Anajane G. Smith

Microchimerism of maternal origin persists into adult life

Recent studies indicate that fetal cells persist in maternal blood for decades after pregnancy. Maternal cells are known to engraft and persist in infants with immunodeficiency, but whether maternal cells persist long-term in immunocompetent offspring has not specifically been investigated. We developed sensitive human leukocyte antigen-specific (HLA-specific) PCR assays and targeted nonshared maternal HLA genes to test for persistent maternal microchimerism in subjects with scleroderma and in healthy normal subjects. Nonshared maternal-specific DNA was found in 6 of 9 scleroderma patients. In situ hybridization with double labeling for X and Y chromosome-specific sequences revealed female cells in peripheral blood samples from 2 male scleroderma patients. HLA-specific PCR also frequently revealed persistent maternal microchimerism in healthy control subjects. The mean age of all subjects with maternal microchimerism was 28 years (range: 9-49 years). With few exceptions, mothers of subjects with persistent maternal microchimerism were HLA incompatible with subjects for class I and class II alleles. These results clearly indicate that HLA-disparate maternal cells can persist in immunocompetent offspring well into adult life. The biological significance of maternal microchimerism and whether it might contribute to autoimmune disease requires further investigation.

Maternal-Fetal Disparity in HLA Class II Alloantigens and the Pregnancy-Induced Amelioration of Rheumatoid Arthritis

BACKGROUND Rheumatoid arthritis frequently remits during pregnancy, for unknown reasons. Since an immune response to paternally inherited fetal HLA can occur during normal pregnancy and since rheumatoid arthritis is an autoimmune disorder with a known HLA class II antigen association, we tested the hypothesis that maternal-fetal disparity in HLA alloantigens might be associated with the pregnancy-induced remission of rheumatoid arthritis. METHODS We studied 57 pregnancies of 41 women with rheumatoid arthritis, 18 prospectively and 39 retrospectively. Serologic and DNA techniques were used to study HLA class I and II antigens. For newborns, typing was performed from cord-blood samples obtained at delivery. For four young children, typing was performed from DNA extracted from hair samples. RESULTS We found significantly more maternal-fetal disparity in HLA-DR and DQ antigens in pregnancies characterized by the remission or improvement of rheumatoid arthritis than in pregnancies characterized by active disease. Further studies using DNA-typing techniques to define allelic variants of HLA-DR and DQ antigens confirmed this observation. Maternal-fetal disparity in alleles of HLA- DRB1, DQA, and DQB occurred in 26 of 34 pregnancies characterized by remission or improvement (76 percent), as compared with 3 of 12 pregnancies characterized by active arthritis (25 percent) (odds ratio, 9.7; P = 0.003). The difference between the two groups was most marked for alleles of HLA-DQA. CONCLUSIONS Amelioration of rheumatoid arthritis during pregnancy is associated with a disparity in HLA class II antigens between mother and fetus. These findings suggest that the maternal immune response to paternal HLA antigens may have a role in the pregnancy-induced remission of rheumatoid arthritis.

The biological significance of HLA‐DP gene variation in haematopoietic cell transplantation

Although it has been over 25 years since HLA‐DP was mapped to the major histocompatibility complex (MHC), its biological functions remain ill‐defined. We sought to test the hypothesis that HLA‐DP functions in a manner similar to that of other class II genes by measuring the risk of clinically severe grades III–IV acute graft‐vs.‐host disease (GVHD) associated with recipient HLA‐DP disparity after haematopoietic cell transplantation. HLA‐DPB1 exon 2 was sequenced in 205 patients who underwent transplantation from HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 allele‐matched unrelated donors. HLA‐DPB1 mismatched recipients experienced a significantly increased risk of acute GVHD compared with HLA‐DP‐identical transplants. Patients who were mismatched for a single HLA‐DPB1 allele had an odds ratio (OR) of 1·0 (0·5, 2·2; P = 0·99) and patients who were mismatched for two alleles had an OR of 2·2 (1·0, 4·9; P = 0·06) for developing acute GVHD. Compared with matched and single‐allele mismatched transplants, patients who were mismatched for two DPB1 alleles had an OR of 2·2 (1·2, 4·1; P = 0·01). HLA‐DP plays an important role in the alloimmune response. A threshold effect of multiple HLA‐DP disparities is evident in determining the risk of acute GVHD after haematopoietic cell transplantation from unrelated donors.

Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain.

We have studied DRB1 sequence polymorphisms associated with DR4 subtypes using DR4-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization. The 5′ amplification primer was designed to hybridize with a unique sequence in the first hypervariable region (HVR) of the DRB1 second ex-on of all known DR4 alleles; the 3′ primer was designed to hybridize with an intron sequence common to all DRB1 alleles. The specificity of the amplification step was demonstrated by double-blind testing of 105 selected DNA samples. Prospective SSOP typing of DR4 alleles was performed in 104 unrelated individuals known to be DR4-positive, including 13 who were DR4-homozygous. A DRB1 subtype corresponding with the previously defined DR4-associated specificities Dw4, Dw10, Dw13.1, Dw13.2, Dw14.1, Dw14.2, Dw15, and DwKT2 could be assigned for each of the 117 DR4 haplotypes tested. In most cases, DR4-homozygous, DRB1-heterozygous individuals could be genotyped with the panel of probes. In the course of our analysis, we identified two new DR4-related alleles, DRB1*04.CB (DRB1*0410)1 and DRB1*04.EC (DRB1*, 0411)2 which were recognized by their novel hybridization patterns. The DRB1 second exon sequence of DRB1*04.CB, is identical to DRB1*0405 except at codon 86 where GTG encodes valine instead of GGT encoding glycine. DRB1*04.EC is identical to DRB1*04.CB except at codon 74 where GAG encodes glutamic acid instead of GCG encoding alanine. Our results provide further evidence that SSOP hybridization is the most effective approach available for subtyping DR4 haplotypes and identifying unrecognized variants. A similar approach should be equally informative for subtyping other DR alleles.

Human Leukocyte Antigen Class II and Cervical Cancer Risk: A Population-Based Study

The critical role of the human leukocyte antigen (HLA) system in presenting peptides to antigen-specific T cell receptors may explain why only some human papillomavirus (HPV)-infected women progress to cervical cancer. HLA class II DRB1 and DQB1 genes were examined in 315 women with invasive squamous cell cervical cancer (SCC) and 381 control subjects. Increased risks of SCC were associated with DRB1*1001, DRB1*1101, and DQB1*0301, and decreased risks were associated with DRB1*0301 and DRB1*13. Of squamous cell tumors, those containing HPV-16 were different from those not containing HPV-16 for 3 alleles: DRB1*0401, DRB1*07, and DQB1*06. Increased risks of SCC were associated with DRB1*0401-DQB1*0301 (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.1-2.7) and DRB1*1101-DQB1*0301 (OR, 2.5; 95% CI, 1.4-4.5), and decreased risks were associated with DRB1*0301-DQB1*02 (OR, 0.7; 95% CI, 0.5-1.0) and DRB1*13-DQB1*06 (OR, 0.6; 95% CI, 0.4-0.9) haplotypes. These results add to the evidence that certain HLA class II alleles or allele combinations, or genes linked to them, make some women more susceptible to SCC.

Murine monoclonal anti-T cell antibodies for treatment of steroid-resistant acute graft-versus-host disease

Four different murine monoclonal anti-T cell antibodies were administered to 15 patients with severe steroid resistant graft versus host disease (GVHD) in a phase I clinical trial in order to evaluate feasibility and toxicity. Antibodies 9.6 (IgG2a) and 35.1 (IgG2a) bind to separate epitopes on the E receptor (Tp50); antibody 10.2 (IgG2a) binds to the murine Lyt-1 homolog (Tp67); and antibody 12.1 (IgG2a) binds to a cell surface antigen with a molecular weight of approximately 100,000 daltons (Tp100). A total of 151 infusions were given, ranging in dose from one to 20 mg, each administered over a one to four hour period. One patient received a total of 259 mg of antibody over a period of 45 days. Six infusions (4%) in two patients were associated with fever or fever and chills. By decreasing the infusion rate, subsequent infusions to these two patients were accomplished without additional reactions. Although most of the patients treated with monoclonal antibodies required platelet support, the number of platelet units given was not significantly different from similar patients not receiving monoclonal antibodies. Six of ten patients receiving intermediate to high doses (5-20 mg) antibody therapy had evidence of at least partial improvement in GVHD in at least one involved organ system. None of the patients became immunized to mouse immunoglobulin. Our results suggest that therapy of GVHD with murine monoclonal anti-T cell antibodies is feasible and that these antibodies apparently can be administered to marrow transplant patients without significant toxicity. Further studies are required to determine which antibodies or combinations of antibodies have optimal anti-GVHD effect.

Remission of Rheumatoid Arthritis During Pregnancy and Maternal-Fetal Class II Alloantigen Disparity

ABSTRACT: Rheumatoid arthritis (RA) is an autoimmune disorder known to be associated with specific class II genes. Although it has been known since 1938 that the majority of women with RA experience disease improvement or remission during pregnancy, the reasons remain unknown. Pregnancy represents an immunologic challenge and maternal immune recognition of the semi‐allogeneic fetus occurs as part of normal pregnancy. We hypothesized that maternal immune response to fetal HLA antigens might be associated with the effect of pregnancy on arthritis activity. To test this hypothesis, we studied HLA antigens in mother‐child pairs comparing maternal‐fetal HLA antigen sharing for pregnancies where arthritis improved with those where disease was active. No significant difference was observed in the two groups for class I HLA antigens. Fetal‐maternal disparity for HLA‐DR and HLA‐DQ antigens was observed significantly more frequently in pregnancies with remission or improvement compared with those in which disease was active. These observations suggest that maternal immune response to fetal paternally‐inherited class II HLA antigens may be important in RA remission observed during pregnancy.

HLA class I and class II profiles of patients presenting with Sydenham's chorea

Abstract Sydenham’s chorea (SC) may occur in rheumatic fever (RF) patients without arthritis and carditis. In this study we typed HLA antigens and alleles in patients presenting with the distinct major clinical manifestations of RF, i.e., chorea, carditis, or arthritis, in population and family studies. We evaluated 91 patients with RF for HLA-A, HLA-B, and HLA-DR antigens; of these, 33 had pure chorea, 26 pure carditis, 16 pure arthritis, and 16 carditis plus arthritis. We also typed 24 SC patients and their unaffected siblings for HLA-DRB1 and HLA-DQB1 alleles using molecular methods. HLA-B49 and HLA-DR1 antigens were overrepresented in the total group of patients with RF and in all the subgroups studied, excluding the SC subgroup in which the frequency of HLA-DR1 antigen was not increased. The frequencies of the HLA-DRB1 and HLA-DQB1 alleles in patients with pure chorea were not significantly different from those observed in controls. Similarly, the frequencies of HLA class II alleles in SC patients did not differ significantly from those observed in unaffected siblings. These findings show that immunogenetic susceptibility to RF varies according to the major clinical manifestation presented by the patient.

Unique sequences for two HLA-DRB1 genes expressed on distinct DRw6 haplotypes.

We have used restriction fragment length polymorphism (RFLP) analysis and DNA sequencing to characterize two distinct DRB1 alleles expressed on DRw52 and DQw7-associated haplotypes but not readily defined by conventional DR serology. These two haplotypes, designated HLA-D “HAG” and “PEV”, react variably with DRw13(w6), DRw14(w6), and the more broad DR “3+6” antisera. Analysis of RFLP revealed that HLA-D “HAG” and “PEV” are associated with different DRw52 variants, and that “HAG” is indistinguishable from DRw18(3) haplotypes. Sequencing of the “HAG” and “PEV” DRB1 genes showed each to represent novel alleles. Nevertheless, these sequences show similarities with the other alleles of the DR5, w6, and w8 family. “HAG” (DRB1*1303) appears to have arisen either from two recombinational events involving at least three DRB1 sequences (DRB1*1101, DRB1*0803, DRB1*0401) or from a single recombinational event together with multiple point mutational events. “PEV” appears to represent a DRB1*1301-1302/DRB1*1101 recombinant allele, with recombination having occured in the region of bases 175 – 198. The results of this study suggest that the DRw52 family haplotypes is derived from a relatively restricted number of ancestral sequences, with diversity among DRB1 alleles within this family arising through gene conversion or recombination events.

Genomics of unrelated-donor hematopoietic cell transplantation

Unrelated-donor hematopoietic cell transplantation is a proven curative modality for hematologic malignancies. The success of unrelated-donor transplantation has been achieved through a better understanding of the immunobiology of the HLA system and through more precise and comprehensive matching of donors and recipients. The extensive polymorphism of HLA genes confers important biological implications affecting engraftment, graft-versus-host disease and overall survival. Although more-complete HLA identity of the donor and recipient is associated with optimal transplant outcome, new information suggests that not every HLA disparity is functionally relevant. Future advances in unrelated-donor transplantation must include the identification of tolerable HLA mismatches, so that more patients may benefit from this therapeutic modality. Furthermore, the role of cytokine-gene polymorphisms and minor histocompatibility genes in transplant outcome requires investigation. Delineation of the function of these markers as transplantation determinants may provide alternative means for optimizing the results of hematopoietic cell transplantation.