Human platelet antigen (HPA)-1a peptides do not reliably suppress anti-HPA-1a responses using a humanized severe combined immunodeficiency (SCID) mouse model.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on ß3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA-1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies.

Quantitation of fetomaternal haemorrhage and F cells in unusual maternal blood samples by flow cytometry using anti-D and anti-HbF.

BACKGROUND: Fetomaternal haemorrhage (FMH) assessment by the Kleihauer-Betke test (KBT) is rapid but semi-quantitative and liable to false positive results. OBJECTIVES: To compare FMH estimated by KBT with flow cytometry (FC) quantitation for 37 patients with massive FMH, obstetric risk factors or technical problems. METHODS: Maternal blood was sent for analysis by FC after KBT. A variety of reagents including anti-haemoglobin F (HbF), anti-D and combined anti-HbF/anti-carbonic anhydrase (CA) were used. RESULTS: Eight cases of massive FMH (>100 mL fetal cells) causing fetal death or severe neonatal anaemia in late gestation were confirmed by FC. Anti-HbF FC identified maternal F cells and fetal cells. In some cases these red cell populations merged but they could be differentiated by anti-CA, labelling F cells only. Using KBT, false positive FMH results were obtained for 12 patients, who had strongly stained cells that were then shown by FC to be maternal F cells. All these patients had increased F cells (>5% of total red cells) whereas only 16% of patients with FMH and 22% of donors had elevated F cells. In contrast, anti-D FC was simple and rapid, quantitating D-positive FMH in all 15 D-negative patients except one with massive FMH of weak D fetal cells. Leucocytes in four samples bound anti-D, variably, giving erroneously high FMH, but they could be eliminated from FC analysis using combined anti-D/anti-CD45. CONCLUSION: FMH quantitation using anti-D by FC is suitable for the majority of maternal samples and could enable accurate targeted dosing of anti-D prophylaxis.

Placental immunology and maternal alloimmune responses.

During pregnancy, women are tolerant of their semi-allogeneic fetus whilst not being immunosuppressed and indeed readily form alloantibodies. This 'Immunological Paradox of Pregnancy' may be explained by an understanding of placental anatomy and immunology. Trophoblast cells form the interface between the fetus and maternal tissues and blood and escape allorecognition because they lack classical human leucocyte antigen (HLA) class I and II molecules. Local immunoregulation, or tolerance, in the decidua is mediated partly by HLA-G(+) extravillous trophoblasts (EVT) that invade the tissue and prevent killing by maternal natural killer cells, cytotoxic T cells and macrophages. Placental hormones orchestrate the composition and regulatory function of maternal immune cells. In contrast, syncytiotrophoblast cells at the surface of chorionic villi, in contact with maternal blood, maintain a state of mild maternal systemic immunity via activation of innate immunity and skewing towards humoral immunity. This enables maintenance of a healthy immune system in pregnant women and robust protective antibody responses to pathogens whilst enabling survival of the fetus. However, this has the unfortunate consequence that pregnant women readily form alloantibodies to incompatible alloantigens on fetal red cells, platelets and leucocytes if fetomaternal haemorrhage (FMH) occurs. The antibodies are initially low affinity but after re-immunization with further FMH become functionally effective, high-titre IgG.

Lessons learnt from many years of experience using anti-D in humans for prevention of RhD immunization and haemolytic disease of the fetus and newborn.

For 40 years prophylactic anti-D has been given to D-negative women after parturition to prevent haemolytic disease of the fetus and newborn. Monoclonal or recombinant anti-D may provide alternatives to the current plasma-derived polyclonal IgG anti-D, although none of them have yet proved as effective in phase 1 clinical trials. The variation in efficacy of the antibodies may have been influenced by heterogeneity in glycosylation of anti-D produced from different cell lines. Some aspects of the conduct of the human studies, most notably the use of low doses of anti-D and target D positive red cells in vivo, may aid the design of the clinical development of other immunomodulatory drugs in order to minimize adverse effects.

Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells.

Cells were isolated from human term placentae by trypsinisation of fragments of chorionic villi and fractionation of cells on a Percoll density gradient into six layers. A panel of 10 monoclonal antibodies to antigens on or in trophoblast cells (placental alkaline phosphatase (PLAP), cytokeratin-7, beta-human chorionic gonadotrophin (beta-hCG), human leucocyte antigen-G (HLA-G)), leucocytes (CD45), monocytes, macrophages, dendritic cells, B cells (HLA class II), mesenchyme cells (vimentin), fibroblasts (fibroblast antigen) and nucleated cells excluding villous trophoblast (HLA class I, CD9) was used to characterise the cells by flow cytometry. For staining intracellular antigens (cytokeratin, vimentin, beta-hCG) the cells were first fixed and permeabilised. The upper two layers from the gradient (density 1.013-1.039 g/ml) contained predominantly PLAP-positive cells or fragments, probably derived from the syncytiotrophoblast. Cytokeratin-positive cells accumulated mainly in the layer of density 1.039-1.052 g/ml and comprised the majority of the cell types identified in this fraction. Few or no cells reactive with antibodies to beta-hCG or HLA-G were identified in any layer. Non-trophoblast cells were heavier, being present mainly at densities 1.052-1.079 g/ml (CD45, HLA class I, vimentin) and 1.066-1.092 g/ml (fibroblast). Fewer than 10% of cells in any layer were HLA class II- or CD9-positive. Further purification of trophoblast cells was by negative immunomagnetic separation with removal of CD45-positive cells and HLA class II-positive cells to less than 1%. On culture of the cells from each layer, those of density 1.039-1.066 g/ml exhibited characteristics of cytotrophoblast cells; they secreted high levels of human chorionic gonadotrophin and formed adherent multinucleate cells. This procedure enabled the selection and enrichment of cytotrophoblast cells and/or syncytiotrophoblast fragments that are suitable for cellular and molecular studies.

Functional interactions of red cells sensitized by IgG1 and IgG3 human monoclonal anti-D with enzyme-modified human monocytes and FcR-bearing cell lines.

Human monoclonal IgG1 and IgG3 antibodies specific for the Rh antigen D (anti-D) were tested for their ability to promote the binding of D-positive red cells to peripheral blood monocytes and Fc receptor (FcR)-bearing cell lines (U937, K562 and Daudi). Monocyte-mediated antibody-dependent cell-mediated cytotoxicity and metabolic (chemiluminescent) responses were also determined. By comparing the activity of different cell lines in rosette assays, and by using murine myeloma IgG2a and IgG1 to block FcRI and FcRII respectively, these functional interactions of sensitized red cells (E-IgG1 and E-IgG3) with monocytes or cell lines were shown to be mediated predominantly and perhaps solely by FcRI. E-IgG3 bound to human monocytes and cell lines to a greater extent than E-IgG1. Rosette formation by E-IgG3 was relatively less susceptible to inhibition by fluid-phase murine IgG2a than was rosette formation by E-IgG1. These findings may be due to the long hinge region of IgG3 which enables it to bridge the gap between two negatively charged cells more efficiently than IgG1. Consistent with this hypothesis was the greatly increased rosette formation achieved by treating monocytes or U937 cells with neuraminidase or bromelain, procedures shown to reduce the zeta potential of these cells. The lytic and metabolic activities of untreated human monocytes were also greater towards E-IgG3 than E-IgG1, red cell binding being a prerequisite for these responses. However, after pretreatment of monocytes with neuraminidase, these responses were greater with E-IgG1 than with E-IgG3. Further, the addition of polybrene to non-specifically enhance cell to cell binding also resulted in greater lysis and chemiluminescence with E-IgG1 than with E-IgG3. These results indicate that, although E-IgG3 are more effective than E-IgG1 in promoting red cell binding to monocytes, E-IgG1 are more efficient at activating the lytic and metabolic processes providing the steric disadvantages of the shorter hinge region of cell-bound IgG1 are circumvented.

Production and use of human monoclonal anti-D antibodies.

Rhesus haemolytic disease of the newborn is a condition which can result in intrauterine or perinatal death. Although the passive administration of therapeutic anti-D post-partum is a most effective method for the prevention of this condition, there is currently a shortage of immune plasma for the preparation of the therapeutic anti-D immunoglobulin product. In addition the availability of anti-D for use in blood grouping has also been reduced. The advances made in recent years in the techniques for the production of human monoclonal antibodies raise the possibility that human monoclonal anti-D-based products may provide solutions to both of these problems. There are now a number of reports of the production of stable cell lines secreting high titre human anti-D. In this review we consider the various strategies used in the production of human monoclonal anti-D-secreting cell lines, the basic properties of these reagents and their potential usefulness in blood grouping, in therapy and as research tools.

Comparison of lysis of bromelain and papain treated red cells in ADCC assays.

Red cells were pretreated with the proteolytic enzymes bromelain or papain prior to use in antibody-dependent cell-mediated cytotoxicity (ADCC) assays with lymphocytes or peripheral blood mononuclear cells (PBMC) as effector cells. At low concentrations of anti-D or anti-A, lysis of papain-treated cells by lymphocytes was greater than that of bromelain-treated cells. Papain digestion resulted in both greater sensitivity to haemolysis by lymphocytes or PBMC and higher agglutination titres of anti-D-sensitised red cells than bromelain. With anti-A, however, although papain also promoted greater haemolysis, it was slightly less effective at red cell agglutination than bromelain.

Haemolysis mediated by anti-D monoclonal antibodies in direct and cold target competition ADCC assays.

Thirteen IgG anti-D human monoclonal antibodies (mAbs) were compared for their ability to mediate lysis of D-positive erythrocytes by PBMC in direct and cold target competition antibody-dependent cell-mediated cytotoxicity (ADCC) assays. In the latter assay, lysis of fluid-phase anti-D-sensitised O Rh D-positive papainised erythrocytes (E-IgG) was inhibited by A (or B) Rh D-negative papainised erythrocytes sensitised by fluid-phase anti-A (or anti-B) mAbs. The competitive and lytic activities of the anti-D mAbs were characterised by the equilibrium dilution (ED) values, which were the reciprocal of the dilution of anti-A (or anti-B) at which lysis of target E-IgG and competitor E-IgG were identical. There was a poor correlation between the number of erythrocyte-bound anti-D molecules and either the sensitivity of E-IgG anti-D to haemolysis in the direct ADCC assay, or to the ED values of the mAbs obtained in the cold target competition ADCC. The discriminatory power of the cold target competition ADCC was better than than of the direct ADCC to detect differences in the lytic potential of the anti-D mAbs.

Rosette formation between immobilised human lymphocytes and erythrocytes sensitised with monoclonal anti-D.

A rapid, reproducible and sensitive assay was developed to investigate the ability of human lymphocytes to form rosettes with erythrocytes sensitised with human monoclonal anti-D. Erythrocytes sensitised with a known number of anti-D molecules per cell were incubated with lymphocytes immobilised on plastic by poly(L-lysine), the resulting rosettes fixed, unbound erythrocytes removed by washing and the cell preparation stained. IgG1 and IgG3 anti-D-coated erythrocytes gave similar rosette formation at sensitisation levels in the range of 5000-15,000 molecules per cell, although at lower sensitisation levels IgG3 gave greater rosette formation than IgG1. A minimum of 500 IgG3 and 1000 IgG1 anti-D molecules per erythrocyte were required for rosetting.

Specific anti-A responses of SCID mice populated with human lymphoid cells from peripheral blood, umbilical cord, bone marrow and spleen after immunization with group A erythrocytes [corrected].

Mice with severe combined immunodeficiency (SCID) were injected i.p. with 10 x 10(6) or 50 x 10(6) human leukocytes obtained from adult peripheral blood, umbilical cord blood, bone marrow and spleen from six group O individuals, together with allogeneic group A erythrocytes. Mice were bled every two weeks and levels of human IgG, IgM and anti-A were determined in the murine plasma. Spleen cells elicited the highest Ig levels (up to 5.2 mg/ml IgG) and umbilical cord the least (0-0.16 mg/ml IgG); maximum IgG levels were obtained at about 6-8 weeks after injection. Anti-A was detected in mice receiving adult peripheral blood or spleen leukocytes and immunizing erythrocytes 4-6 weeks after injection. Mice injected with the higher dose of leukocytes gave the best anti-A responses, but were more likely to develop tumours after 8 weeks.

Comparison of the ability of red cells sensitized with a bispecific anti-D x anti-Fc gamma RIII Fab fragment to activate human K cells and peritoneal macrophages through Fc gamma RIII.

The functional activity of Fc gamma RIII on human K cells from peripheral blood was compared with that of Fc gamma RIII on peritoneal macrophages (PM) separated from the waste material of patients undergoing peritoneal dialysis. Fc gamma R function was assessed in vitro using human monoclonal IgG1 anti-D (AB5) or a bispecific antibody comprising Fab fragments of AB5 chemically linked to Fab fragments of monoclonal anti-Fc gamma RIII, 3G8 (AB5 x 3G8). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, K cells mediated the lysis of papainized red cells sensitized with the AB5 x 3G8 bispecific antibody but not with AB5. In contrast, red cell lysis by PM was not promoted by AB5 x 3G8 although AB5 was active. However, this lysis, being inhibited by monomeric IgG, was presumably mediated via Fc gamma RI. AB5 x 3G8 also failed to promote the binding and phagocytosis of both papainized and native red cells by PM although 99% of red cells and over 90% of peritoneal cells bound the bispecific antibody. In marked contrast to K cells therefore, Fc gamma RIII on PM was unable to mediate functional interactions with red cells sensitized with anti-D x anti-Fc gamma RIII bispecific antibody.

Optimisation of human anti-tetanus toxoid antibody responses and location of human cells in SCID mice transplanted with human peripheral blood leucocytes.

A strategy for the production of human antibodies to tetanus toxoid (TT) is described. Human peripheral blood lymphocytes (PBL) were injected into severe combined immunodeficient (SCID) mice (termed hu-PBL-SCID) and the mice subsequently immunised with purified TT. By using low immunising doses of antigen, PHA activated PBL and PBL from donors who were recently immunised with TT, we established ongoing antibody responses to TT with specific recall IgG responses of up to 22 IU ml-1 in the murine plasma, which was greater than that in the donors' serum. Total IgG concentrations of up to 6 mg ml-1 were detected over a 32 week period. Lower levels of IgM and IgM anti-TT were also detected over this time. Large cellular infiltrations of human CD45+ and CD20+ cells were detected by immunocytochemistry in the mesenteric membranes, mesenteric lymph nodes and the pancreas 5 weeks after PBL were engrafted into a SCID mouse. Human cells were also observed in the lungs, liver, thymus and spleen. Cells isolated from the tissues were cultured with Epstein-Barr virus and the resulting B-cell lines produced Ig in vitro up to 7 weeks, with IgG and IgM anti-TT detected transiently in a culture of cells from the mesenteric membranes.

Case report: anti-Cra in pregnancy.

A 39-year-old Grenadian multiparous patient presented in the 12th week of pregnancy. Her red cells were found to have the rare Cr(a-) (ISBT Number 202001) phenotype within the Cromer complex, and her serum contained anti-Cra. To date, anti-Cra has not been implicated in hemolytic disease of the newborn (HDN), but there are very few published reports on this topic. This case provided an excellent opportunity for study. The patient's serum showed no detectable functional activity in in vitro antibody-dependent, cell-mediated cytotoxicity assays, and no increase in the strength of the antibody during the pregnancy. The newborn infant showed no clinical signs of HDN, and was of normal weight. This case study suggests that anti- Cra is not implicated in HDN.

Monoclonal anti-D for prophylaxis of RhD haemolytic disease of the newborn.

The incidence of Rh D haemolytic disease of the fetus and newborn has been dramatically reduced by the prophylactic administration of anti-D immunoglobulin to Rh D-negative women. This preventive treatment depends on adequate supplies of anti-D derived from plasma of immunised donors, and replacement with monoclonal anti-D would be advantageous. Two monoclonal antibodies, BRAD-3 (IgG3) and BRAD-5 (IgG1) have been produced from EBV-transformed B-lymphoblastoid cell lines in Bristol and extensively characterised. Both have shown good (but differing) functional activities, determined by study of interactions of anti-D coated red cells with effector cells bearing IgG Fc receptors. Phase I clinical trials using Rh D negative male volunteers were undertaken in Bristol. The plasma half lives of BRAD-3 and BRAD-5 were characteristic for their IgG subclass, and both anti-D mediated accelerated circulatory clearance of D-positive red cells infused two days after i.m. injection of the antibodies. BRAD-3 and BRAD-5 were then shown to protect the volunteers from mounting a primary anti-D response to these D-positive red cells, and thus they may be suitable for Rh D prophylaxis of Rh D-negative women.

Coordinator's report: an assessment of the functional activity of human Rh monoclonal antibodies after their evaluation by nine laboratories.

The functional activity of 55 anti-D and 26 MAbs of other Rh specificities was determined as part of the Third International Workshop and Symposium on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens. Most MAbs were IgG1 (45 anti-D, 16 Rh). There was a large range of anti-D and IgG concentrations of the anti-D MAbs (0.4-1680 IU/ml, 1.5-400 micrograms/ml). Correlation between quantitative data from different laboratories was good. Six laboratories tested the anti-Ds in monocyte ADCC assays. Methods varied greatly, especially the effector cells used, the methods of red cell sensitisation, the effector to target cell ratio and the assay incubation times. There was some correlation of results between most laboratories, but results were better when similar assays were performed. The extent of monocyte-mediated haemolysis was related to the number of molecules of IgG anti-D bound to the red cells. Monocyte phagocytosis assays resulted in a greater variability in results between the four evaluating laboratories, though IgG3 MAbs were found more active than IgG1, and mostly promoted red cell adherence rather than phagocytosis. Two monocyte chemiluminescence assays found comparatively low activity of the IgG3 MAbs; the most active IgG1 anti-Ds were MAbs 93 and 95. Correlation between different monocyte-mediated assays was generally poor unless the assays were performed by the same laboratory. Results of a macrophage ADCC assay showed that the IgG3 anti-D's promoted greater haemolysis than most IgG1 MAbs. Only two IgG1 anti-D MAbs (70 and 104) mediated high haemolysis in this assay. Lymphocyte ADCC assays were utilised in four laboratories. The IgG3 antibodies exhibited low activity but one IgG1 anti-D (104) consistently mediated high haemolysis. There was no relationship between quantitation and haemolysis in this assay. A few MAbs exhibited low or no activity in most assays, mainly due to low quantitation. Most of the MAbs of non-D specificity mediated low functional activity which in most cases was not due to low quantitation. However two MAbs, 24 (anti-CcEe) and 25 (anti-CD), consistently showed good activity. This study highlighted the great variability in assay methodology between the nine participating laboratories, which resulted in some apparent differences in functional activity of some of the MAbs. The use of standardised methods as well as fewer MAbs tested and quantitated at different concentrations may help to determine the relevant factors contributing to IgG function.

Characterization and functional activity of human Rh monoclonal antibodies.

For quantification of the number of molecules of IgG anti-D bound to RBC, a combination of flow cytometry plus Sol-ELISA was found more accurate than Sol-ELISA alone. However, some Rh MAbs gave very low values of cell-bound IgG using flow cytometry. Monocyte-mediated haemolysis correlated well with the number of molecules of red cell-bound IgG1 anti-D. The monocyte phagocytosis assay was found more sensitive but less quantitative than the monocyte ADCC assay. Most monoclonal antibodies mediated little haemolysis by lymphocytes in ADCC assays, and haemolysis was not related to IgG concentration, titres or cell-bound IgG.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers.

Prophylactic anti-D is a very safe and effective therapy for the suppression of D-immunization and prevention of haemolytic disease of the foetus and newborn. The primary mode of action of anti-D is rapid clearance of fetal D-positive red cells from the maternal circulation, mediated by interactions with immunoglobulin G Fc receptors on macrophages in the spleen. Many anti-D monoclonal antibodies (mAb) have been produced by a variety of methods. Twelve anti-D mAbs were tested in eight studies for their ability to mediate clearance of autologous red cells, and 13 antibodies studied in seven trials of the clearance of D-positive red cells injected into D-negative subjects. Antibodies produced by human B-cell lines, mouse-human heterohybridomas and Chinese hamster ovary cells varied in their activity with none being quite as effective as polyclonal anti-D. However, clearance mediated by recombinant anti-D produced by rat YB2/0 cells was extremely rapid, faster than polyclonal anti-D, but with haemolysis and some hepatic accumulation of red cells observed in one study. Two human anti-D mAbs prevented D-immunization. In contrast, anti-D mAbs from heterohybridomas increased the incidence and rapidity of anti-D responses. It is hypothesised that unnatural glycosylation of monoclonal anti-D produced by some cell lines may have caused these unexpected results. In some antibodies, unusual oligosaccharides on anti-D may have affected binding to Fc receptors resulting in reduced red cell clearance. For others, non-human glycoforms of anti-D might have bound to innate immune recognition molecules promoting pro-inflammatory reactions. These extensive data on the clinical activity of monoclonal anti-D produced by cell lines derived from four species will inform the future development of monoclonal anti-D for RhD prophylaxis.

Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism.

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.