Immunodeficiency in RFM/(T6xRFM)F1 mouse chimaeras with lethal host-versus-graft syndrome.

Rather than central tolerance, the perinatal inoculation of related F1 hybrid spleen cells into inbred mice may result in host-versus-graft (HVG) reactions manifested as transient autoimmunity, or as a lethal immunodeficiency syndrome. RFM/(T6xRFM)F1 chimaeras with lethal disease die in 30 days with lymphosplenomegaly, immune complexes and impaired immune responses. The present studies used in vitro proliferation assays to show that the HVG reaction caused hyperplasia sufficient to account for the lymphosplenomegaly, while also causing severe impairment of splenic and nodal cell responses to concanavalin A (Con A) and to bacterial lipopolysaccharide (LPS). By 25 days, HVG mice could not distinguish between self and non-self as judged by mixed lymphocyte reactions (MLR) to RFM, (T6xRFM)F1 and third party A/J cells. There were no indications that host cells reactive to F1 donor cells had undergone clonal deletion, anergy or expansion. Flow cytometry revealed that donor T lymphocytes achieved stable engraftment, mostly in the nodes, despite the HVG reaction. Taken together with previous observations, these studies showed that HVG reactions in young parent F1/chimaeras can result in an immunodeficiency state which is characterized by an early appearing, profound and persistent impairment of both host and donor T and B cell functions. The results suggest that HVG reactions can contribute directly to immune deficits seen after clinical allogeneic bone marrow transplantation.

Use of polymorphic DNA probes to test the lymph nodes of infants and fetuses for spontaneous chimerism.

Spontaneous chimerism is rare, but it has been observed in infants who have received intrauterine transfusions, or in those infants who have severe combined immunodeficiency disease. The site of maternal cell engraftment has not been identified in these cases, but it is apparently not the bone marrow. Lymph nodes have not been investigated. Based on recent experimental data that showed that foreign cells grow in the lymph nodes, but not in the marrows of perinatally inoculated mice, the decision was made to use highly specific polymorphic DNA probes to assay for the presence of maternal DNA in the nodes that were removed from three infants and two fetuses. No evidence for the engraftment of maternal cells was detected at a 5% level of sensitivity. These results were in accord with findings from previous work that has shown that the placenta and host-resistance mechanisms combine to make spontaneous chimerism a rare event. The results also showed that similar tests for maternal cell engraftment in lymph nodes and other sites, not previously evaluated, are feasible and perhaps indicated in infants with congenital and acquired immunodeficiencies, such as severe combined immunodeficiency disease and acquired immunodeficiency syndrome.

A background-free assay for cells forming antibody to glucose oxidase coupled with an enzyme-linked immunosorbent assay (ELISA) to quantitate antibody secretion.

We describe an immunocytochemical assay for cells forming antibody to glucose oxidase (GO). The method is specific in that only cells containing intracytoplasmic antibody capable of binding the immunogen (GO binding cells; GOBC) are stained. The method is sensitive because there is no GO activity in mammalian tissues. This lack of background readily permits detection of one GOBC among 10(6) nucleated lymphohemopoietic cells. The technique is reliable because purified chemicals are used. Although it is not possible to determine the Ig class of antibody formed by an individual cell, as can be done with the hemolytic plaque assay, the amount and class of secreted antibody to GO can be quantitated by an indirect enzyme-linked immunosorbent assay (ELISA), which is also described. GO is immunogenic and stimulates the formation of large numbers of GOBC in the popliteal lymph nodes after injection with adjuvant into the footpads of mice, but 1-mg doses injected IV or IP are lethal because of its enzymatic activity, which causes hypoglycemia and methemoglobinemia.

Unfractionated spleen cells but not natural killer (NK) cells from RFM donors prevent the progression of host-versus-graft disease in murine RFM/(T6 x RFM)F1 chimeras.

Host-versus-graft (HVG) syndrome is the fatal allogenic disease which develops in susceptible strains of inbred mice following their perinatal inoculation with related F1 hybrid spleen cells. Deaths are caused by pathogenic immune complexes. It is thought that the antibody components of these complexes are produced by F1 donor B cells stimulated by the allogenic HVG reaction. To complement previous work that showed that lethal disease could be prevented if the HVG response was suppressed, the present studies tested whether or not it could also be prevented by augmenting HVG reactivity with the adoptive transfer of spleen cells syngenic with the host. The data show that unfractionated RFM spleen cells given on Days 13-14 prevented lethal disease in 86% of RFM/(T6 x RFM)F1 chimeras. Successful therapy was associated with the suppression of formation of nephropathic-immune complexes, and with the rejection of F1 donor cells or their gradual replacement by host cells. RFM spleen cells enriched for NK activity by a new improved method not only failed to prevent HVG disease but appeared to exacerbate it. This was also true of spleen cells that had been activated in vitro for 3 days with IL-2, a procedure that greatly enhanced their cytolytic activity against YAC-1 targets. It is suggested that therapy with NK cells failed, even after IL-2 activation, because they had no effect on proliferating and antibody-forming F1 donor cells that had engrafted in large numbers in the lymph nodes of the RFM hosts.

Apparent difference in the roles of spleen and lymph nodes in the pathogenesis of host-versus-graft disease in murine parent/F1 chimeras.

Host-versus-graft (HVG) disease is the often fatal immunodeficiency syndrome that can be induced in susceptible strains of mice by the perinatal inoculation of semiallogenic spleen cells. To determine the distribution and engraftment of the donor cells in the spleens and lymph nodes of RFM hosts, sequential tests were done for the presence of (T6 X RFM)F1 cells marked by their ability to form donor-specific antibodies to horseradish peroxidase (HRP) and by the T6 chromosome. Quantitation of cells with intracytoplasmic antibody that bound HRP (HRPBC) and of metaphases with the T6 marker showed that greater than 90% of donor cells were located in the hyperplastic lymph nodes. The pattern of sequential changes in numbers of HRPBC corresponded with the rise and fall in titers of antibodies to HRP. The marked differences in localization of donor cells suggested that host nodes and spleens played different roles. The lymph nodes became the main sources of donor antibodies and the principal repositories of (T6 X RFM)F1 cells capable of replication. The spleen evidently served as a major site of host resistance to engraftment. This was attributed to the ability of host cells there to inhibit selectively the proliferation of the semiallogenic donor cells. It is also proposed that counts of HRPBC measured the vigor of the HVG reaction in host spleen and lymph nodes, because the appearance of virtually all these antibody-forming cells was the result of the maturational effect of the allogenic reaction on F1 donor B memory cells.

Sensitized (T6 x RFM)F1 donor B cells contribute to hypergammaglobulinemia and to poor primary responses in RFM mice with allogenic host versus graft disease.

Experimental host versus graft (HVG) disease is the fatal immunodeficiency syndrome which is induced in susceptible strains of inbred mice by the perinatal inoculation of related F1 hybrid spleen cells. The allogenic HVG reaction results in severe T-cell depletion, but hyperplasia of B cells, of which some are F1 donor in origin. To investigate the role of F1 donor B cells in the development of hyperglobulinemia in HVG mice which respond poorly to primary antigenic challenge, antibodies to horseradish peroxidase (HRP) of (T6 x RFM)F1 donor B-cell origin were used as markers for the engraftment of primed donor B cells in RFM hosts, and as sequential measures of the allogenic reaction on them. F1 donor B cells sensitized to HRP survived different stages of the HVG reaction after inoculation on Day 1 or Day 8 after birth. Tests for the anti-HRP antibody output of RFM host cells, and engrafted HRP-primed and unprimed (T6 x RFM)F1 donor cells suggested that the hyperglobulinemia seen in HVG mice was caused principally by antigen-primed, F1 donor B cells stimulated by the allogenic effect, with or without further exposure to the antigen(s) to which the donors had been sensitized prior to transplantation. The poor primary responses were attributed to the engraftment of the many donor B cells already committed, to the immunological immaturity of the host B cells, and to the lack of T-cell help for adult unprimed F1 donor B cells. Taken together with previous work, the data also suggest that antigen-primed donor B cells were engrafted in preference to equally histoincompatible donor T cells and unprimed donor B cells.

Immunoperoxidase in lymphomas induced by host-versus-graft (HVG) reaction.

It appears that the development and type of lymphoid neoplasm in mice depends on the ratio of T cell subsets and the repeated stimulation of the lymphoid systems as well as a genetic predisposition. Mice with HVG disease developed lymphomas in an average of 462 days versus 567 days for normal mice. Murine leukemia virus was detected in the HVG mice perinatally at 3 weeks of age and three months earlier in the noninjected mice. The immunoglobulins were IgA and IgM or IgM alone and IgG in three cases. No IgA was detected.

Sensitized (T6 X RFM)F1 donor B cells are engrafted and synthesize antibodies without further antigenic stimulation in RFM recipients with allogenic host-vs-graft disease.

Host-vs-graft (HVG) syndrome is the fatal disease of altered immunity that may be induced in susceptible strains of inbred mice by the perinatal inoculation of semiallogenic spleen cells. The allogenic HVG reaction causes severe depletion of both the donor F1 and host T lymphocytes, but the B cell system is hyperplastic and serum immunoglobulins (Ig) are markedly elevated. Death is caused by glomerulonephropathy and coagulopathy, both thought to be due to immune complexes. Previous work indicated that RFM host B cells are poor antibody producers, and therefore are an unlikely source of the high levels of serum Ig. The present studies were undertaken to determine if (T6 X RFM)F1 donor B cells could survive the allogenic HVG reaction and then contribute to the hyperglobulinemia. Spleen cells from (T6 X RFM)F1 donors marked with the distinctive T6 chromosome, and pre-sensitized against horseradish peroxidase (HRP) or sheep red blood cells (SRBC), were inoculated into RFM perinates. Cytogenetic studies revealed that replicating (T6 X RFM)F1 donor cells were present in low numbers in the spleens, but high numbers were found in the nodes of RFM hosts. After the transplantation of F1 cells sensitized to HRP, plasmacytoid cells with intracytoplasmic antibody to HRP were detected in the spleens and nodes of RFM recipients. Hemolytic plaque-forming cells and serum hemolysins were detected in RFM hosts that were perinatally inoculated with spleen cells from F1 donors sensitized to SRBC. The results suggested that F1 donor B cells had been engrafted, and then proliferated and matured into antibody-synthesizing cells. Their maturation was attributed to the allogeneic HVG effect, because there was no further exposure of the sensitized F1 donor B cells to antigen after transplantation, and because similar levels of mature F1 donor B cell activity were not found in T6/(T6 X RFM)F1 chimeras, which never develop HVG disease but become highly tolerant. These studies suggest a possible explanation for the seeming paradox of how hyperglobulinemia can develop in HVG mice that respond poorly to primary challenge with thymus-dependent antigen. It is proposed that primed, F1 donor B cells are stimulated into excessive production by the allogeneic reaction, whereas both donor and host B cells are poor primary responders because of the T cell deficiency induced by the same reaction.

Expression of murine leukemia viruses in RFM mice with host versus graft disease after perinatal inoculation of (T6 X RFM)F1 lymphohemopoietic cells.

Host versus graft disease is the fatal syndrome of altered immunity that follows the perinatal inoculation of related F1 hybrid spleen cells to susceptible strains of inbred mice. The allogenic reaction results in severe depletion of T-lymphocytes, but causes hyperplasia and hypersecretion of B-cells. Among the long-term survivors of acute host versus graft reactions, there is a high incidence of nonthymic lymphomas associated with ecotropic murine leukemia virus that may be of donor F1 origin. The present studies were done to determine whether ecotropic murine leukemia virus played any role in the pathogenesis of acute host versus graft disease in RFM mice perinatally inoculated with (T6 X RFM)F1 spleen cells. In RFM/(T6 X RFM)F1 chimeras, N-tropic murine leukemia virus can be detected as early as 3 days. The progression of the disease was accompanied by increasing viral expression. The inoculation of N-tropic virus of F1 donor origin into RFM neonates failed to induce disease, although the virus proliferated. Detection of progressively rising titers of antibody to murine leukemia virus linked the virus to the development of hyperimmunoglobulinemia by virtue of its ability to serve as a replicating source of antigens. These and other studies provided evidence that the seemingly paradoxical appearance of hyperimmunoglobulinemia in T-cell-deficient mice with the host versus graft syndrome is due, at least in part, to the stimulation of presensitized F1 donor B-cells, which are not destroyed in the allogenic reaction, as are the T-cells. Another unusual finding was the detection of polytropic murine leukemia virus in 25-day-old RFM/(T6 X RFM)F1 chimeras. It is suggested that the allogenic host versus graft reaction favored the formation of recombinants.

Expression of retrovirus-related antigen in pregnancy. I. Antigens cross-reacting with simian retroviruses in human foetal tissues and cord blood lymphocytes.

Antisera to Mason-Pfizer or Baboon Endogenous Virus possessed complement-dependent cytotoxicity for cell lines chronically infected with these viruses, with some degree of cross-reaction. When appropriately absorbed with virus-free cells, the antisera were not cytotoxic for lymphocytes of adult males but lysed lymphocytes of neonates in about half of the 30 cord blood samples tested and were also cytotoxic for one third of 26 trophoblast suspensions prepared from healthy placentae. Detection of retrovirus-related antigens was no more frequent in trophoblast from 9 pre-eclamptic placentae. These viral antigens were also demonstrated in cultures of foetal tissues, but only after a period of culture and only when the cells were treated with 5-iododeoxyuridine. The results demonstrate that retroviruses are not only expressed in vivo in the placenta but can also be induced in cultures of foetal cells. Retrovirus-related antigens also seem to be present on foetal lymphocytes.

Sequential changes in serum immunoglobulin levels in young RFM mice with host-versus-graft disease.

RFM mice perinatally inoculated with (T6 X RFM)F1 spleen cells develop raidply progressive host-versus-graft (HVG) disease. They are usually dead by 30 days with hyperimmunoglobulinaemia, immune complexes, plasmacytosis and marked T-cell deficiency. In the present studies, sequential quantitative analyses of serum immunoglobulins (Igs) were done, and search was made for high titred antibodies presumed to be major components of the excessive Ig levels. Based on the Ig changes, three stages of disease could be identified. In the first period, which extended from 7 to 10 days, the early appearance of IgA and IgM correlated with previous discoveries of the precocious appearance of germinal centres and enhanced antibody response. The second period, from 10 to 25 days, was characterized by rapid increases in all serum Ig levels in patterns which suggested a variable and selective loss of control of production of the individual Ig classes. IgG1 levels at 25 days averaged twenty-three times the highest adult control value. Failure of IgG2 and IgA levels to surpass adult maxima seemed only to reflect hastened maturation. Lack of success in finding high titred specific antibodies coupled with the previous evidence of poor primary antibody responses suggested the alternative possibility that most of the Igs were non-specific. The third stage, from 25 days to death, saw the apparent decline in IgG1 and IgG2 levels, and the progressive increase in the percentage of HVG mice with IgM levels above the adult maximum. It is proposed that the apparent divergence between Ig and antibody forming capacity is related to the severe disruption of the T-cell system induced by the HVG reaction.

Hyperimmunoglobulinaemia, T-cell deficiency and plasmacytosis in RFM mice with host versus graft disease induced by the perinatal inoculations (T6XRFM)F1 spleen cells.

Host versus graft (HVG) syndrome may be induced in parental strain mice by perinatal inoculations of F1 hybrid spleen cells. The principal manifestations of the disease include thrombocytopaenia, intravascular fibrin deposits, intestinal haemorrhage, hepatic infarcts, lymphosplenomegaly and renal disease. Immune complexes have been shown to be the cause of the renal lesions, and have been implicated as the triggers for disseminated intravascular coagulation. In the present studies of RFM mice perinatally inoculated with (T6 x RFM)F1 spleen cells (RFM/(T6 x RFM)F1 mice), quantitative determinations of serum immunoglobulins (Ig) revealed marked elevations of IgG1, IgG2, IgA and IgM. Electrophoretic analyses revealed the polyclonal pattern which typically follows chronic antigenic stimulation. However, IgG1 levels which reached 29 to 72 times control values suggested disruption of homeostatic mechanisms which control circulating Ig levels. Because antibody responses to histocompatibility antigens were present only occasionally, and then in low titre, it seemed unlikely these antigens were the principal causes of hypergammaglobulinaemia and plasmacytosis. Morphological studies indicated that the elevated levels of Ig seen in end-stage HVG syndrome correlated well with marked plasmacytosis, the third morphological finding in a sequence that included the precocious development of germinal centres and subsequent depletion of thymic-dependent (T) lymphocytes. The fact that spleen cells from RFM/(T6 x RFM)F1 mice were severely impaired in their capacity to cause graft versus host disease in related (T6 x RFM)F1 and unrelated C3H mice provided strong evidence that the HVG reaction resulted in T-cell depletion, rather than specific immunoincompetence.

Intravascular fibrin deposits, hepatic infarcts and thrombocytopenia in parent/F mouse chimeras with host-versus-graft syndrome.

Host-versus-graft (HVG) disease is the fatal result of the allogenic reaction which occurs in parental strain mice perinatally inoculated with F(1) hybrid spleen cells. The principal manifestations of the syndrome in RFM/(T(6) X RFM)F(1) mice are thrombocytopenia, intestinal hemorrhage, hepatic necrosis, lymphoproliferative disorders and renal disease due to immune complexes. The discovery of intravascular fibrin deposits in the present studies establishes disseminated intravascular coagulation (DIC) as an intermediary mechanism of HVG disease. It is suggested that the characteristic declines in blood platelet levels, intestinal hemorrhages and hepatic infarcts are triggered principally by immune complexes. Cellular infiltrates of the liver, granulocytosis and hypergammaglobulinemia are other abnormalities which are regularly found in HVG mice and which are also thought to predispose to DIC.