Altered Peripheral Blood Leucocyte Phenotype and Responses in Healthy Individuals with Homozygous Deletion of FHR1 and FHR3 Genes.

A homozygous 83-kb deletion encompassing the genes for complement factor-H-related proteins 1 and 3 (FHR 1, FHR3) is known as a risk factor for some immune inflammatory disorders. However, the functional relevance of this FHR1/3 deletion is relatively unexplored. Globally, healthy populations of all ethnic groups tested show an 8-10% prevalence of homozygosity for this deletion polymorphism. We have begun to compare the peripheral leucocyte phenotype and functionality between FHR1/3-/- and FHR1/3+/+ healthy adult individuals. We report that the two groups show significant differences in their peripheral blood innate leucocyte subset composition, although the adaptive immune subsets are similar between them. Specifically, FHR1/3-/- individuals show higher frequencies of patrolling monocytes and lower frequencies of classical monocytes than FHR1/3+/+ individuals. Similarly, FHR1/3-/- individuals show higher frequencies of plasmacytoid dendritic cells (pDCs) and lower frequencies of myeloid DCs (mDCs) than FHR1/3+/+ individuals. Notably, classical monocytes specifically showed cell-surface-associated factor H (FH), and cells from the FHR1/3-/- group had somewhat higher surface-associated FH levels than those from FHR1/3+/+ individuals. FHR1/3-/- monocytes also showed elevated secretion of TNF-α, IL-1ß, and IL-10 in response to TLR7/8 or TLR4 ligands. Similarly, FHR1/3-/- mDCs and pDCs showed modest but evident hyper-responsiveness to TLR ligands. Our findings, that the FHR1/3-/- genotype is associated with significant alterations of both the relative prominence and the functioning of monocyte and DC subsets, may be relevant in understanding the mechanism underlying the association of the genotype with immune inflammatory disorders.

Characterization of genetic predisposition and autoantibody profile in atypical haemolytic-uraemic syndrome.

We previously reported that Indian paediatric patients with atypical haemolytic-uraemic syndrome (aHUS) showed high frequencies of anti-complement factor H (FH) autoantibodies that are correlated with homozygous deletion of the genes for FH-related proteins 1 and 3 (FHR1 and FHR3) (FHR1/3-/- ). We now report that Indian paediatric aHUS patients without anti-FH autoantibodies also showed modestly higher frequencies of the FHR1/3-/- genotype. Further, when we characterized epitope specificities and binding avidities of anti-FH autoantibodies in aHUS patients, most anti-FH autoantibodies were directed towards the FH cell-surface anchoring polyanionic binding site-containing C-terminal short conservative regions (SCRs) 17-20 with higher binding avidities than for native FH. FH SCR17-20-binding anti-FH autoantibodies also bound the other cell-surface anchoring polyanionic binding site-containing region FH SCR5-8, at lower binding avidities. Anti-FH autoantibody avidities correlated with antibody titres. These anti-FH autoantibody characteristics did not differ between aHUS patients with or without the FHR1/3-/- genotype. Our data suggest a complex matrix of interactions between FHR1-FHR3 deletion, immunomodulation and anti-FH autoantibodies in the aetiopathogenesis of aHUS.

Do follicular dendritic cells regulate lupus-specific B cells?

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. In this review we describe recently published results in which a B cell receptor-knock-in mouse strain specific for nucleolar self-antigens was bred with mice deficient in complement C4 and discuss the implications for the lupus field. Absence of C4 leads to a breakdown in the elimination of autoreactive B cell clones at the transitional stage. This is characterized by a relative increase in their response to a range of stimuli, entrance into follicles and a greater propensity to form self-reactive germinal centers. In this review, a model is proposed in which, in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells and follicular dendritic cells, resulting in secretion of Type I interferon. This allows for the maturation and activation of self-reactive B cell clones leading to increased spontaneous formation of germinal centers and subsequent generation of autoantibodies.

Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner.

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.

Endocytosis and recycling of immune complexes by follicular dendritic cells enhances B cell antigen binding and activation.

Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.

Recruitment of memory B cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus.

Successful recall Ab responses require recruitment of quiescent memory B cells to secondary lymphoid organs. However, the cellular dynamics of memory cells responding to local antigenic challenge at lymphoid sites distal from the initial Ag encounter are not well understood. We show in this study that memory B cells generated following s.c. immunization in one footpad generate secondary responses to soluble Ag given i.p. but not to Ag given s.c. in the contralateral footpad unless LPS is coadministered. Memory B cells do not express CD62L, and CD62L(-ve) cells cannot enter lymph nodes unless LPS-mediated inflammation is induced there. Functional TLR4 is required on the B cells, as well as on non-B cells, in the lymph node to achieve full recruitment. Furthermore, splenectomized mice fail to respond to such inflammatory s.c. challenge in contralateral footpads, unlike lymphadenectomized mice lacking the original draining lymph nodes. Splenectomized mice also fail to respond to i.p. challenge with soluble Ag. Together, these data indicate that, unlike the central memory pool of T cells, which circulates through resting lymph nodes, the majority of long-lived memory B cells are spleen resident and require inflammatory signals for mounting recall responses at distal challenge sites.

Modulation of antigen presentation and B cell receptor signaling in B cells of beige mice.

Binding of Ag by B cells leads to signal transduction downstream of the BCR and to delivery of the internalized Ag-BCR complex to lysosomes where the Ag is processed and presented on MHC class II molecules. T cells that recognize the peptide-MHC complexes provide cognate help to B cells in the form of costimulatory signals and cytokines. Recruitment of T cell help shapes the Ab response by facilitating isotype switching and somatic hypermutation, and promoting the generation of memory cells and long-lived plasma cells. We have used the beige (Bg) mouse, which is deficient in endosome biogenesis, to evaluate the effect of potentially altered Ag presentation in shaping the humoral response. We show that movement of the endocytosed Ag-BCR complex to lysosomes is delayed in Bg B cells and leads to relatively poorer stimulation of Ag-specific T cells. Nevertheless, this does not affect Bg B cell activation or proliferation when competing with wild-type B cells for limiting T cell help in vitro. Interestingly, Bg B cells show more prolonged phosphorylation of signaling intermediates after BCR ligation and proliferate better to low levels of BCR cross-linking. Primary Ab responses are similar in both strains, but memory responses and plasma cell frequencies in bone marrow are higher in Bg mice. Further, Bg B cells mount a higher primary Ab response when competing with wild-type cells in vivo. Thus, the intensity and duration of BCR signaling may play a more important part in shaping B cell responses than early Ag presentation for T cell help.