Humoral immune response to 2,4-dinitrophenyl -- keyhole limpet hemocyanin in antigen-free,germ-free and conventional BALB/c mice

The B cell immune response to 2,4-dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen-free, germ-free and conventional BALB/c mice. The numbers of total and of DNP-specific IgM-, IgG- and IgA-secreting cells in the spleen were determined by enzyme-linked immunosorbent plaque assays after primary, secondary and hyperimmunization. Three days after primary immunization a peak of DNP-specific IgM-secreting cells was seen in conventional mice only. However, this specific response was accompanied by a rise in the total number of IgM-secreting cells. At day 6 after primary immunization the total number and the frequency of DNP-specific IgG-secreting cells were higher in antigen-free mice, compared to germ-free and conventional mice. After secondary immunization in conventional mice only, a considerable bystander IgG response was seen together with the DNP-specific IgG response. At the end of the secondary response 90% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 63% and 14%, respectively. After hyperimmunization, the absolute number of DNP-specific IgG-secreting cells in the spleen was 5-fold and 11-fold higher in antigen-free mice then in germ-free and conventional mice, respectively. Approximately 48% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 17% and 12%, respectively. The results show that the exogenous antigenic load of animals influences the immune response to newly introduced antigens. The higher absolute and relative numbers of antigen-specific IgG-secreting cells after hyperimmunization in antigen-free mice compared to germ-free and conventional mice may provide a better source for antigen-specific B cells that eventually can be used for hybridoma production.     

Spleen cells from antigen-minimized mice are superior to spleen cells from germ-free and conventional mice in the stimulation of primary in vitro proliferative responses to nominal antigens

T lymphocytes from mice reared under conditions of differential exposure to food, environmental and microbial antigens were compared for phenotypic shifts that may be associated with prior exposure to antigens as well as functional variations in the ability to respond to antigens de novo. While the intra-epithelial CD8 T cell compartment was found to differ significantly in the type of T cell receptor predominantly expressed, CD4 T cells from various lymphoid organs of conventionally reared specific pathogen-free (CL-SPF) mice showed only subtle phenotypic differences from cells obtained from antigen-minimized germ-free (AF) and germ-free (GF) mice. Cells derived from mice exposed to a reduced antigen load exhibited primary in vitro proliferative responses to antigens such as dinitrophenyl-keyhole limpet hemocyanin which were significantly enhanced when compared with similar responses of cells from conventional mice. In cell mixing experiments, differences in the reactivity of T cells from the spleens of AF, GF and CL-SPF mice were dependent on the source of the spleen cells employed as antigen-presenting cells (APC). Experiments in which the T cell population was held constant revealed that, as APC, spleen cells from AF mice were most often superior to spleen cells from GF mice which were in turn considerably better than a similar population from SPF mice. We conclude that the enhanced primary reactivity of spleen cells from AF mice to nominal antigen in vitro is likely to be the result of a difference in the function and/or regulatory activities of the cell population employed as APC in this investigation.     

Influence of indigenous microbiota on experimental toxoplasmosis in conventional and germ‐free mice

Toxoplasmosis represents one of the most common zoonoses worldwide. Its agent, Toxoplasma gondii, causes a severe innate pro‐inflammatory response. The indigenous intestinal microbiota promotes host animal homoeostasis and may protect the host against pathogens. Germ‐free (GF) animals provide an important tool for the study of interactions between host and microbiota. In this study, we assessed the role of indigenous microorganisms in disease development utilizing a murine toxoplasmosis model, which includes conventional (CV) and GF NIH Swiss mice. CV and GF mice orally inoculated with T. gondii had similar survival curves. However, disease developed differently in the two animal groups. In CV mice, intestinal permeability increased and levels of intestinal pro‐inflammatory cytokines were altered. In GF animals, there were discrete epithelial degenerative changes and mucosal oedema, but the liver and lungs displayed significant lesions. We conclude that, despite similar survival curves, CV animals succumb to an exaggerated inflammatory response, whereas GF mice fail to produce an adequate systemic response.     

The relation of idiotype expression to isotype and allotype in the anti-p-azobenzenearsonate response

The distribution of idiotype-positive plaques in mice of various strains injected with T-independent (TI) and T-dependent conjugates of p-azobenzenearsonate (Ars) has been investigated. With maturation of the responses there is a progressive loss of dominance of the major cross-reactive idiotype (CRI) in A/J and C.AL-20 mice. The extremes are represented by the predominantly CRI⁺ IgM plaque response to a single injection of the TI antigen, Ars-coupled Brucella organisms (Ars-Br), and the low frequency of CRI⁺ plaques in the polyisotypic response elicited by Ars-KLH (keyhole limpet hemocyanin) in adjuvant. The secondary response to Ars-Br was characterized by an intermediate display of idiotype-positive plaques in IgM, the IgG subclasses and IgA. Detailed analyses of the distribution of CRI⁺ plaques between isotypes in 60 mice hyperimmunized with Ars-KLH revealed that the expression of idiotype in any of the IgG subclasses and IgA was constant and independent. This is interpreted to indicate random assembly of V_H with Cγ and Cα genes during the switch. BALB/c and C3H mice also express a predominant idiotype during primary and secondary responses to Ars-Br as detected by our heterologous anti-A/J CRI serum. These strains only very rarely maintain the idiotype after hyperimmunization with Ars-KLH. We conclude that hyperimmune responses which probably involve multiple modes of regulation give a poor indication of the germ-line V gene repertoire.     

Stimulation of enterocyte enzymatic activities,MHC class II expression and other immunological factors after oral treatment with Nocardia delipidated cell mitogen in germ-free rats

Nocardia delipidated cell mitogen (NDCM) was given intragastrically (200 μg/animal) to 2-month-old germ-free (GF) and conventionally (CV) reared AVN rats. On day 4, enzymatic activities of enterocyte brush border membrane vesicles (BBMV) isolated from jejunal scraping were measured. The results indicated that activities of sucrase, lactase and glucoamylase in BBMV were stimulated following NDCM treatment. The most pronounced increase of enzymatic activities was found in GF rats. In contrast to CV rats, GF rats do not express MHC class II on epithelial cells during their life. NDCM treatment induced MHC class II expression in enterocytes from GF rats. The levels of IgA and IgG in sera from NDCM-treated CV rats did not change significantly. The level of IgG in sera of GF rats was found to be enhanced after NDCM treatment. Markedly increased incorporation of ³H-thymidine by spleen lymphocytes stimulated with Con A in vitro was observed only in NDCM-treated GF rats. ³H-uridine incorporation into Con A-stimulated lympocytes of GF rats was decreased after NDCM treatment.     

Influence of bacteria from the duodenal microbiota of patients with symptomatic giardiasis on the pathogenicity of Giardia duodenalis in gnotoxenic mice

Recent studies have shown that the intestinal microbiota is essential for the pathogenicity but not for the multiplication of Giardia duodenalis in the intestinal lumen. The microbial components responsible for this phenomenon are not known. Twenty-eight facultative and three strictly anaerobic micro-organisms were isolated from the dominant duodenal microbiota of five patients with symptomatic giardiasis. The bacterial combinations from each patient were associated with groups (GN) of germ-free mice. Five days after the association, when their faecal populations ranged from 10(7) to 10(9) cfu/g, all groups were inoculated intragastrically with 10(5) viable trophozoites of G. duodenalis strain BT6. Two groups of germ-free (GF) and conventional (CV1) mice were also infected. Gnotobiotic animals were killed 10 days after infection and GF and CV1 animals were killed 10, 20 and 30 days after infection. More marked pathological alterations were detected in CV1 mice when compared with GF animals. Gnotobiotic animals showed intermediate pathological alterations between CV1 and GF mice. The CV1 and GF groups became infected by day 3 and faecal cyst levels were similar in both groups throughout the experiment. Total and G. duodenalis-specific IgA levels in the intestinal fluid and G. duodenalis-specific IgM and IgG levels in the serum increased during the infection and were higher in CV1 animals at all times tested when compared with GF mice. The present results confirm the stimulatory activity of the intestinal microbiota on the pathogenicity of G. duodenalis, and some combinations of microbial components of the dominant duodenal ecosystem from patients with symptomatic giardiasis can partially develop this function. However, none of these combinations was able to stimulate the protozoan pathogenicity in the same manner as the entire intestinal microbiota.     

Regulation of delayed-type hypersensitivity. VII. The role of I-J subregion gene products in the inhibition of delayed-type hypersensitivity to major histocompatibility antigens by specific suppressor T cells

Suppressor T cells for delayed-type hypersensitivity (DTH) to alloantigens are induced by injecting mice intravenously with a high dose of X-irradiated allogeneic cells. Using the same protocol, suppression of DTH directed against H-2 subregion products could also be induced, provided that the H-2 incompatibility between the cells used for the induction of suppression and the recipients includes the I-J subregion genes. Thus, the I-J subregion difference is both necessary and sufficient for the induction of suppression of DTH to the whole or part of the H-2 gene products. The suppression appears to be mediated by antigen-specific suppressor T cells which recognize the allo-I-J molecules and are able to suppress the DTH response to other H-2 subregion gene products in an associative recognition manner. T cells from (B10 × BALB/c)F₁ mice suppressively primed against B10.A(5R) cells (directed against J^kE^k antigens), when adoptively transferred to normal syngeneic recipients, were capable of suppressing the hosts' DTH response to B10.A(4R) cells (K^kA^k antigens) when the recipients were challenged with [B10.A(4R) × B10.A(5R)]F₁ cells (K^kA^k × J^kE^k). The recipients express normal DTH reactivity to B10.A(4R) cells when challenged with a mixture of B10.A(4R) and B10.A(5R) cells (K^kA^k + J^kE^k). These results provide direct evidence that when functioning as alloantigens, the I-J determinants preferentially induce suppressor T cells which specifically impair the immune response to the I-J molecules as well as other H-2 gene products if they are physically associated with the I-J determinants. The role of I-J subregions as the suppressor genes is discussed in terms of the possible application in transplantation immunity and the host's defence against infectious diseases.     

Aberrant IgG isotype generation in mice with abnormal behaviors

BTBR T+tf/J (BTBR) mice were recently cited as a suitable animal model for the study of autism because of their behavioral characteristics and immunological changes similar to those reported from autistic subjects. The BTBR mouse was reported to have significantly higher levels of serum IgG, brain IgG deposits and anti-brain IgG than highly social C57BL/6 mice, suggesting involvement of aberrant immune responses in the occurrence of autism. Up-regulation of IgG production was investigated here, with a focus on the pattern of IgG isotype distribution compared with that in FVB/NJ (FVB) mice, another highly social control strain. The results indicated that levels of serum IgG₁, IgG_2b and IgG₃ in post-natal day 21 BTBR mice was significantly higher than FVB mice, regardless of sex, resulting in higher IgG₁:IgG_2a ratios in BTBR mice than in FVB mice (statistical significance in males). A similar outcome regarding the IgG₁:IgG_2a ratio was observed in culture supernatants of bone marrow cells from these hosts. A presence of brain-reactive IgG in the sera of BTBR was higher than in FVB mice; levels of brain-reactive IgG against whole brain homogenates were higher in BTBR than in FVB mice, with significant differences seen in the striatum and substantia nigra regions. Levels of IgG₁ deposited in the cerebellum, cortex, hippocampus or striatum of both BTBR male and female mice were significantly higher than in FVB counterparts. Overall, these results suggest that alterations in IgG isotype production or deposition in the brain could be implicated in the aberrant immune reactivities of BTBR mice.     

Genetic deletion of the adenosine A2A receptor in mice reduces the changes in spinal cord NMDA receptor binding and glucose uptake caused by a nociceptive stimulus

Mice lacking the adenosine A_2A receptor are less sensitive to nociceptive stimuli, and A_2A receptor antagonists have antinociceptive effects. We have previously shown a marked reduction in the behavioural responses to formalin injection in A_2A receptor knockout mice. This may be due to the presence of pronociceptive A_2A receptors on sensory nerves, and if so spinal cords from A_2A receptor knockout mice may have altered neurochemical responses to a nociceptive stimulus. We tested this hypothesis by studying two parameters known to change with spinal cord activity, NMDA glutamate receptor binding and [¹⁴C]-2-deoxyglucose uptake, following intraplantar formalin injection in wild-type and A_2A receptor knockout mice. In naïve untreated A_2A knockout mice [¹⁴C]-2-deoxyglucose uptake in all regions of the spinal cord was significantly lower compared to the wild-type, similar to the reduced NMDA receptor binding that we have previously observed. Following formalin treatment, there was an decrease in [³H]-MK801 binding to NMDA receptors and an increase in [¹⁴C]-2-deoxyglucose uptake in the spinal cords of wild-type mice, and these changes were significantly reduced in the A_2A knockout mice. In addition to altered behavioural responses, there are therefore corresponding reductions in spinal cord neurochemical changes induced by formalin in mice lacking adenosine A_2A receptors. These observations support the hypothesis that activation of A_2A receptors enhances nociceptive input into the spinal cord and suggests a possible role for A_2A antagonists as analgesics.     

An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl₄) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and α‐smooth muscle actin (α‐SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl₄ and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl₄ mice, CV were enlarged, with marked sinusoidal‐free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and α‐SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl₄ and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.     

Inducing protective antibodies against ring-infected erythrocyte surface peptide antigen of Plasmodium falciparum using immunostimulating complex (ISCOMs) delivery

In the present study, synthetic peptides (EENVEHDA)₂ [(oc)₂] and (DDEHVEEPTVA)₂ [(un)₂] of ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum were linked with palmitic acid and entrapped in immunostimulating complexes (ISCOMs). The immunogenicity of the peptide(s) and mixture of peptides were studied in mice with different genetic background. Peptide(s) entrapped in ISCOMs using a low-dose immunization strategy generated high-titer as well as high-affinity antibodies. Interestingly, no genetic restriction of the immune response was observed in any of the strains studied. The IgG subclass pattern with the peptide(s) showed predominately IgG2a/2b isotypes, while with the mixed peptide formulation, (un)₂-specific IgG isotype pattern showed induction of both IgG1 and IgG2a/2b isotypes. These cytophilic antibodies inhibited the ring as well as schizont stage and total parasite growth during in vitro merozoite reinvasion inhibition study. In the mixed peptide preparation, the same pattern of immune response was achieved as that of individual peptide(s) using ISCOMs delivery. Therefore, the entrapment of otherwise poorly immunogenic synthetic peptides in ISCOMs resulted in increased immunogenicity followed by strong secondary response and can be adopted for developing subunit immunogen formulation against malarial parasite. Received: 10 February 2000     

Cytotoxic T cell responses to haptenated cells II. On the role of H-2 genes

Cytotoxic T cell (CTL) responses to cells coupled with high or low doses of the hapten 3-(p-sulfophenyldiazo)-4-hydroxyphenyl acetic acid (SP) were studied. CTL responses to densely coupled cells were obtained with all mouse strains tested. Only mice carrying the K^k allele responded to sparsely coupled cells. Mice expressing I-A^kbut not K^k were nonresponders. (Responder × nonresponder) F₁ hybrids responded in vitro to sparsely coupled cells from responder but not from nonresponder mice. However, responder mice could be cross-primed in vivo with SP-coupled cells from nonresponder mice. CTL responding to sparsely coupled cells were restricted to K^k. Cross-reactive lysis of target cells not carrying the K^k allele was dependent on the hapten density and the H-2 haplotype carried by the target cells.     

Effects of microflora on the neonatal development of gut mucosal T cells and myeloid cells in the mouse

Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut-homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM-1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM-1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3(+)beta(7) (+) cells were present in similar numbers in CV and GF intestine at birth. They were CD62L(-) in CV mice and were accompanied by further CD3(+)beta(7) (+)CD62L(-) T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus-derived T cells seed the intestinal lamina propria before birth via beta-MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool.     

Tolerance induction as a multi-step process

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule K^b were deleted in mice carrying K^b but not in mice expressing the mutant K^b-molecule K^bm1 [TCR (H-2^{bm1 × k}) mice]. These T cells were tolerant in vivo but could be activated in vitro by the K^b antigen. This in vitro reactivity was abolished after the tolerant T cells encountered K^b-positive cells that had been intravenously injected. Furthermore, in TCR (H-2^{bm1 × k}), mice expressing K^b only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with K^b on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.     

Altered Th1/Th2 balance associated with the immunosuppressive/protective effect of the H-2Ab allele on the response to allo-4-hydroxyphenylpyruvate dioxygenase

The H-2A^b allele exerts a dominant down-regulatory effect on the anti-allo-HPPD (4-hydroxyphenylpyruvate dioxygenase) antibody response, through a hitherto unknown mechanism. In the present study, the allo-variable peptide bound to responder H-2A^k molecules with higher affinity than to H-2A^b ones, arguing against the operation of an affinity hierarchy. Quantitative polymerase chain reaction revealed differences in cytokine mRNA expression between suppressed and high-responder mice. Lymph node cells of responder but not suppressed mice contained high levels of interleukin (IL)-4 mRNA as early as 11 h post-immunization and continued to do so for at least 8 days; this early burst was paralleled by a small burst in transforming growth factor (TGF)-β mRNA level. Differences in IL-12 mRNA were not detected, although an early IL-12 effect could not be excluded. Interferon (IFN)-γ appeared to contribute to the suppression at later time points. Early treatment of responder mice with anti-IL-4 monoclonal antibody (11B11) down-regulated the antibody response. The proliferative T cell response from hyperimmunized mice was reduced but still detectable in the presence of an H-2A^b allele. Thus, in the presence of this allele, the Th1 response is enhanced and that of Th2 cells suppressed, apparently as a result of the bias of H-2A^b-restricted T cells in favor of the Th1 subset.     

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β₂GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β₂GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β₂GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.     

Selective,Prolonged Alteration of Complement-Mediated Immune Clearance after Acute Exposure of Mice to Ethanol

A selective and prolonged alteration in complement-mediated immune clearance was found in mice given a single intraperitoneal injection of ethanol. Rate constants for the separate components of complement- and IgG Fcγ-mediated clearance were determined using a branched series, first-order reaction sequence model and measurements of the disappearance of radiolabeled IgG-opsonized murine erythrocytes from the circulation of BALB/c mice. The rate constant governing immune clearance mediated by IgG Fcγ receptors (k₃) decreased to 16% of control at 1 hr after ethanol injection but returned to normal in 72 hr. A >50% decrease in complement-mediated clearance occurred, with a nadir of complement-mediated sequestration (k₁) and complement-dependent phagocytosis (k₄) at 1 hr (P < 0.001). In this case, however, k₁ and k₄ rate constant values did not return to control levels until 6 weeks after the injection of ethanol. The rate constant governing C3b deactivation and release of deactivated, sensitized cells back to the circulation before they undergo phagocytosis (k₂) was initially normal, but decreased in Week 6 and remained low to the end of the observation period at 22 weeks (P < 0.0001). These changes resulted in a major reduction in overall complement-mediated immune clearance up to 4 weeks after the ethanol injection. The change to normal rates for sequestration and phagocytosis coupled with decreased deactivation and release at 6 weeks postinjection resulted in a small increase in overall complement-mediated clearance that persisted through Week 22.     

Participation of calbindin-D28K in nociception: results from calbindin-D<Subscript>28K</Subscript> knockout mice

Since calbindin-D_28K (CB-D_28K)-positive neurons have been related to nociceptive sensory processing, we have hypothesized that altered CB-D_28K expression could alter nociceptive transmission. We have used +/+ and −/− knockout (KO) mice for CB-D_28k in different behavioral models of pain and sensory responses at the caudalis subdivision of the trigeminal spinal nucleus in order to understand how this protein may participate in nociception. Behavioral responses to formalin injection in the hind paw or at the whisker pad or in the hind paw glutamate or i.p. acetic acid tests showed an increase of the pain threshold in CB-D_28k −/− mice. KO mice showed a diminution of the inhibitory activity at Sp5C nucleus and a marked reduction of GABA content. Sp5C neurons from CB-D_28k −/− mice did not change their spontaneous activity or tactile response after formalin injection in the whisker pad. In contrast, Sp5C neurons increased their spontaneous firing rate and tactile response after formalin injection in their receptive field in CB-D_28k +/+ mice. The results of this study demonstrate the active role played by CB-D_28k in nociceptive sensory transmission. The lack of this calcium binding protein, associated to deficient GABAergic neurotransmission, translates into dysfunction of sensory processing of nociceptive stimuli.     

Development and cytolytic function of intestinal intraepithelial T lymphocytes in antigen-minimized mice.

Intraepithelial T lymphocytes in the small intestine (IEL) consist of alpha beta T-cell receptor (TCR)-bearing T cells (alpha beta-IEL) and gamma delta TCR-bearing T cells (gamma delta-IEL). Development and cytolytic activation of alpha beta-IEL sharply attenuate in germ-free (GF) mice fed a natural diet (Nat-GF), but the number and cytotoxicity of gamma delta-IEL are comparable between conventional (CV) and Nat-GF mice. In this report, we compared the properties of IEL in Nat-GF mice and GF mice fed antigen-minimized diet (AgM-GF mice) of C57BL/6 strain to evaluate an influence of gut antigenic load on IEL development. Numbers of alpha beta-IEL and gamma delta-IEL in AgM-GF mice were less by 1.9- and 1.4-fold than those in Nat-GF mice, respectively. Significant decreases in the proportions of CD4+8-, CD4-8 alpha beta +, and CD4+8+ subsets and a resultant increase in the ratio of CD4-8 alpha alpha + subset were evident in alpha beta-IEL of Nat-GF mice compared with CV mice, but the subset constitution of alpha beta-IEL was similar between Nat-GF and AgM-GF mice. In contrast, relative composition of gamma delta-IEL was not different between CV, Nat-GF, and AgM-GF mice. alpha beta-IEL displayed low cytolytic activity in Nat-GF mice and were almost deprived of their cytotoxicity under the antigen-minimized condition. While gamma delta-IEL were strongly cytolytic in Nat-GF mice their cytolytic activity was remarkably reduced in AgM-GF mice. These results indicate that gamma delta-IEL are activated independently of microbial colonization in the gastrointestinal tract but their activation occurs in response to the exogenous antigenic substances other than live micro-organisms.     

Genetic control of the expression of immunoglobulin isotypes in the responses of mice to sheep red blood cells

Studies have been carried out on the genetic control of the expression of individual Ig classes in the responses of A/J (A) and C57BL/6J (B6) mice to sheep red blood cells (SRBC). An isotopic anti-immunoglobulin test employing Ig class-specific reagents was used to measure levels of IgM, IgA, IgG₁ IgG_2a, IgG_2b and IgG₃ anti-SRBC antibodies in sera of hyperimmunized mice. Compared with B6 mice, A mice are low IgM and IgA responders but high responders with respect to all IgG subclasses. In (B6 × A)F₁ mice low responsiveness was dominant for all Ig classes indicating that IgM and IgA responses are controlled by genes different from those which control IgG subclass responses. Analysis of the responses of [(B6 × A)F₁ × A] and [(B10. A × A)F₁ × A] backcross mice indicates multigenic control of IgG subclass responses. Expression of IgG_2a and IgG₃ responses appear to be regulated largely by single genes whose action is modified by genes at other loci; control of IgG₁ and IgG_2b is, clearly, polygenic with no evidence for major control by any single gene. The magnitude of all IgG subclass responses appears to be regulated by a gene(s) associated with or linked to the IgC_H locus. Analysis of the responses of H-2-congenic mice shows that major histocompatibility genes do not have a strong influence on the patterns of IgM and IgG responses in A or B6 mice. The overall results indicate that while there is some common control of all IgG classes, there is also separate polygenic control of individual Ig classes.