To drive or be driven: the path of a mouse model of recurrent pregnancy loss.

This review is an example of the use of an animal model to try to understand the immune biology of pregnancy. A well-known model of recurrent spontaneous pregnancy loss is put in clinical, historical, and theoretical context, with emphasis on T cell biology.

Impact of mouse pregnancy on thymic T lymphocyte subsets.

It has been reported that fetal lymphoid progenitor cells are acquired during gestation and are able to develop in the maternal mouse thymus into functional T cells. Moreover, previous pregnancies increase the number of fetal cells in the mother. In the present study, we investigated whether mouse pregnancy induces changes in T lymphocyte subsets in the maternal thymus. We determined the T lymphocyte subsets in two allogeneic cross-breedings, namely CBA/J×BALB/c (normal) and CBA/J×DBA/2 (abortion prone), and investigated the effects of the age and parity of the female, as well as pregnancy outcome, on thymocyte populations. In addition, hormonal effects were evaluated in a syngeneic combination (CBA/J×CBA/J). We found that during pregnancy both hormonal and allogeneic stimuli induced a reduction in the CD4(+)CD8(+) subset with an increase in the CD4(+)CD8(-) population. Only young females of the normal combination exhibited an increase in the CD4(-)CD8(+) population. All young mice showed an increase in CD4(+)CD25(+)FoxP3(+) T cells. Interestingly, the γδT thymus pool was increased in all females of the normal allogeneic pregnancy only, suggesting the participation of this pool in the observed beneficial effect of multiparity in this cross-breeding. Our results demonstrate that allogeneic pregnancies induce important variations in maternal thymocyte subpopulations depending on the age of the female and the male component of the cross-breeding.

Morphofunctional characteristics of the immune system in CBA and C57Bl/6g mice.

We studied peculiarities of morphofunctional organization of the immune system in C57Bl/6g and CBA mice differing by their susceptibility to various types of infectious agents. The revealed differences in the structure of lymphoid organs, T lymphocyte subpopulation ratio and their differentiation into Th1/Th2 cells after mitogen stimulation drove us to a conclusion on genetically determined regularities in the development of the immune response in these animal strains.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

A T helper cell for anti-viral cytotoxic T-cell responses.

We demonstrate here: (a) the existence of T helper (Th) cells that augment the generation of virus-specific cytotoxic T cells in vitro, (b) that the helper cells carry the theta and Ly 1 membrane antigens, (c) that activation of the Th effect is specific for viral antigens, and (d) that the delivery of help is not H-2 restricted.

Induction of partial chimerism in nonirradiated B-lymphocyte-deficient CBA/N mice.

Nonirradiated B-lymphocyte-deficient CBA/N mice given T6T6 chromosome-marked normal CBA/CaHN spleen cells became lymphoid chimeras exhibiting donor-type mitoses. Normal CBA/CaHN recipients did not exhibit significant numbers of donor-type mitoses. The lymphoid cell chimerism in the CBA/N host appeared in spleen, lymph nodes, and Peyer's patches, but not in marrow or thymus. Stimulation of CBA/N-recipient spleen cells in vitro suggested that the chimerism involved donor T6T6 cells which were responsive to the B-lymphocyte mitogen, lipopolysaccharide, but not to the T-lymphocyte mitogen, phytohemagglutinin. These data indicate that stable, long-term chimerism of a specific class of lymphocytes is possible in nonirradiated, B-lymphocyte-deficient CBA/N mice.

Tolerance to rat monoclonal antibodies. Implications for serotherapy.

The antiglobulin response is a major complication of mAb therapy. It has been suggested that, in clinical practice, this might be avoided by using human or chimeric mAbs, or by prior induction of tolerance to the therapeutic mAb. In this study, we show that it is possible to induce tolerance in mice to the constant regions of rat IgG2b mAbs by both classical deaggregation methods and by anti-L3T4 mAb therapy. Mice tolerant to IgG2b constant region determinants failed to make an antiglobulin response when immunized with a number of mAbs of the same isotype that had no binding specificity for mouse cells, but produced vigorous antiidiotypic responses to cell-binding mAbs. Binding of antibodies to hemopoietic cells rends their idiotypic determinants major immunogens even in the presence of tolerance to constant region epitopes. These findings suggest that the use of human or chimeric mAbs will not be sufficient to eliminate the antiglobulin response, and that additional methods need to be investigated.

Antibody-defective, genetically susceptible CBA/N mice have an altered Salmonella typhimurium-specific B cell repertoire.

CBA/N mice, which express the X-linked immunodeficiency gene xid, are susceptible to Salmonella typhimurium. The basis for this susceptibility is currently unknown. However, previous studies (10) from this laboratory have provided evidence that susceptibility may be due to a defective anti-S. typhimurium antibody response. In that report we hypothesized that the defective antibody response may be a reflection of an altered S. typhimurium-specific B cell repertoire. In the studies described here, we have investigated this hypothesis using a modification of the in vitro splenic focus system. The frequency and characteristics of salmonella-specific B cells in normal, innately resistant, CBA/Ca mice have been compared with those of salmonella-susceptible, anti-S. typhimurium antibody-defective CBA/N mice. The results show that CBA/N mice express no primary or secondary S. typhimurium-specific B cell precursors after stimulation with an acetone-killed and dried (AKD) preparation of S. typhimurium strain TML. However, after three immunizations, the CBA/N tertiary frequency of 15.4 per 10(6) splenic B cells was similar to the primary precursor frequency in immunologically normal CBA/Ca mice, but 23-fold lower than the tertiary precursor frequency in CBA/Ca control mice. Moreover, CBA/N mice had an altered isotype distribution pattern after stimulation with AKD-TML. Greater than 70% of the tertiary CBA/N TML-specific B cells secreted IgG2, in contrast to either nonimmune or primed control mice. In addition, 80% of the CBA/N TML-specific B cells secreted only a single isotype, whereas the majority of B cells from primed normal mice secreted multiple isotypes. Fine specificity analysis of the TML-specific B cells indicated that the array of antigenic determinants to which CBA/N B cells could respond was restricted. Although the majority of primed CBA/Ca and primed CBA/N B cells were specific for LPS, the fine specificity pattern exhibited by CBA/N B cells was similar to that observed in unprimed normal mice, i.e., the vast majority were specific for the O antigen region of the LPS molecule. In contrast, a major portion of the LPS-specific B cells in primed CBA/Ca mice were directed against the KDO/lipid A region of the LPS molecule. Therefore, it appears that CBA/N mice lack or are unable to stimulate the B cell subset that predominates in primed, normal mice. Taken together, these studies indicate that the basis for susceptibility of CBA/N mice to S. typhimurium is multifactorial and suggests that the inability of some animals to respond to some infectious agents may be related to holes in their B cell repertoire.

CBA/N X-linked B-cell defect prevents NZB B-cell hyperactivity in F1 mice.

NZB mice and their F1 hybrids produce excessive polyclonal IgM and autoantibodies of both IgM and IgG classes. CBA/N mice and CBA/N-mothered F1 males fail to make antibody to many T-independent antigens and have low levels of serum IgM; further, these mice lack a population of splenic B cells characterized by a low-to-intermediate density of surface IgM. We have studied male CBA/N, NZB, CBA/N X NZB, NZB X CBA/N, and CBA/J mice; female CBA/N X NZB mice; and males of several control crosses of NZB and CBA/N mice. We have found that the CBA/N X-linked defect of T-independent immune response is completely expressed in CBA/N X NZB mice. In marked contrast to NZB mice and to NZB mice and to NZB F1 hybrids bearing at least one normal X chromosome, the CBA/N X NZB males failed to respond to two T-independent antigens, had small numbers of splenic IgM-producing cells, barely detectable splenic IgM production, and splenic B-cell surface-Ig patterns resembling those of CBA/N mice. These data suggest that the NZB B-cell abnormality resulting in excessive IgM production occurs almost exclusively in that population of B cells affected by the CBA/N X chromome-linked defect. Preliminary studies suggest that CBA/N X chromosome retards the spontaneous development of anti-erythrocyte autoantibodies in CBA/N X NZB males. Castration, known to accelerate autoimmune disease in certain NZB F1 males, appears to have no influence on the immune functions examined in this study.

Ontogeny of B cells in CBA/N mice. Evidence for a stage of responsiveness to thymus-independent antigens during development.

This paper deals with the CBA/N mice, a strain bearing a genetic defect in their B-cell compartment. By using a previously described system we have been able to show that the immature cells of CBA/N mice are functionally indistinguishable from normal immature cells, in that both can be triggered to respond to thymus-independent (TI) antigens, provided they are supplied with helper T cells. When the maturation is completed, CBA/N B cells are unable to respond to TI antigens (like lipopolysaccharide and polyvinyl pyrrolidine) irrespective of the presence of helper T cells, whereas normal mature B cells have grown able to respond without any help. These data allow us to reject the hypothesis that CBA/N mice are arrested at an immature stage and clearly support the idea that they have deviated during development so that only thymus-dependent B cells develop.

Influence of the sex-linked defect in CBA/N mice on autoimmune responses to isologous erythrocytes. Ability to overcome the defect with age.

Normal mice spontaneously develop plaque-forming cells (PFC) specific for antigens on modified self erythrocytes (bromelain-treated mouse erythrocytes [BrMRBC] antigens). Our study demonstrates that the sex-linked defect that results in the inability of CBA/N mice to respond to several T-independent antigens (TI-2 antigens) also regulates the autoantibody response to BrMRBC antigens. Thus, in CBA/N homozygous mice and male F1 offspring of CBA/N-mothered crosses, e.g., (CBA/N X NZB)F1 males, such PFC are absent. To examine whether specific autoreactive B cells are present in defective mice, the latter were stimulated either nonspecifically with the mitogen LPS or by infection with lethal malaria (17XL Plasmodium yoelii) known to induce anti-BrMRBC PFC specifically. The results indicate that modest antibody responses to self antigens could be induced in young (5- to 7-wk old) defective mice and that these responses increased as a function of age. The data is consistent with the view that the defect in CBA/N mice does not result from an absence of functional anti-BrMRBC B cells but rather from low frequencies of the specific precursors, which can be triggered and expanded with age probably by environmental stimulations.

In vitro cell-mediated immune responses to the male specific(H-Y) antigen in mice.

C57BL/10 female mice were primed to the male specific antigen H-Y, either by grafting with syngeneic male tail skin or by i.p. injection of syngeneic male spleen cells. Primed female spleen cells, either unseparated or filtered through nylon wool to remove most of the B lymphocytes, were then cultured for 5 days in vitro with irradiated syngeneic male spleen cells and assayed against 51Cr-labeled target cells. Both unseparated and nylon wool filtered female cells displayed significant cytotoxic activity restricted to male target cells. Pretreatment of sensitized female cells with antitheta serum and complement just before assay abolished cytotoxic responses. We were unable to demonstrate cell-mediated cytotoxic responses into two nonresponding strains, CBA and B10.A, which fail to reject male isografts. The cytotoxic activity of C57BL/10 female cells was restricted to male target cells histocompatible with C57BL/10 over at least a portion of the major (H-2) histocompatibility complex. We conclude that secondary in vitro cytotoxic responses against the H-Y antigen are mediated by cytotoxic T lymphocytes, and that the H-Y target cell antigen may be specified by the H-2 complex.

Analysis of H-2 determinants recognized during the induction of H-Y-immune cytotoxic T cells by monoclonal antibodies in vitro.

Monoclonal antibodies directed to the D region of H-2(k) when present during in vitro culture inhibit the generation of CBA/H and C3H.H-2(o) H-Y-immune cytotoxic T cells . Monoclonal antibodies directed to the I-A(k) and I-E(k) region specifically inhibited induction of CBA/H H-Y-immune cytotoxic T cells only when they were present simultaneously in culture. These findings show T helper cell requirement for CBA/H H-Y-immune cytotoxic T cell induction, and suggest that two I region-coded restriction antigens for T helper cells are involved.

Primary in vitro cytotoxic T cell response to non-major histocompatibility complex alloantigens in normal mice.

We have shown for the first time that it is possible to consistently generate a primary in vitro cytotoxic T cell (Tc) response to non-major histocompatibility complex alloantigens using responder cells from a normal mouse strain. This was achieved by carrying out, in the generating phase, a limiting dilution procedure in which it appears that suppressor cells that inhibit Tc activation or expansion are too dilute to manifest their effect. Moreover, the response was observed in mouse serum-(MS) as well as fetal calf serum- (FCS) supplemented media, an important finding in the light of the anomalous nonspecific effects induced by FCS. The cytotoxic response produced in MS-supplemented media was shown to be highly specific in both the generating and effector phases, whereas the responses in FCS had a strong nonspecific component.

Identification of a B-cell surface structure involved in antigen-dependent triggering: absence of this structure on B cells from CBA/N mutant mice.

CBA/N mice have an X-linked B-cell maturation defect which is reflected in part in an absence or dysfunction of a subclass of mature B cells. We have immunized the defective male offspring of the mating (CBA/N female X BALB/c male) with BALB/c spleen cells. The resulting antiserum (alphaLyb3) selectively reacts with a component on the surface of a portion of B cells from a panel of H-2 different mouse strains. Binding of alphaLyb3 serum to this B-cell subclass results in substantial (10- to 20-fold) enhancement of the antibody response to low doses of SRBC. Both binding and enhancing activity are removed by absorption with B cells from B6 and BALB/c, but not CBA/N mice. Absorption of the serum with bone marrow cells, T cells, or thymocytes from Lyb3+ strains does not remove activity. Since the enhanced plaque-forming cell (PFC) responses are specific for the immunizing antigen, and since no PFC response is produced by injection of the antiserum alone, this enhancement probably reflects a second signal produced by specific interaction between antibody and the surface Lyb3 component. Moreover, this signal can partially replace the requirement for T cells in the production of antibody to a "thymus-dependent" antigen. These findings (taken in conjunction with the previously described immune defects in CBA/N mice and other studies of B-cell maturation) suggest to us that Lyb3 is a cell surface component expressed selectively on a mature B-cell subclass. This component is important in B-cell triggering by antigen and fails to develop in CBA/N mice, due to a dysfunction of a regulatory gene on the CBA/N X chromosome.

Defective colony formation by B lymphocytes from CBA/N and C3H/HeJ mice.

B cells from CBA/N mice did not form colonies in semisolid agar cultures under circumstances where normal B-cell clonal proliferation is linear with respect to the number of functional cells cultured. This was no due to the unresponsiveness of CBA/N cells to mitogens, and under appropriate liquid culture conditions many CBA/N lymphocytes differentiated to plasma cells containing large amounts of IgM in response to LPS. On the other hand, the same cells proliferated and matured poorly in liquid cultures prepared at low-cell density. The frequency of granulocyte-macrophage progenitors and multipotential hemopoietic stem cells in bone marrow, ability of peritoneal macrophages to elaborate soluble enhancing factors, and levels of serum inhibitors were normal in CBA/N mice. Together with the results of cell-mixing experiments, these findings confirm the selective and intrinsic nature of the CBA/N deficiency. It is suggested that the B-cell cloning technique may be of value in selectively enumerating and assessing functional capability of thymus-independent B cells. C3H/HeJ mice which have previously only been known to be hyporesponsive to certain forms of lipopolysaccharide had a subnormal incidence of colony-forming B cells.

Subclass restriction of murine antibodies. II. The IgG plaque-forming cell response to thymus-independent type 1 and type 2 antigens in normal mice and mice expressing an X-linked immunodeficiency.

Antigens have been classified previously into three categories, thymus-dependent (TD), thymus-independent type (TI) 1, and TI-2, based upon thymic dependence and ability to stimulate an immunodeficient strain of mouse, CBA/N. Here we demonstrate that the different antigen classes elicit IgG antibodies of different subclasses. TD antigens stimulate predominantly IgG1 antibodies, with smaller amounts of IgG2 and IgG3 being expressed. TI-1 antigens stimulate almost no IgG1 antibodies and equal amounts of IgG2 and IgG3. TI-2 antigens elicit predominantly IgG3 antibodies. Mice expressing the CBA/N phenotype are known to be nonresponsive to TI-2 antigens. This was confirmed in this study. In addition, we demonstrate that the IgG3 component of the response to TI-1 antigens is virtually absent in mice expressing the CBA/N phenotype, which supports our previous finding that the CBA/N defect may be restricted to a B-lymphocyte subpopulation containing most of the precursors of IgG3-secreting cells.

B-cell differentiation in the CBA/N mouse. I. Slower maturation of mitogen and antigen-responsive B cells in mice expressing an X-linked defect.

The effect of age on the mitogenic and antigenic responsiveness of B cells is examined in spleen cell cultures of CBA/N and (CBA/N X DBA/2) F1 mice. Spleen cells from young male F1 mice (4- to 6-wk old) show lower mitogenic responses to lipopolysaccharide, a lower frequency of sheep erythrocytes (SRBC)-reactive B-cell precursors, and a lower percentage of Ig-bearing cells than age-matched female F1 mice. The expression of all three functions were found to increase with the age of the F1 male mice. Whereas male F1 mice at 60 wk of age showed an equivalent percentage of Ig-bearing spleen cells and a similar mitogenic responsiveness to LPS when compared to adult female F1 mice, the frequency of SRBC-reactive B-cell precursors remained threefold lower. These findings reveal that there is a slower maturation of B cells in mice expressing the X-linked defect and suggests that the defect has differential effects on the mechanisms of antigen and mitogen activation of B cells.

Reaginic antibody produced in mice with contact sensitivity.

Mice produced reaginic antibody within 1 wk of painting with the contact sensitizing agent picryl chloride. The titers, measured by passive cutaneous anaphylaxis in rats, increased after repeated applications of picryl chloride. In contrast, serum agglutinins did not increase after two applications of picryl chloride. Reagin was also elicited by another contact sensitizing agent, oxazolone. Some strain variation of the response to picryl chloride was found, with CBA mice being good responders and BALB/c and C57BL/10 mice being poor responders.

Antibody response of immunodeficient (xid) CBA/N mice to Escherichia coli 0113 lipopolysaccharide, a thymus-independent antigen.

CBA/N mice, which possess an X-linked immunodeficiency (xid), produce a convincing antibody response to lipopolysaccharide derived from Escherichia coli 0113 (LPS 0113), a thymus-independent antigen. The antibody response produced was shown to be specific for the O-polysaccharide moiety of LPS 0113, rather than lipid A or lipid-A-associated protein. The relevance of this finding to the nature of the genetic defect of xid-mice is discussed.