Fig. 5.1
The HLA gene cluster is localized on chromosome 6. HLA class I genes (HLA-A, B, C) encode HLA class I heavy chain which pairs with non-polymorphic protein β2-microglobulin on the cell membrane. HLA class II (HLA-DP, DQ and DR) are also dimers comprised of α chains and β chains. Some individuals may also express antigen DR52, DR53 and DR51, of which the β chain is encoded by gene DRB3, DRB4 and DRB5, respectively. MICA and MICB genes are also localized in this region
HLA class I polypeptides (HLA-A, HLA-B, or HLA-C) function as a dimer when noncovalently bound to a non-polymorphic polypeptide called β2 microglobulin. This dimer presents peptides to the T cell receptor present on T cells. The HLA class I polypeptide is organized as α1, α2, α3, transmembrane and cytoplasmic domains. The α1 and α2 domains of the HLA class I polypeptide are highly polymorphic and form the antigen recognition site. HLA class II polypeptide (HLA-DR, DQ or DP) also forms a dimer: α chain and β chain. The HLA class II α chain is encoded by genes HLA-DRA1, DQA1 or DPA1. The β chain is encoded by genes HLA-DRB1, DQB1 or DPB1. Both α chain and β chain can be polymorphic for HLA-DQ and DP. For HLA-DR, only HLA-DRB1 is polymorphic. The HLA class II polypeptide is organized as α1 and α2, transmembrane, and cytoplasmic domains. The antigen recognition site of HLA class II peptides is contributed by α1 domains of both α chains and β chains. HLA class I molecules predominantly present endogenous peptides derived from defective folded proteins or virus proteins synthesized inside the cell, while HLA class II molecules present peptides derived from protein/pathogens in extracellular compartments to T cells. The non-classic HLA gene, including MICA, is also localized in this region. MICA can be stress induced and bind to and activate NK cells against stressed or damaged cells. MICA has limited polymorphism and is not associated with β2 microglobulin. Although MICA cannot present peptides to the immune system, it can be recognized by the recipient’s adaptive immune system. The presence of MICA antibodies has been shown to be associated with transplant coronary artery disease in heart transplantation.
These HLA genes are localized in a 4 Megabase region in chromosome 6 and tend to transfer together from parents to their offspring. Each individual carries one copy of genes at each HLA locus on each of two chromosome 6, and these two chromosomes segregate during meiosis. Therefore, there is ¼ chance that two siblings share the same HLA gene content. It isn’t always easy to find a matched donor carrying compatible HLA alleles, especially for highly sensitized patients. The frequency of HLA alleles varies among different ethnic groups. For example, HLA-B46 has a high frequency in East Asian populations, such as in Thai populations where the frequency is as high as 14%, while it may hardly be detected in other ethnic groups [2]. Because some HLA alleles exist at much higher frequencies in certain populations, it would likely be easier to find a donor carrying these alleles in that particular population than others.
Alloantigen Presentation/Cell Mediated Rejection
In order for the recipient’s adaptive immune system to recognize mismatched alloantigens, they need to be presented as peptides by the HLA antigens on the recipient’s antigen presenting cells which are recognized by the recipient’s CD4+ T helper cells. Activation of the recipient’s CD4+ T helper cells is prerequisite to initiate CD8+ T cell mediated cytotoxic response, and B cell mediated humoral response against alloantigens. Alloantigens can be presented to the recipient’s T cells through three pathways: the indirect, direct, and semi-direct pathways (Fig. 5.2). In the indirect pathway, alloantigens are presented in a similar way as antigens derived from pathogens. The recipient’s antigen presenting cells capture alloantigens which are shed from the graft, and present these antigens on the context of the recipient HLA class II to the recipient CD4+ T helper cells. Alloantigens targeted by de novo donor specific antibody usually are mainly presented through the indirect pathway [3]. Because in the indirect pathway, alloantigens have to be captured and processed first by the recipient’s antigen presenting cells before being presented to CD4+ T helper cells, it takes longer, compared to the direct pathway of recognition to alloantigens. The recipient immune system usually takes more than 2 weeks to develop do novo donor specific antibodies.
Fig. 5.2
In the indirect pathway, recipient antigen presenting cells (APC) present donor-derived peptides on the context of recipient HLA class II to recipient CD4+ T helper cells. This pathway is important for initiating antibody mediated rejection. In the direct pathway, donor derived APC present allopeptides restricted on donor HLA class II to recipient CD4+ T helper cells, and allopeptides restricted on donor HLA class I recipient CD8+ cytotoxicity cells. This pathway is usually responsible for acute cellular mediated immune responses to intact donor HLA class I antigens. In the semidirect pathway, recipient APC capture membrane fragments bearing intact HLA class I antigens from donor cells, and present intact class I antigens to recipient CD8+ T cells. Allopeptide restricted on recipient HLA class II are also presented to recipient CD4+ T helper cells by the same APC
Compared to the indirect pathway, alloantigens are presented directly by donor-derived antigen presenting cells to the recipient’s CD4+ T helper cells in the direct pathway. These passenger antigen presenting cells in the allograft are transplanted into the recipient along with the graft. These passenger cells can interact with the recipient’s CD4+ T helper cells directly and present alloantigens restricted by the donor HLA molecules to CD4+ T helper cells to initiate adaptive immune response. In this pathway, antigen presenting cells don’t need to process new antigens, and immune response is activated relatively fast. This pathway usually is responsible for the acute cellular mediated immune response [4].
Immune response to alloantigens can also be initiated by the third pathway, the semi-direct pathway. In this pathway, the recipient antigen presenting cells, mainly dendritic cells obtain donor HLA: peptide complexes by capturing the membrane from the donor passenger antigen presenting cells or endothelial cells. These recipient dendritic cells then can present HLA alloantigens to both CD8+ cytotoxic T cells as an intact protein and to CD4+ T helper cells as processed allopeptide simultaneously. This semi-direct pathway explains how CD8+ cytotoxic T cells can target HLA alloantigens expressed on the graft. This semi-direct pathway may be critical for the CD8+ T cell mediated cytotoxic response for mismatched HLA antigens between the donor and recipient [5].
T Cell Mediated Response: Effector T Cells and the Memory Response
Murine models have shown that rejection of different organs may depend on certain T cell subsets. Studies in heart have shown that rejection can occur in the absence of CD8+ T cells but not in the absence of CD4+ T cells, suggesting that class II expression on the graft is sufficient to mediate rejection [6]. Distinct effector phenotypes, Th1, Th2, and Th17 have been described; however, cytokines are pleotropic and their role in the clinical rejection process remains somewhat controversial. Naïve T cells may be polarized into distinct helper T cell subsets based on cytokine signatures, the signature signal transducer and activator of transcription (STAT) molecules which sense the extracellular cytokine environment. Briefly, the Th1 cells secrete IL-2, IFN-γ, and TNF; Th2 cells secrete IL-4, IL-5, IL-10 and IL-13; Th17 cells secrete IL-17. Subsets of T cells, which can be either CD4+ or CD8+, can inhibit the immune response of other T cells and are termed regulatory T cells (Tregs). Although these various Th subsets were thought to be stable, more recent reports indicate these subsets may be flexible in their T cell phenotypes [7].
Naïve T cells proliferate through the autocrine growth factor IL-2 and can differentiate into various types based their encounters with different cytokines. The CD4+ cells are often termed T helper cells and CD8+ cells are frequently termed cytotoxic cells. However, cells of both phenotypes can be helper or cytotoxic based on their MHC antigen specificity toward class I versus class II. That is, CD4+ cells can be helper or cytotoxic. When exposed to IL-12, activated T cells can differentiate into a predominantly IFN-γ producing phenotype and are designated in the Th1 category. Activated T cells that are exposed to IL-4 predominantly differentiate into the Th2 cells that produce IL-4, IL-5, IL-10 and IL-13. Upon exposure to TGF-β and IL-6, they can differentiation into Th17 cells producing IL-17 (A and F) and IL-22 [8, 9]. The Th1 and Th17 cells have been associated with autoimmunity while the Th2 cells are often associated with asthma and allergies. The Th1 IFN-γ producing cells are often associated with acute allograft rejection along with the presence of IL-17. The Th2 cells have also been associated with the rejection process.
After an initial antigenic challenge, a second stimulation by the same foreign antigen triggers a memory response characterized by a faster kinetics of lymphocyte activation for both the T and B cell compartments. After an initial response where the antigen is cleared, the number of effector cells peaks at about 1 week, after which about 90% of the effector cells die. The remaining population is long-lived memory T cells with distinct phenotype and function. These memory T cells have a lower activation threshold allowing them to respond quickly upon restimulation. These effector memory T cells express homing receptors that allow for migration to non-lymphoid sites of inflammation [1].
B cells go through an affinity maturation process which depends on the interactions with the APC and the activated T cells environment. Some of the B cells differentiate into antibody secreting plasma cells while others become memory B cells which persist for long periods of time. The secondary response for the memory B cells is also shorter (3–5 days) compared to the primary response (7–10 days). The antibodies produced by a B cell memory response have higher affinity and are usually characterized by subgroups such as IgG, IgA and IgE versus IgM. Important to transplantation is the varied effectiveness of specific immunosuppressive drugs for removing antibody producing cells depending on their characteristics.
Antibody Production and Biology
Despite the improvement of immune suppressing regimens, antibody mediated rejection remains a major obstacle to long term graft survival. With the help from CD4+ T helper cells, naïve B cells with an alloantigen bound on their B-cell antigen receptor are primed and differentiate to plasma cells secreting antibodies against the antigen that is bound to the B cell antigen receptor. Naïve B cells can also differentiate to memory B cells which can rapidly differentiate into plasma cells upon recurrent exposure to the initial antigens. Plasma cells can survive in niches mainly in bone marrow for long periods of time. Both memory B cells and long lived plasma cells provide long-term humoral immunity [10].
CD20 protein is widely expressed on the surface of B cells during B-cell ontogeny, and is necessary for B-cell activation [11]. Anti-CD20 antibodies, Rituximab or Obinutuzumab, are used to treat lymphoma and autoimmune disorders by depleting B cells through antibody dependent cell cytotoxicity. CD20 antibodies are also used for desensitization or antibody mediated rejection for solid organ transplant. However, the expression of CD20 is lost after the B cells differentiate into plasma cells. Therefore CD20 antibody therapy would be ineffective to remove antibodies after B cells differentiate to antibody-secreting plasma cells. This may be the reason why CD20 antibody treatments are not always effective to desensitize or treat antibody-mediated rejection. Another drug used for desensitization or treatment of antibody mediated rejection is Bortezomib. Bortezomib is a proteasome inhibitor originally used to treat myeloma. Bortezomib is used to inhibit antibody production on the premise that plasma cells which synthesize a large amount of antibodies, and need to degrade incorrectly folded proteins, might be more sensitive to the inhibition of proteasome.
Antibody production can be stimulated by sensitizing events, such as pregnancy, transfusion and previous transplant. Alloantibodies damage the graft mainly through three ways. The first is complement dependent cytotoxicity. Upon binding to antigens on cells of the graft, alloantibodies recruit C1q, the first complement component activated in the classic complement pathway, through the Fc fraction of IgG [12]. There are 4 isotypes of IgG antibodies: IgG1, IgG2, IgG3 and IgG4. The affinity of these IgG to C1q is IgG3> IgG1> IgG2> IgG4. IgG3 and IgG1 alloantibodies may be more potent than IgG2 and IgG4 to activate the classic complement pathway. The presence of donor specific IgG3 antibodies against HLA is associated with high risk of antibody mediated rejection in renal transplant [13]. C1q binding to alloantibodies sequentially activates complement components C4, C3 and then C5, which in turn can lead to the formation of membrane attack complex. Membrane complex composes a pore in the cell membrane and causes cell death. Unintended activation of complement is detrimental to the tissue and organ; the activation of complement is tightly controlled by many negative regulators [14]. Even if alloantibodies are produced, complement may not necessarily be activated on the graft due to these negative regulations.
C4d, a split product of complement C4, produced after the activation of the classic complement pathway, is covalently linked to the cell membrane. Its half-life is 12–31 days in vivo [15]. These characteristics make positive C4d staining on the biopsy a useful marker for diagnosis of antibody mediated rejection in kidney and heart transplant. In the classic complement activation pathway, activation of complement C1 stimulates complement C4 to transform into active form C4b through proteolytic cleavage. The activity of C4b is negatively regulated by complement 4-bindig protein (C4BP). C4BP prevent C4b from activating the downstream complement cascade by degrading C4b through proteolytic cleavage. One of the cleavage products is C4d [14]. Thus C4d deposition on the graft is dependent on both the activation of complement C1 and the presence of negative regulator of C4BP. When C4BP negative regulator is missing, or its activity is low, C4d will not be generated and detected even if the complement is fully activated, which might be one of the reasons why a biopsy diagnosed with antibody mediated rejection is stained C4d negative.
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