Kenneth D. Beaman

Cloning of a cDNA for a T cell produced molecule with a putative immune regulatory role

An expression cDNA library was constructed from the helper T cell hybridoma, A.1.1, which has been shown to produce constitutively proteins involved in the down regulation of the immune response. From this library we identified and characterized a cDNA clone, J6B7, by screening with a polyclonal antibody specific for secreted immune regulatory proteins. The mRNA for J6B7 is expressed specifically in some T cells, but not in the thymoma BW5147 or liver cells. J6B7 is 2937 nucleotides in length and contains one open reading frame encoding for a peptide of predicted Mr of 98,042. The nucleotide and deduced amino acid sequences of J6B7 did not reveal significant homology to any published sequences. Hybridization and translation experiments reveal that the J6B7 can hybrid select mRNA from total RNA isolated from either A.1.1 cells or thymic tissue which can be translated in vitro to a peptide which is bound by a monoclonal antibody (mAb) specific for antigenic determinant(s) shared by immune regulatory proteins. Furthermore, the in vitro translated proteins obtained from A.1.1 cells and thymus showed significant suppression of a mixed lymphocyte reaction (MLR) in a dose dependent manner, reaching maximum suppression of 71% and 89%, respectively. These results suggest that the cDNA, J6B7, codes for an immune regulatory protein.

Down-regulation of maternal antiphospholipid antibodies during early pregnancy and pregnancy outcome

OBJECTIVE We investigated the hypothesis that maternal autoimmune responses to phospholipid antigens measured before and during pregnancy are not related to successful pregnancy outcome. STUDY DESIGN One hundred twenty-three women with recurrent spontaneous abortions were serially tested for antiphospholipid antibodies during their pregnancies. RESULTS In 72 women with recurrent spontaneous abortions and without antiphospholipid antibodies before the pregnancy, the incidence of antiphospholipid antibody production at the time of pregnancy termination was significantly higher in those who miscarried the index pregnancy than those who were delivered of a live-born infant. In 51 antiphospholipid antibody-positive women with recurrent spontaneous abortions there were dramatic increases in titers of anticardiolipin antibody and antiphosphatidylserine antibody in those who miscarried the index pregnancy (p < 0.005). In women who were delivered of a live-born infant, the titers remained stable or decreased during pregnancy. CONCLUSIONS Down-regulation of antiphospholipid antibody production during early pregnancy is associated with favorable pregnancy outcome.

T-cell suppressor factors play an integral role in preventing fetal rejection

In previous studies we showed that the blocking of T-cell suppressor factors (TsF) with monoclonal antibody could completely ablate pregnancy, and demonstrated the presence of TsF in fetal and maternal tissues. In our current study we used a monoclonal antibody specific for TsF to determine the time during gestation when TsF is most integral in the maintenance of pregnancy. Significant decreases in the number of viable pregnancies when monoclonal antibody was administered on days 3, 4 and 5 were demonstrated. In addition, ELISAs were used to measure the levels of TsF in tissues from pregnant and non-pregnant mice. Increased levels of TsF in the uterus and the lymph nodes draining the uterus were observed when compared to the same tissues of non-pregnant animals. These data strengthen the hypothesis that TsF is in part responsible for the fetal specific immune supression during pregnancy in allogeneic mice and more clearly define the importance of TsF in the implantation of the embryo.

Antibodies to phospholipids and nuclear antigens in non-pregnant women with unexplained spontaneous recurrent abortions

In a collaborative study of 73 non-pregnant Kuwaiti women with unexplained spontaneous recurrent abortion (RSA), 30 control healthy non-pregnant multiparous Kuwaiti women and 20 North American women who received elective abortion(s), autoantibodies to 6 phospholipids and 9 nuclear antigens were measured. Women with recurrent spontaneous abortions demonstrated 3 times higher incidence of antibodies to phospholipids (30.1%) than controls (10% each) (P = 0.029). The incidence of both IgM and IgA class antiphospholipid antibodies were significantly higher than those of controls. The incidence of antibodies to cardiolipin in women with recurrent spontaneous abortions (12.3%) was significantly higher than those of controls (P = 0.035) and incidence of IgM but not IgG anticardiolipin antibody was significantly higher in women with RSAs than in controls (P = 0.053). The incidences of anti-polyinosinic acid (P = 0.035) and anti-histone 1 antibody (P = 0.052) were significantly higher in women with recurrent spontaneous abortions than controls. There was no significant difference in the incidence of autoantibodies between primary and secondary aborters. However, women with a history of second trimester abortions showed a higher incidence of antiphospholipid antibodies than women with first trimester abortions only. Recurrent spontaneous abortion is associated with autoantibodies to phospholipid epitopes including IgA antiphospholipid antibodies.

MHC class II compatibility in aborted fetuses and term infants of couples with recurrent spontaneous abortion

Maternal-fetal histocompatibility for alleles at HLA class II loci, HLA-DQA1 and HLA-DQB1, was examined in 40 abortuses and 31 liveborn children of 68 couples with a history of idiopathic recurrent spontaneous abortion (RSAB) who underwent leukocyte immunization prior to the index pregnancy. Significantly more couples with RSAB shared two HLA-DQA1 alleles as compared with fertile control couples (0.18 vs. 0.03, respectively; P = 0.031). There were no differences in HLA sharing between couples with RSAB who experienced a repeat abortion in the index pregnancy as compared with couples with RSAB who were delivered of a liveborn child. Non-significant deficits of abortuses who were compatible for alleles at the HLA-DQA1 (6 observed vs. 8.5 expected; P = 0.225) and the HLA-DQB1 (7 observed vs. 9.2 expected; P = 0.254) loci were observed. A significant deficit of HLA-DQA1 compatible liveborn children was observed (1 observed vs. 5.5 expected; P = 0.0069). The overall deficit of HLA-DQA1 compatible fetuses (7 observed vs. 14.0 expected; P = 0.0018) after approximately 8 weeks gestation suggests that HLA-DQA1 compatible fetuses may be aborted early in pregnancy, prior to the time when fetal tissue can be recovered for genetic studies.

Intravenous immunoglobulin infusion therapy in women with recurrent spontaneous abortions of immune etiologies

We have investigated clinical effectiveness of intravenous immunoglobulin G infusion (IVIg) on antiphospholipid antibody titers in five women with evidence of antiphospholipid antibody-associated recurrent spontaneous abortions and one with antinuclear antibody who became refractory to conventional autoimmune treatment during pregnancy and experienced pregnancy complications. Three women developed intrauterine growth retardation and three had complicated twin pregnancies with rising autoantibody titers. Antiphospholipid antibody and antinuclear antibody titers were tested pre and 2 weeks after each IVIg infusion. We report that: (i) IgG antiphospholipid antibody titers were significantly suppressed after each IVIg infusion (P < 0.05); (ii) IgM antiphospholipid antibody titers were also significantly suppressed after each IVIg infusion (P < 0.0001); (iii) decreased titers of autoantibodies paralleled increased levels of maternal IgG which lasted for at least 30 days; the autoantibodies showed a definite rise again prior to the next infusion; (iv) antinuclear antibody titers were effectively suppressed; and (v) rising autoantibody titers combined clinical manifestation of intrauterine growth retardation and women with complicated twin pregnancies. We conclude that IVIg infusion effectively suppresses IgM and IgG autoantibodies to phospholipids and antinuclear antibody in autoimmune women with a history of recurrent spontaneous abortions and refractory to conventional anticoagulation or immunosuppressive treatment.

Sequence analysis of porcine immunoglobulin light chain cDNAs

A porcine cDNA library was constructed using poly(A)+ RNA isolated from the spleen of an adult Minnesota miniature swine. Screening the library with antisera specific for porcine immunoglobulin light chains resulted in the selection and isolation of two recombinant clones, PLC18 and PLC3, which encode for kappa and lambda light chains, respectively. These cDNAs contain sequence information for a portion of the variable region and all of the constant region. The lengths of the constant regions are 105 amino acids for lambda and 108 amino acids for kappa. The deduced amino acid sequences of porcine immunoglobulin light chains share a high degree of homology with similar sequences from other species in both the fourth framework region and the constant region.

Characterization of antibodies induced by paternal lymphocyte immunization in couples with recurrent spontaneous abortion

This study was designed to identify and characterize the allo- and autoantibodies induced following successful paternal lymphocyte immunization to prevent recurrent spontaneous abortion. Firstly the titers of maternal anti-paternal antibodies in women with successful pregnancies as determined by the flow cytometry crossmatch (FCXM) were highly variable; however, in all cases, the initial pre-immunization titers were negative and the post-immunization titers were positive by the FCXM in successfully treated women. Secondly, the specificities of maternal alloantibodies to paternal HLA antigens (immunogen) were evaluated. No all predicted antibodies to mismatched paternal HLA antigens were found by microlymphocytotoxicity (MCX) assays and the specificities varied. Thirdly, antibodies in post- but not preimmunization sera reacted with two lymphoid cell lines, SupT1 and SB; in addition, the rise and fall of the titers of these sera with paternal cells seemed to be reflected with the cell lines by the FCXM. Fourthly, autoantibodies to activated lymphocytes were detected and seemed to correlate with successful immunization since women who had another abortion following immunotherapy lacked these autoantibodies. These findings suggest that the antibody response following successful immunotherapy is complex and needs to be studied further to understand the mechanism of this treatment.

New Horizons in the Evaluation and Treatment of Recurrent Pregnancy Loss

During the past 15 years we have evaluated couples with unexplained recurrent spontaneous abortion (RSA), couples with normal pregnancies, and women who electively terminate their pregnancies early in gestation and have found major differences in the alloimmune and autoimmune status in women with unexplained RSA (1–3). In studies of over 1500 couples in this latter category we have shown (i) a higher incidence of HLA-DR and HLA-DQ antigen sharing compared to fertile controls (4); (ii) a higher incidence of HLA-DR and -DQ homozygosity in the male partners (5); (iii) a markedly decreased level of maternal alloantibody to paternal T and B lymphocytes compared to fertile control couples (2, 6); (iv) a strikingly higher incidence of antiphospholipid antibodies to phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) that increases in incidence and titer with each subsequent pregnancy loss in three distinct populations of women studied (United States, Kuwait, and Colombia) (3, 7); and (v) an 89% incidence of subsequent pregnancy loss following lymphocyte immune therapy in autoimmune women untreated for the autoimmune abnormalities (2).

Inhibition of binding of anti-CD3 antibodies to paternal lymphocytes correlates with failure of immunotherapy for treatment of recurrent spontaneous abortions

A two color flow cytometry crossmatch (FCXM) was used to evaluate the induction of anti-lymphocyte antibodies in 34 women undergoing immunotherapy for recurrent spontaneous abortions. All women had anti-lymphocyte antibodies that reacted with T-cells when analyzed by FCXM. However, inhibition of the binding of anti-CD3 to paternal CD3 lymphocytes in the presence of maternal antipaternal lymphocyte antiserum was found for some couples following lymphocyte immunotherapy for spontaneous recurrent abortions. Ten couples who had another spontaneous abortion following immunotherapy showed inhibition. In contrast, eight couples who did not show inhibition of the binding of anti-CD3 T lymphocytes to paternal lymphocytes by maternal anti-lymphocyte antiserum had live births. Women of the remaining 16 couples were either pregnant and awaiting birth or not pregnant. Thus, by FCXM it may be possible to predict those couples who will have successful pregnancies following this treatment.