concentrations are sustained 24 to 48 hours after SAA concentration is rapidly decreasing to normal. The role of Interleukin 1, known to have an essential role in SAA production, was investigated for SAP production. Purified mouse IL 1 and rabbit IL 1 produced a minimal elevation of SAP concentration above normal values, especially when compared with their effects on SAA concentration. BCG infection was shown to synergistically augment SAA induction by LPS and was shown to enhance IL 1 production by macrophages in response to LPS. Unlike SAA synthesis, BCG-preinfection fails to synergistically augment the LPS-induced SAP response. BCG infection alone produced highly elevated and sustained increases in SAP concentration, whereas, the effect on SAA concentration was minimal. Macrophages appear to play an important role in SAP acute phase elevation, but the mechanism in different from that of SAA elevation.

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. II. Cellular requirements for T cell transformation mediated by a soluble Mycoplasma mitogen.

The mitogenic property of supernatants from M. arthritidis cultures (MAS) is shown to be nonsedimentable, nondialyzable, labile to 56 degrees C for 1 hr, and active against spleen cells from both normal and germfree mice. Both viable M. arthritidis and MAS were active for T lymphocytes because anti-Thy-1 antiserum and C eliminated responsiveness. Spleen cells enriched for T lymphocytes by passage over nylon columns lost responsiveness unless supplemented with a radioresistant adherent cell population that was shown to bear Ia antigens. Evidence is also presented that the genetic control of T lymphocyte responses to the mycoplasma mitogen was exercised at the level of the Ia-bearing adherent cells. Thus adherent cells from positive responder mouse strains, but not those from nonresponder mouse strains, restored the responses of T cells from F1 hybrids between responder and nonresponder strains.

Genetic control of lipopolysaccharide-induced mobilization of CFUs. Dissociation between early and delayed mobilization of CFUs in complement C5-deficient mice and LPS non-responder mice.

Lipopolysaccharide (LPS)-induced mobilization of CFUs from haemopoietic tissues into the circulation has a biphasic pattern. The first rise occurs within 30 min of LPS injection, the second 4-7 days later. This second rise coincides with an increase of the CFUs number in the spleen from about 3000 to about 50,000. We have investigated the relationship between the two peaks by making use of complement C5-deficient mouse strains and the LPS non-responder mouse strains C3H/HeJ and C57BL/10ScCr. These latter two strains lack a serologically identifiable structure ('LPS-receptor') which is present in all LPS-responder strains. After injection of eleven different mouse strains with LPS, the numbers of circulating CFUs increased rapidly in all strains, except in the C5-deficient A/J, AKR/J, DBA/2J and B10.D2/oSn mice. On the other hand, the delayed LPS-induced accumulation of CFUs in blood and spleen occurred in all mouse strains tested, including the C5-deficient strains, but not in the LPS non-responder strains C3H/HeJ and C57BL/10ScCr. These results show that (a) early LPS-induced mobilization of CFUs is dependent on the availability of C5, in contrast to the delayed CFUs accumulation in blood and spleen, (b) the presence of the LPS receptor is not required for early CFUs mobilization by LPS and (c) recognition of the mobilizing agent by a specific receptor is required for the delayed accumulation of CFUs in blood and spleen.

Bacterial phagocytosis by macrophages from lipopolysaccharide responder and nonresponder mouse strains

The phagocytic capacity of macrophages from C3H/H3J mice was assessed against lipopolysaccharide-producing (Escherichia coli) and -nonproducing (Staphylococcus aureus) bacteria. Despite their gene-coded unresponsiveness to lipopolysaccharide endotoxin and lymphokines and their defective tumoricidal activity, proteose peptone-induced C3H/HeJ macrophages did not display a defective phagocytic capacity, but rather displayed an enhanced phagocytosis of both bacterial strains compared with macrophages from closely related C3H/HeN mice. Unstimulated peritoneal resident C3H/HeJ macrophages, on the other hand, displayed a normal phagocytic activity toward E. coli and enhanced phagocytosis toward S. aureus.

IgE Responses to Synthetic Polypeptide Antigens

Abstract The serum IgE response to the synthetic polypeptide antigen L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) was investigated. The present study details the experimental conditions required to elicit optimal IgE anti-GAT antibody titers, the genetics controlling this response, and the ability of anti-suppressor cell treatments to affect the magnitude and duration of IgE anti-GAT levels in responder and nonresponder strains of mice. Significant IgE anti-GAT titers were obtained in BALB/c (IgG responder) mice after administration of 1 to 100 µg of GAT in adjuvant (Maalox or Maalox-pertussis), with optimal secondary IgE titers after challenge with 1 or 10 µg doses. All IgE responses were transient in comparison to IgG antibody levels. IgE anti-GAT immunity was found to be regulated by a gene(s) in the I-A/I-B subregion of the H-2 complex, in agreement with earlier studies on serum IgG or cell-mediated immunity. Mice bearing the H-2ja, p, q, s haplotypes were nonresponders, and treatment of SJL (H-2s) or DBA/1 (H-2q) mice with cyclophosphamide or low dose irradiation to remove suppressor cells failed to permit IgE anti-GAT responses, while markedly augmenting IgE anti-TNP responses in the same mice, and IgE anti-GAT responses in BALB/c mice. However, the presence of functional GAT-specific IgE B cells in nonresponder strains was established by demonstrating IgE anti-GAT responses to GAT complexed with methylated serum albumin in these mice. The implications of these data with respect to H-2 linked Ir gene function and class-specific regulation of antibody responses are discussed.

Macrophage activation for tumor cytotoxicity: Genetic variation in macrophage tumoricidal capacity among mouse strains

Development of activated tumoricidal macrophages following Mycobacterium bovis, strain BCG infection in vivo or lymphokine treatment in vitro was examined with more than 20 mouse strains. Peritoneal macrophages from 8 of 22 strains failed to develop tumoricidal capacity by 7 days after intraperitoneal BCG infection. Macrophages from 6 of 6 in vivo nonresponder strains also failed to develop tumoricidal capacity after in vitro treatment with lymphokines. Identification of nonresponder mouse strains should provide a useful resource for analysis of intermediary reactions in macrophage activation.

Idiotypic Analysis of Anti-Gat Antibodies

Abstract The humoral response to poly-(L-glutamic acid60, L-alanine30, L-tyrosine10), GAT, in mice is further characterized by both idiotype and fine specificity analyses. The common idiotype on murine anti-GAT antibodies (CGAT) was identified in anti-GAT antisera from seven additional strains of mice. These data confirm that the CGAT idiotype can be induced in all inbred strains of mice. Using a partially inbred strain of mice selected for their ability to respond to poly-(L-glutamic acid50, L-tyrosine50), GT, we demonstrated that the GT copolymer is capable of inducing antibodies that express the CGAT idiotype. In contrast, antisera directed against poly-(L-glutamic acid60, L-alanine40), GA, bind GAT but lack CGAT idiotype. These results indicate that GAT molecules contain determinants either similar or identical to those on GT molecules, which are responsible for the induction of CGAT idiotypic antibodies. We also demonstrated that both GAT responder and nonresponder strains of mice can produce anti-GAT antibodies with similar fine specificity patterns and CGAT idiotype. In addition, we demonstrate that antibodies of the IgM, IgG1 and IgG2 immunoglobulin classes express the CGAT idiotype.

Suppressor Cell Induction in Vitro

Abstract The capacity of responder and nonresponder strains of mice to generate suppressor cells and factors to two antigens under MHC linked Ir gene control was investigated. Eight different H-2 types (H-2b,d,f,k,p,q,r,s) as well as seven independently derived strains (B10, BALB/c, CBA/Ca, A/St, DBA/2, P/J, SJL) were tested, and all yielded suppressor factor (SF) to (T,G)-A--L and GAT. This indicated that the genetic control of SF production was different from that of helper cell induction. Unlike previous reports of GAT suppressor extracts the GAT-specific suppressor factors acted equally on both responder and nonresponder strains. As reported earlier with in vitro induced protein- (KLH) specific suppressor factors, GAT and (T,G)-A--L specific suppressor factors failed to show any genetic restriction in their function. The implications of these results for the general mechanism of Ir gene control are discussed.

Refractoriness to Migration Inhibitory Factor of Macrophages of LPS Nonresponder Mouse Strains

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.

H-2 control of the magnitude and heterogeneity of the immune response.

AbstractThe antibody responses to the random linear terpolymer poly(LLyu54, LLys36, LAla10), and its 2,4 dinitrophenyl conjugate, DNP‐GLA, were analyzed. Dominant immune response genes (Ir genes) mapping to the I region of the murine major histocompatibility complex control the magnitude of the anti‐GLA and DNP‐GLA antibody responses. Mice of the H‐2f haplotype were found to be high responders. These animals produce significantly higher titers of anti‐GLA antibody to GLA than intermediate H‐2‐congenic responder mice of the H‐2b,a,d,k,s,u,v or z haplotypes. In contrast, animals with H‐2 haplotypes j,n,p,q or r were found to be nonresponders to GLA. The relative magnitude of each strain's response to GLA was also reflected in the relative magnitude of its anti‐DNP antibody response to DNP‐GLA. High, intermediate and nonresponder mice could be further distinguished by the number of antigen‐specific plaque‐forming cells generated in the response to GLA, and in the relative amounts of IgM, IgG1 and IgG2 anti‐GLA antibodies produced in both primary and secondary anti‐GLA responses. Both intermediate and nonresponder strains of mice, however, could respond to GLA that was chemically coupled with an immunogenic carrier, chicken gamma globulin, to generate antigen‐specific plaque‐forming responses that were similar in magnitude to that produced by high responder strains to GLA.Isoelectric focusing studies revealed that GLA intermediate responder mice generated anti‐DNP antibody responses to DNP‐GLA of very restricted heterogeneity. H‐2‐congenic high responder animals, however, produced anti‐DNP antibody responses upon DNP‐GLA immunization that were significantly more heterogeneous than these restricted anti‐DNP‐GLA responses of intermediate responder animals. Thus, genes located within the major histocompatibility complex can influence the degree of heterogeneity of the anti‐DNP antibody response to DNP‐GLA. The potential mechanisms responsible for the Ir gene‐controlled differences in the magnitude or heterogeneity of the immune response to GLA or to its DNP conjugate, DNP‐GLA, are discussed.

Immunosuppressive factors from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10. IV. Lack of strain restrictions among allogeneic, nonresponder donors and recipients.

The synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) fails to stimulate development of GAT-specific antibody responses in nonresponder mice but stimulates development of GAT-specific suppressor T cells that inhibit the development of normal anti-GAT plaque-forming cell responses to GAT complexed to methylated bovine serum albumin (MBSA). Extracts from lymphoid cells of GAT-primed but not control, nonresponder (DBA/1) mice contain a T-cell factor (GAT-TsF) that also specifically suppresses responses to GAT-MBSA by normal syngeneic spleen cells. The experiments reported in this communication demonstrate that: (a) extracts from all GAT-primed nonresponder mice tested contain GAT-TsF; (b) non-H-2 genes do not restrict the production of GAT-TsF; (c) all nonresponder strains of mice regardless of their non-H-2 genes are suppressed by GAT-TsF from all other strains bearing the nonresponder H-2p,q,s haplotypes; (d) suppression of GAT-MBSA responses by both syngeneic and allogeneic nonresponder spleen cells is mediated by a molecule encoded by the H-2 gene complex; and (e) both syngeneic and allogeneic nonresponder mice are suppressed by purified GAT-TsF that lacks immunoreactive GAT.

Immunosuppressive factor(s) extracted from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) II. Cellular source and effect on responder and nonresponder mice.

The synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) fails to stimulate development of GAT-specific antibody responses in nonresponder strains of mice, but does stimulate the development of GAT-specific suppressor T cells that inhibit the development of normal anti-GAT antibody responses to GAT complexed to methylated bovine serum albumin (GAT-MBSA). Furthermore, extracts prepared from lymphoid cells of GAT-primed, but not control, nonresponder mice inhibit the development of antibody responses to GAT-MBSA by normal nonresponder mice. This suppression is specific, dose-dependent, and can be readily analyzed in vitro. The suppressive factor is a T-cell product. An extract from GAT-primed DBA/1 mice inhibits the response to GAT-MBSA by spleen cells from histoincompatible strains of mice that are nonresponders to GAT, but not strains that are responders to GAT.

Reconstitution of genetically regulated responses against random and ordered synthetic polypeptides by methylated bovine serum albumin as analyzed by isoelectric focusing

In previous publications it was shown by avidity measurements, cross-reactivity patterns and genetic analyses, that the tetrapeptide T-T-G-G is the immuno-dominant epitope of the synthetic polypeptide (T, G)-A--L. In the present study this close immunological relationship between the random multichain copolymer (T, G)-A--L and the ordered analogue (T-T-G-G)-A--L is extended by two additional criteria. First, the immune response against (T-T-G-G)-A--L in H-2k nonresponder mouse strains can be reconstituted to high antibody levels by complexing this antigen to methylated bovine serum albumin, as was tested earlier for (T,G)-A--L. The antibodies elicited upon reconstitution in both antigenic systems are directed mainly against the same determinant, T-T-G-G. Second, isoelectric focusing analysis of specific antisera developed with radiolabeled antigen revealed restricted 7 S IgG antibody populations in high responder and reconstituted high and low responder mice. The spectra were found to be of similar complexity in the (T,G)-A--L and in the (T-T-G-G)-A--L system. From these data it was concluded that the repertoires of specific B cells to T-T-G-G are very similar in high and low responder strains, and the defect in the H-2k low responder systems should be located at the level of T-B cell cooperation.

Genetic control of the immune response: ability of antigens of defined amino acid sequence to be recognized by the Ir‐1 gene system

Mice were injected with a series of (T,G)-A--L[poly (L Tyr, L Glu)-poly DL Ala)--poly (L Lys)]-like compounds with side chains of homogeneous sequences: T-A--L, GT-A--L, GGT-A--L, and TG-A--L. T-A--L was not immunogenic. However, T-A--L was able to bind antibodies to (T, G)-A--L 509, and this binding could not be blocked by A--L. When complexed with bovine serum albumin, T-A--L, was immunogenic in both responder and nonresponder strains of mice. GT-A--L and GGT-A--L were both immunogenic and elicited the characteristic responder-nonresponder difference induced by (T,G)-A--L. TG-A--L was also immunogenic, but there was considerable overlap in the response of responder and nonresponder strains. On the average, responder mouse serum had a slightly higher antigen-binding capacity than nonresponder mouse serum. In contrast to antibodies against GGT-A--L, antibodies against TG-A--L bound heterologous antigens poorly. These data, along with the results of other investigators, are consistent with the hypothesis that there are multiple Ir- 1 genes which recognize different sequences. The specificity of the Ir- 1 genes is extraordinary. The polypeptides TG-A--L, TGTG-A--L and GTTG-A--L do not appear to be recognized by these genes.