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As outlined in figure 1.4, B cells can be stimulated to undergo proliferation (clonal expansion) and further differentiate into antibody secreting plasma cells, and long-lived memory B cells. The current understanding of how this process is performed and regulated is outlined below.

The B cell lineage contains a variety of cells with differing functions and a wide variety of surface (CD) markers. B1 cells, characterized by the surface marker CD5, are able to produce low-affinity ‘natural antibody’ independent of T-cell help, whereas as the B2 cells are involved more in the adaptive immune response. These B2 cells are initially formed in the bone marrow, and are then released as immature B cells whereupon they continuously circulate through secondary lymphoid organs such as the lymph node and spleen until they encounter antigen (figure 1.7). Upon activation, the B cell then interacts with its cognate T-helper cell [47]. This interaction involves the processing and presentation of antigen by the B cell, which then presents the antigen, complexed with the MHC class II molecule, for recognition by the TCR (figure 1.6).

A number of factors make B cells vitally important antigen presenting cells (APCs). Firstly, as outlined in figure 1.4, B cells have the ability to clonally expand and therefore rapidly amplify a specific immune response. Secondly, B cells are able to take up antigen extremely efficiently via their cell surface B cell receptor (BCR).

Figure 1.7: B cell differentiation. B1 cells are recently discovered B cells capable of producing low affinity natural antibody [47]. These B1 cells may differentiate into CD24+/27+/38+ regulatory B cells which play an important role in regulating T cell mediated responses. B2 cells are typically immature B cells produced in the bone marrow which migrate to the germinal centres where they encounter antigen, interact with T cells, then differentiate into either memory B cells or antibody producing plasma cells.

In addition, B cells are powerful mediators and regulators of specific T cell responses. B cells produce cytokines which act upon T cells, for example the activated B cell produces interferon-gamma (IFN-γ) which mediates T cell polarization and

Natural'an)body' B1'Cell' CD5' Regulatory' B'Cell' CD5' IL810' CD24' CD38' CD27' B1'Cells' B2'Cells' Immature'B'cell' Bone'Marrow' T8helper' Cell' Germinal'Centre' Plasma'cell' Memory'B'cell'

differentiation into memory T-cells [48]. Regulatory B-cells, thought to derive from the B1 lineage and characterized by surface expression of markers CD24, CD27, and CD38 (figure 1.7), also inhibit T-cell specific responses primarily due to release of interleukin- 10 (IL-10). These regulatory B-cells are thought to constitute around 5% of circulating B cells [49].

The fate of the activated B cells can follow one of two paths. They can either develop into extrafollicular plasmablasts that produce low affinity antibody, or they can enter the germinal centre where they undergo somatic hypermutation and class switch recombination (described in more detail in section 1.5). Within the germinal centre, B cells with higher affinity for antigen are positively selected and are then able to differentiate into either memory B cells or antibody producing plasma cells (figures 1.4 and 1.7). A small proportion of these B cells migrate to the bone marrow where they reside as long-lived plasma cells. These cells occupy a limited number of niche sites within the bone marrow and although they are unable to proliferate, they are able to act as long-term ‘factories’ constantly producing IgG [47]. The structure, function, and relevance to transplantation of the IgG molecule is discussed in detail in section 1.5.

The importance of the B cell in the immune response to transplantation is highlighted by the fact that a number of immunosuppressive strategies have been developed which target B cell functions and effector mechanisms. Direct B cell depletion can be achieved crudely via splenectomy, or more selectively via the administration of monoclonal antibodies which directly bind to markers expressed on the B cell surface. Anti-thymocyte globulin (ATG) and the anti-CD52 antibody alemtuzumab (Campath-1H) are both used to deplete B cell populations, although both of these agents will also deplete T cells [47]. The anti-CD20 monoclonal antibody rituximab is more specific to B cells but has the therapeutic limitation that the terminally differentiated antibody producing plasma cell does not express CD20 [50].

The development of potent anti-CD19 antibodies may offer an improved efficacy as CD19 is expressed both earlier in the B cell lineage compared to CD20, but has also been shown to deplete bone marrow residing long-lived plasma cells by up to 50% [51, 52].

Specific depletion of the plasma cells is an attractive target for specific immune depletion and the proteasome inhibitor Bortezomib is currently being assessed for its ability to both deplete HLA-specific antibody pre-transplant, but also to treat rejection episodes post-transplant [53-55]. When plasma cells manufacture IgG a proportion of the protein will be mis-folded, and under normal conditions this mis-folded protein is degraded within the proteasome and then recycled within the cell. Bortezomib prevents the degradation of mis-folded proteins which then build up within the cells and lead to apoptosis of the plasma cell. Recent studies have shown that Bortezomib can lead to modest reductions in circulating levels of HLA-specific antibodies in sensitized patients [56], but it appears unlikely that Bortezomib will be suitable for use as a desensitizing agent when given in isolation [57, 58]. Further studies have suggested that Bortezomib may have potential to treat AMR post-transplant [47].