Immunological competence and host-specific tolerance of antibody-facilitated bone marrow chimeras.

We have generated murine antibody-facilitated (AF) bone marrow chimeras in the genetic combination P1----(P1 X P2)F1 by the simultaneous injection of P1 bone marrow cells and anti-P2 MHC monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These mice have normal life spans and appear to be healthy, with no overt signs of graft-versus-host disease. We have undertaken an extensive survey of the ability of stable, long-term AF chimeras to generate immune responses in vitro and in vivo. Both T and B lymphocyte functions have been analyzed in proliferative and effector cell assays. The AF chimeras respond normally to mitogenic as well as antigenic stimuli, and exhibit normal capacities for cellular collaboration in the generation of immune responses. However, splenic lymphocytes from AF chimeras are substantially and specifically hyporesponsive or nonresponsive to host, P2-encoded, alloantigens in in vitro assays of cell-mediated immunity. This host-specific tolerance is exhibited by the cytotoxic T lymphocyte lineage; T helper cells necessary for the generation of a cytotoxic response may also have decreased reactivity to host determinants. We conclude that our protocol for the production of AF chimeras does not compromise the immune system of chimeric animals but does allow the maintenance of host-specific tolerance, after stable equilibrium has been attained.

Characterization of hemopoietic stem cell chimerism in antibody-facilitated bone marrow chimeras.

We have previously described a model for bone marrow transplantation that involves preparation of the host with monoclonal antibody against class I or class II antigens instead of irradiation or cytotoxic drugs. This allows engraftment and subsequent repopulation of the host by donor tissue. We have previously reported on chimerism in the peripheral blood of P1----(P1 X P2)F1 animals. In this report, we describe the examination of the bone marrow and spleen stem cell chimerism of these antibody-facilitated (AF) chimeras, by determining, with an isozyme assay, the phenotype of methylcellulose colonies grown from stem cells. We have found a correlation between peripheral blood chimerism and the stem cell constitution of both spleen and bone marrow. The peripheral blood chimerism also correlates with the level of chimerism in macrophages derived from peritoneal exudate cells. These findings indicate that assaying the peripheral blood of such chimeras provides an excellent indication of the degree of chimerism at the stem cell level and stands in sharp contrast to the level of chimerism in certain lymphoid compartments, as described in our accompanying article.

Characterization of immune effector cells present in early murine decidua.

It has been suggested that murine decidual cells act as an important immunoregulatory population localized to the pregnant uterus. We have examined early murine decidua to determine if immune effector cells occur in the decidual environment in proximity to the conceptus. High levels of natural killer (NK) cell activity were found consistently in decidual cell suspensions with peak activity occurring on Day 6.5 of gestation. NK activity declined as pregnancy proceeded and was not significant by Day 12.5 of gestation. Decidual cell suspensions did not appear to contain significant numbers of functional B or T effector cells. No antipaternal T-cell response could be demonstrated even in the decidua of immune mice. Lack of T-cell responses was attributed to the absence of T cells from decidua rather than to their inactivation because precursors of cytotoxic T lymphocytes (pCTL) could not be detected in decidual cell suspensions. Furthermore, the levels of pCTL detectable in spleen cell suspensions could not be reduced by mixing spleen cells with 7.5-day decidual cells. These results suggest that B cells and T cells may not occur in early decidua while NK cells are present and regulated independently.

Characterization of host lymphoid cells in antibody-facilitated bone marrow chimeras.

We have produced stable murine antibody-facilitated (AF) chimeras by the simultaneous injection of P1 bone marrow cells and anti-P2 monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These AF chimeras are healthy, long-lived, and exhibit no overt signs of graft-versus-host disease. They are immunocompetent and tolerant of host, P2-encoded alloantigens. Donor cell engraftment and takeover, monitored by glucosephosphate isomerase isozyme patterns, is usually complete (greater than 95%) in the peripheral blood, bone marrow, and hemopoietic stem cell compartments of long-term (greater than 3 months posttransplantation) AF chimeras. We report here, however, that splenic, lymph node, and thymic leukocytes of AF chimeras represent donor/host chimeric populations. Spleen cell populations of AF chimeras exhibit substantial chimera-to-chimera variation in the preponderant residual host cell type(s) present. Our interpretations of the implications of these findings are discussed.

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy.

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.

Antibody-facilitated chimeras. Stem cell allotransplantation using antihost major histocompatibility complex monoclonal antibodies instead of lethal irradiation for host conditioning.

Complete hemopoietic takeover between semiallogeneic adults can be accomplished by the administration of antihost major histocompatibility complex (MHC) monoclonal antibody (mAb) and donor stem cells. The recipients of such treatment are termed antibody-facilitated chimeras, and they have been produced in (BALB/cCr----(BALB/cCr X C3H/HeJ)F1 and DBA/2J----(DBA/2J X C3H/HeJ)F1 strain combinations. Donor stem cells can be derived from spleen, bone marrow, or T-cell-depleted bone marrow. Engraftment by donor hemopoietic cells can be facilitated by mAbs directed toward class I (anti-H-2Kk) or class II (anti-H-2I-Ak) MHC antigens of the host. By monitoring isozymes of glucose phosphate isomerase, it can be shown that the establishment of donor hemopoiesis is stable, persisting for more than a year without graft-versus-host disease. The effect of anti-MHC mAb on pluripotential stem cells was evaluated by the (colony-forming units-spleen) (CFU-S) assay. The number of CFU-S in target (H-2k) bone marrow was reduced by in vitro pretreatment with anti-H-2Kk mAb, but not with anti-H-2I-Ak mAb. In contrast, in vivo administration of either anti-H-2Kk or anti-H-2I-Ak mAb resulted in a marked decrease in the CFU-S capacity of relevant strain mice. These observations suggest that the target of the in vivo mAb treatment may be pluripotential stem cells--or perhaps the hemopoietic microenvironment.

Placenta as a selective barrier to cellular traffic.

Transplacental passage of cells from mother to fetus during murine pregnancy was examined by using glucose phosphate isomerase (GPI) or fluorescein tagging as markers. Female mice of strains (BALB/cCr X C3H/HeJ)/F1 or (A/J X C57BL/6J)F1 (both Gpi-1a/b) were mated to the corresponding Gpi-la males (BALB/cCr or A/J, respectively). Cells of the liver, blood, and/or spleen in the offspring were typed at days 15, 16, 17, or 18 of gestation, the day of delivery, or 1 day postpartum. Only two of 172 Gpi-1a/a mice obtained from these matings showed evidence of maternal cell trafficking. Sensitivity of the assay was 1% Gpi-1a/a population. Fluorescein-labeled BALB/cCr peripheral red blood cells (RBC) or white blood cells (WBC) were injected i.v. into syngeneically mated BALB/cCr mothers on day 18. After 24 hr, the blood or liver of the neonates was formalin-fixed and examined in the fluorescence-activated cell sorter (FACS). Some RBC crossed the placenta, but WBC were usually not detected in fetal liver in significant numbers. This technique was very sensitive, and we estimated that no more than 225 WBC could enter the fetus via this route. Thus, we conclude that passage of significant numbers of maternal WBC into the fetus is rare, and perhaps this passage is related to a placental abnormality.

Suppression of cellular immune responses by mouse amniotic fluid.

Mouse amniotic fluid (AF) strongly inhibited the in vitro generation of cell mediated cytolytic responses to allogeneic EL4 tumor cells. Potent immunoregulatory capabilities of AF in utero are suggested by in vitro suppression of immune responses by concentrations below physiological levels and by the inhibition of lytic function late in effector cell differentiation. Finally, suppression was not contingent on the parity of the female or the source of effector cells or of the AF.

Pregnancy and lactation induced suppression of cell mediated immunity.

The effect of pregnancy and lactation on effector or killer cell responses of syngeneically bred BALB/c (H-2d) female mice to the EL4 (H-2b) tumor and C57BL/6 (H-2b) female mice to the P815 (H-2d) tumor was evaluated with cell mediated cytolysis (CMC). Virgin age correlated females served as controls. Maternal spleen cell responses to the tumor were depressed at 10-15 days of pregnancy and during lactation at 1-5 days and 10-20 days postpartum. One to five days after weaning the maternal response began to increase again. In this study the greatest suppression of the response occurred at 1-5 days postpartum. The route of tumor sensitization, i.e. intraperitoneal or subcutaneous, did not make a difference in the depression of the maternal response. Serologic evaluation of splenic cell populations with anti-Thy 1.2 serum did not indicate differences which could account for the suppressed maternal CMC response.

An in vitro and in vivo analysis of murine immunocompetence during pregnancy and lactation.

Spleen cells from pregnant and lactating BALB/c mice were depressed in their cytolytic capabilities after in vivo immunization with the allogeneic EL4 lymphoma. However, in vitro spleen cells from both syngeneic (BALB/c X BALB/c) and allogeneic (BALB/c X C57BL/6J) matings responded with proliferative and cytolytic responses which were comparable to virgin controls. Upon secondary in vitro stimulation, in vivo primed maternal cells had responses which were similar to virgin controls. In addition, the in vivo sensitized maternal spleen cells adoptively immunized in irradiated allogeneic recipients responded like the virgin controls. In these studies, suppressor cells could not be detected in either nonimmune or immune maternal spleen cell populations.

Alterations in thymus and spleen cell populations and immune reactivity during syngeneic pregnancy and lactation.

Maternal thymus and spleen cell composition and mitogenic responsiveness were examined in the mouse during syngeneic pregnancy and lactation. The results indicated significant changes in thymus cell populations and in thymus cell in vitro mitogen, mixed-leukocyte and in vivo graft-versus-host (GVH) responses during pregnancy and lactation. In the spleen, in vitro mitogen responses were not altered, however, in vivo GVH reactivity was decreased during lactation.