The Rhesus (Rh) D antigen is important in transfusion medicine and is the major target in haemolytic disease of the newborn (HDN). The aims of this project were to characterise the helper-T (Th) cell response that drives anti-D alloantibody production in HDN as a first step towards developing an improved or alternative strategy to the current programme of prophylaxis, based on the manipulation the T-cell response. Peripheral blood mononuclear cells (PBMC) were obtained from 22 individuals, who had developed anti-RhD alloantibodies following natural or deliberate immunisation, and from 22 RhD negative and from 12 RhD positive control donors who had not been immunised with RhD. A panel of 69 overlapping synthetic 15-mer peptides, spanning the sequence of the RhD protein, was screed for the ability to stimulate recall proliferation and cytokine production from T-cells in cultures of the PBMC. T cells from all 22 of the alloimmunised donors proliferated in response to RhD peptides and typically multiple peptides were stimulatory. In contrast, only a few minor responses were observed in the control donors. RhD peptides 6, 13, 17, and 28 were identified as immunodominant peptides that stimulated proliferation in over 50% of the alloimmunised donors. Each of these peptides were promiscuous in their ability to stimulate T-cells from donors of the common HLA allotypes in this study. Each immunodominant peptide contains multiple core epitopes and multiple sets of MHC/TCR contacts. Preliminary findings indicate that neither peptides shorter than 15mer length nor analogues can be designed to boost or tolerise alloimmunised donors. The RhD peptides induced a complex pattern of cytokine production from alloreactive T cells. Both IFN-gamma and IL-4 could be produced to the RhD peptides indicative of a Th0 response. In addition, particular peptides elicited the production of the potentially inhibitory cytokines IL-10 or TGFb and not proliferation.

No description available.

Antibodies passively administered together with their specific antigen can enhance or suppress the specific antibody response. This phenomenon is known as antibody feedback regulation. Whether this modulation causes up- or downregulation of the antibody response depends both on the antibody isotype and the antigen used. IgG antibodies passively administered together with particulate antigens, e.g. erythrocytes, can completely prevent the induction of an antibody response to the antigen. The suppressive capacity of IgG has been routinely used in the clinic since the 1960’s in RhD-prophylaxis to prevent hemolytic disease of the fetus and newborn. Although studied for decades, the underlying mechanism of IgG-suppression has remained elusive. The main focus of this thesis has been to elucidate the mechanism behind IgG-suppression of antibody responses in vivo in mouse models using intravenous immunization with specific IgG together with native or haptenated sheep red blood cells, SRBC. We show that IgG-suppression of IgM and long-term serum IgG-responses operates independently of activating FcγRI, III, IV, or the inhibitory FcγRIIB, thus confirming and extending previous findings. Moreover, we demonstrate for the first time that C1q, C3 and CR1/2 are dispensable for IgG-suppression of antibody responses. These findings strongly argue against the involvement of Fc-dependent mechanisms as the explanation for IgG-suppression. Interestingly, GC formation occurs in IgG-suppressed mice although the antibody response to surface SRBC epitopes are completely suppressed. The data suggests that these GCs develop in response to intracellular SRBC epitopes as well as to the passively administered suppressive IgG. Moreover, we demonstrate that passively administered IgG suppresses several parameters of an antibody/B cell response including antigen specific GC and non-GC B cells, extra-follicular antibody secreting cells, long-lived plasma cells and induction of immunological memory. Before the onset of the present study, two mechanisms appeared compatible with the majority of experimental findings: IgG-mediated antigen clearance and epitope masking. Herein we show that the contribution of IgG-mediated antigen clearance is negligible and that suppression of IgG-responses is strictly epitope specific. This provides compelling evidence that a very important mechanism underlying IgG-suppression is epitope masking.