See also Drugs/agents which inhibit DC maturation

DC are derived form bone-marrow progenitors (CD34+) and are present at two stages in the body. DC differentiation involves the change from multipotential hematopoietic progenitor cells (HPCs) to immature DCs (iDCs). Immature DC are present in most tissues and have the role of sentinel. Immature DC have a high capacity to capture antigens and transport them from peripheral tissues to the secondary lymphoid organs. Thus iDCs have a big capacity for Ag-uptake but a relatively poor ability to activate T cells. This differentiation is induced in vitro by exogenous cytokines such as GM-CSF and TNF-alpha for CD34+ HPCs, GM-CSF and IL-4 for monocytes, CD40 receptor cross-linking for CD34+ HPCs, or calcium ionophore alone for monocytes.

The next stage involves maturation of iDCs to mature DCs (mDCs), which exhibit enhanced Ag-presentation, up-regulated surface major histocompatibility comnplex (MHC) molecules, as well as co-stimulatory and adhesion molecules. Mature DC are present in secondary lymphoid organs and present antigens to T cells. Antigen presentation by DC to T cells occurs in the context of cell surface MHC class II molecules and co-stimulation signals, and to become fully potent APC, DC must thus undergo maturation.

After internalization, most exogenous antigens are processed through an endosomal and lysosomal pathway in which proteins are cleaved into peptides and loaded onto MHC class II molecules. Alternatively, exogenous antigens can be released into the cytosol, gaining access the the proteasome, the main non-lysosomal protease, that generates peptides and transfers them to the endoplasmic reticulum, where they are loaded onto MHC class I molecules. This exogenous MHC class I presentation is also refered to as “cross-presentation”.

What Drives DCs to Mature

In vivo, maturation (second stage) is probably triggered by “danger signals” which may include  a variety of stimuli including live bacteria and components (lipopolysaccharide (LPS), DNA), viral infection and inflammatory cytokines.

DC differentiation is plastic. Different cytokine microenvironments, which can relate to tissue localization or to prior interaction with microorganisms, induce DCs to differentiate towards different states that can polarize T cells towards different functions. Studies have shown that immature DCs mature in the following ways:

• Microbial signaling through toll-like receptors (TLRs) is an effective way to mature DCs to their immunogenic state. Immature DCs can be considered as immunological sensors that analyze the nature of the microorganisms and other danger signals through cross-linking of their pattern recognition receptors (PRR). The majority of the known PRR recognize bacterial pathogen associated molecular patter (PAMP), such as Toll-like receptor (TLR) 2 for peptidoglycan, TLR4 for lipopolysaccharide, TLR5 for flagellin or TLR9 for CpG.
• Different cytokine microenvironments, which can be related to the tissue localization or to prior interaction with microogranisms induce DCs to differentiate towards different states that can polarize T cell towards different functions. Different microbial stimuli induce different responses from the same DC population. Several soluble immunosuppressive factors on the maturation of DC form monocytes or CD34+ myeloid stem cell progenitors have been shown. Among these factors are a number of cytokines that are often produced by malignant cells such as TGF-?1, IL-6, vascular endothelial growth factor (VEGF) and IL-10.
• NF-kB: TLR ligation results in NF-kB activation and this pathway is important in DC maturation. Translocation of the NFkB family members Rel B and p50 from cytoplasm to nucleus is required for myeloid DC maturation. Antigen-exposed myeloid DCs, in which RelB function is inhibited, lack cell surface CD40 expression and prevent priming of immunity, and suppress a previously primed imune response. DCs in which RelB nuclear translocation is inhibted through prevention of IkB phosphorylation, DCs generated from RelB deficient mice, and DCs generated from CD40 deficient mice similarly confer suppression. TLRs trigger DC maturation using a NFkB dependent pathway. Indirect activation of DCs by proinflammatory cytokines (Il-1, Il-18, TNF-alpha) and chemokines is also NFkB dependent.
• T cell-derived signals. Activated CD4+ T-helper cells upregulate  CD40 ligand. Signaling through the CD40 receptor activates DCs.
• Antigens with which DCs interact:
• Recent studies in mice indicate that DCs also mature through interaction with natural killer T cells. This process is dependent on NK T-cell activation with the synthetic glycolipid antigen alpha-galacto-sylceramide, presented by CD1d molecules on the DCs.

Molecular Mechanisms of Maturation

The molecular mechanisms of DC maturation are not well understood but involve several routes such as NF-kB pathway and the p38 mitogen-activated protein kinase (MAPK) pathway. In vitro, bacteria-induced DC maturation involves (a) ERK kinase, allowing for DC survival, and (b) NF-kB, allowing for DC maturation characterized by increased expression of costimulatory and MHC-class II molecules, release of chemokines, and migration. This coordinated process leads to high T cell stimulatory capacity as well as IL-12 release, all of which result in the induction of protective immune responses.

Numerous publications have shown that all TLR agonists tested to date can lead to increased expression of CD40, CD80 and CD86 in at least one DC subset. NF-KB pathway  is a major transcription factor controlling the expression of these markers and thus it has been reasonable to assume that NF-kB activation following TLR signalling is sufficient to promote DC maturation. This has been put into question by the work of Hoshino et al., however, who showed a marked decrease in CD40 upregulation in STAT-1 -/- BM-DC treated with CpG or LPS compared to wild type DC. This result suggests that DC maturation in response to TLR ligation is, to a large extent, dependent on secondary production of cytokines such as type I interferons (IFN-I), which signal in an autocrine or paracine manner. Consistent with these results, IFN-I is a potent stimulus for DC maturation and IFN-I receptor (IFN-IR) is required for the adjuvanticity of Complete Freund’s adjuvant which contains several TLR ligands in the form of killed Mycobacteria. TLR agonists and/or IFN-I may further promote DC maturation via induction of TNF, IL-15 or other inflammatory cytokines, which also promote upregulation of B7 and CD40 expression on DC.

The loss of receptors specific for inflammatory chemokines is mediated by distinct mechanisms. Following stimulation with LPS, the loss is very rapid (up to 80% and 60% of surface CCR1 and CCR5 expression in 3 h). Thus this rapid down regulation cannot result from changes in transcription or mRNA stability. One proposal is that the rapid loss is mediated by a novel mechanisms that involves the production of chemokines by maturing DC, leading to homologous desensitization of the cognate receptors. Evidence for this mechanisms includes (1) maturing DC produce a number of chemokines that act on their own receptors, (2) receptor down regulation is prevented by brefeldin A and cycloeximide and (4) receptor levels can be reconstituted in mature DC by reculturing them in fresh medium without stimuli.

Importance of relB: On a molecular level DC maturation is guided by relB, a subunit of the NFkB transcription factor. RebB has been shown to play a major role in DC function by regulating CD40 and MHC expression. Upon stimulie exerted by TNF-alpha, LPS or virus-derived IL-1, relB translocates to the nucleus and promotes transcription of CD40, CD80/86 and MHC genes, all of which are indicators of DC activation. Blokage of this translocation can lock DCs in an immature state, as indicated by results using REelB-deficient mice. Most of the pharmaceuticals that inhibit DC maturation also interact with the relB pathway. For example, there is evidence that mycophenolate, mofetil, glucocorticoids and vitamin D3 all downregulate NFkB expression. RelB has even been suggested as a useful marker to qualify DC as Treg-inducing DCs. Evidence derives from observations showing that nuclar relB is absent in steady-state DCs located in peripheral tissues, whereas relB becomes upregulated in the nucleus in DCs residing in inflamed or lymphoid tissues.

P38 MAPK: LPS reportedly activates the p38 MAPK pathway in DC and this coincides with DC maturation. However, activation of p38, although necessary, is not sufficient to stimulate DC maturation, indicating that alternative pathways are required. See also P38 as an inhibitor of DC maturation The activation of the NF-kB pathway is a likely candidate. Activation of p38 may reusult in the modulation of chromatin structure and enhanced accessibility of NF-kB to the relevant elements in the promoters of cyotkine genes and other genes presumably involved in DC maturation.

JNK: was reportedly not to be involved in controlling DC maturation.

Changes which occur as DCs mature

Maturation of DC results in a complete reprogramming of the cell, with downregulation of endocytic activity, upregulation of MHC, adhesion and costimulatory molecules, as well as a striking switch in chemokine receptor usage. Some changes that take place on DC maturation include the following:

• increased formation of stable MHC-peptide complexes. There is upregulation of cell surface MHC molecules, which in the case of both MHC I and II is due to increased biosynthesis, and in the case of MHC II is due to a prolongation of the half-life of MHC peptide complexes.
• higher expression of membrane molecules: DCs express on their plasma membrane high levels of coreceptors such as CD80 (B7-1), CD86(B7-2), CD40, CD54, OX40L, 4-1BBL, etc. that bind costimulatory receptors on T-cells.  CD95 is also up-regulated on mature DC.  For example, LPS has been shown to up-regulate expression of cell-surface MHC class II (Iab), CD40, CE54, CDE80 and CD86 (Chen, 2002). DC-LAMP is also known to be up-regulated upon differentiation/maturation of DCs.
• synthesis of cytokines that influence T cell proliferation and differentiation such as IL-12.
• altered production of chemokines Maturing DCs entering the draiming lymph nodes will be driven into the paracortical area in response to the production of MIP-3? and/or 6Ckine by cells spread over the T cell zone. The newly arriving DCs might themselves become a source of these chemokines, allowing an amplifciation or persistence of the chemotactic signal. Because these two chemokines can attract mature DCs and naive T lymphocytes, they are likely to play a key role in helping Ag bearing DCs to encounter specific T cells. Upon encounters with T cells, which can take place not only in the wsecondary lymphoid organ but also the site of tissue injury, DCs receive additional maturation signals from CD40 ligand, RANK/TRANCE, 4-1BB and OX40 ligand molecules which induce the release of chemokines such as IL-8, factalkine, and macrophage derived chemokines that attract lymphocytes.
• Alterned production of chemokine receptors that intensify movement of DCs into lymphatic vessels and lymphoid organs. Depending on their tissue of origin, immature DC express a variety of chemokine receptors (CR), most of which bind to inflammatory chemokines. For example, infiltration of graft tissues by immature DC allows these DC to take up and process graft Ag for presentation to naive T cells in draining secondary lymph nodes (DLN). The internalization and processing of Ag initiates DC maturation, which involves decrease of CR for the inflammatory chemokines and increase of the CR for the constitutive chemokines.

See also specific diseases for references to CRegs  and Drug Targetting under Pharmacology

CRegs for the Treatment of Diseases

CR1: Levin (US 6,169,068) teaches a method for treating diseases involving complment by pulmonary administration of complement inhibitor proteins such as a soluble complement receptor type 1 (sCR1). 

CD46 (MCP): Sweigard teaches that adenovirus mediated delivery of CD46 attenuates the AP pathway and has implications for age related macular degeneration (Gene Therapy 18, 613-621 (2011).

CD59: 

–AMD: Bora discloses that a recombinant membrane targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice (J Biol Chem 285, 2010, 33826-33833). Cashman also discloses that a non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration (PLOS ONE, e 19078, 6(4) 2011, 1-9).

Ramo evaluates adenovius delivered human CD59 as a potential therapy for AMD in a model of human membrane attack complex formation on murin RPE (Invest Opthalmol Vis Sci 49(9): 4126-4136 (2008).

–Atherosclerosis: Wu teaches that CD59 protects against atherosclerosis by restricting the formation of complement membrane atttack complex (Circ Res 104: 550-558), 2009). 

CReg Fusion Constructs for the Treatment of Diseases

–CD55 (DAF) and CD46 (MCP) fusion proteins: have been shown to be more effective thatn DAF, MCP or DAF and MCP at inhibiting C3 deposition via the alternative pathway. Chimeric proteins in which MCP, DAF and MCP-DAF hybrids have also been produced with cell surface loalizing domains which target the molecules to cell surfaces, thereby increasing the concentraiton of these molecules on cell surfaces where they can act to inhibit complement-mediated cell lysis. MCP-DAF hybrids may contain the entire sequence of both molecules or may contain just the SCR regions form each MCP and DAF.  (Innis, WO 96/34965). Ko (US 5,679,546 and 5,851,528) and Wijesuriya (US 2006/0154336) also disclose chimeric genes and proteins which express the biological activities of both MCP and DAF. The proteins are referred to as “Complement Activation Blocker” (CAB) proteins.

–CD46 and CD55 and CD59 fusion construct: Kumar-Singh (US 13/692734) teaches a construct having the complement regulatory domains of each of CD46, C55 and CD59 with the native secretory signal of CD59 at the N-terminus. The SCR domains of CD46 and CD55 are involved in complement control and the serine threonine proline (STP) region is heavily glycosylated. The majrority of amino acids of the STP region were removed and the amino acid sequences of the full-lenght SCR doamins for each regulator was retained. Glycine linkers were added to separate each complement regulatory domain. 

–CR2 fusion constructs with CRegs: (see Complement Receptor Drug Targetting under Pharmacology)

–DAF-CD59: Fodeor (5627264) teaches chimeric complement inhibitors having C3 inhibitory activity and a second functional domain having C5b-9 inhbitory activity such as DAF-CD59 fusion consructs.

–Antibody-CD59 constructs (see also drug delivery under pharmacology): Zhang (J. Clinical Invest. 1999, 103(1)) teaches antibody-CD59 chimeric fusion proteinswhich provided targeted cells with effecitve protection from complement mediated lysis.

There are 3 distinct pathways of complement activation. The classical system is antibody dependent whereas the other 2 pathways, lectin and alternative, are antibody independent and are initiated by reaction of complement proteins with surface molecules of microorganisms rather than antibody. All 3 pathways generate C3, and C5 convertases and bound C5b, which is converted into a membrane attack complex (MAC). The MAC complex forms a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse freely across the membrane. Hydrolysis of C3 by C3 convertase enzymes of the classical, lectin and alternative pathways is the major amplification step, generating large amounts of C3b, which forms part of the C5 convertase. C3b also can diffuse away from the activating surface and bind to immune complexes of foreign cell surfaces, where it functions as an opsonin by phagocytic cells bearing C3b receptors.

Nomenclature: Classical pathway components are labeled with a C and a number (e.g., C1, C3). Because of the sequence in which they were identified, the first four components are numbered C1, C4, C2 and C3. Alternative pathway components are lettered (e.g., B, P, D). Cleavage fragments are designated with a small letter following the designation of the component (e.g., C3a and C3b are fragments of C3). Inactive C3b is designated iC3b. Polypeptide chains of complement proteins are designated with a Greek letter after the component (e.g., C3alpha and C3beta are the alpha and beta chains of C3. Cell membrane receptors for C3 are abbreviated CR1, CR2, CR3, and CR4.

(1) classical pathway See outline

(2) lectin pathway see outline

(3) alternative pathway (AP):  See outline

See also Regulation of the Complement system .

See also the use of complement receptors for drug targeting (under “pharmacology” and “drug delivery”).

There are three known gene superfamilies of complemen receptors: The short consensus repeat (SCR) modules that code for CR1 and CR2, the beta-2 integrin family members CR3 and CR4, and the immunoglobulin Ig-superfamily member CRIg.

Complment Receptor 1 (CR1) (alsko known as human C3b/C4b receptor or CD35):

Structure: CR1 is a 180-210 kDa glycoprotein consisting of 30 short consensus repeats (SCRs). The structure of the C3b binding site, contained with SCR 15-17 of CR1 (site 2), has been determiend by MRI.

Function: CR1 plays a major role in immune complex clearance. High affinity binding to both C3b and C4b occurs through two distinct sites, each composed of 3 SCRs. The main function of CR1 is to capture ICs on erythrocytes of transport and clearance by the liver. There is a role in phagocytosis for CR1 on neutrophils, but not in tissue macrophages. In addition to its role in clearance of immune complexes, CR1 is a potent inhibitor of both classical and alternative pathway activation through its interaction with the respective convertases. See also Regulation of the Complement system.

Several soluble fragments of CR1 have also been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNA being expressed. The soluble CR1 fragments were functionally active and inhibited in vitro the consequences of complement activation such as neutrophil oxidative burst, complement mediated hemolysis and C3a and C5a production. (WO 94/17822).

Complement receptor type 2 (CR2) (also known as CD21): 

Structure: CR2 contains an extracellular portion having 15 or 16 repeating units known as showrt consensus repeats (SCR domains). The SCR domains have a typical framework of highliy ocnserved residues including 4 cysteines, two prolines, one tryptophane and several other partially conserved glycines and hydrophobic residues. In the ful lenght human CR2 protein sequence, amino acids 1-20 comprsie the elader peptide, amino acids 23-82 comprise SCR1, 91-146 comrpise SCR2, 154-210 comprise SCR3, 215-271 compise s SCR4. The active site (C3d binding site) is located in SCR1-2 (the first two N-terminal SCR domains). These SCR domains are separated by short sequences of variable lenght that serve as spacers. (Gilkeson, WO2007149567). 

Functions: CR2 serves as a receptor for breakdown products of the complement protein C3 such as  C3b, iC3b and C3d cleavage fragments via a binding site located within the first two amino-terminal short consensus repeats (SCRs 1 and 2) of the CR2 protein. Consequently, the SCR1-2 domain of CR2 discriminates between cleaved (i.e., activated) forms of C3 and intact circulating C3. CR2 is present on mature B lymphocytes, CD4 and CD8 positive peripheral T lymphocytes, early thymocytes, epithelial cells and follicular dendritic cells. It plays a role in B cell activation, the generation of immunologic memory, Ig class switching and the regulation of homotypic and heterotypic adhesion. Natural ligands of CR2 include the iC3b and C3d fragments of complement C3. C3d coats foreign antigens and the subsequent cross-liking of CR2 bound C3d to B cell receptors amplifies a signal transduction cascade through the CR2/CD19/CD81 co-activation complex of B cells. 

CR2 is not an inhibitor of ocmplement and it does not bind C3b, unlike the inhibiotrs of complement activation (e.g., DAF, MCP, CR1 and Crry). (Tomlisnon, WO/2004/045520). 

Human CR2 is also the obligate receptor for the Epstein-Barr virus (EBV) through its interactions with the gp350/220 viral membrane protein. CR2 also serves as a receptor for CD23 and has been shown to be essential for normal humoral immunity to T dependent antigens as well as to possibly play an important role in the maintenance of B cell self tolerance and the development of autoimmunity. CR2 has also been shown to mediate the interaction of C3-bound HIV-1 as an immune complex with B cells in a fashion that promotes transfer of virus and infection of CD4T cells.

CR3 and CR4: 

Functions: are transmembrane heterodimers which are involved in adhesion to extracellular matrix and to other cells as well as in recognition of iC3b. They belong to the integrin family and perform functions not only in phagocytosis, but also in leukocyte trafficking and migration, synapse formation and costimulation. Integrin adhesiveness is regulated through a process called inside-out signaling, transforming the integrins form a low to a high affinity binding state. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm.

CRIg (huSTIgMA, Z39Ig, PRO362): 

Structure: A human CRIg protein was first cloned from a human fetal cDNA library using degenerate primers recognizing conserved Ig domains of human JAM1. Sequencing of several clones revealed an open reading from of 400 amino acids. Blast searches confirmed similarity to Z39Ig, a type 1 transmembrane protein. The novel human protein was originally designated as a “single transmembrane Ig superfamily member macrophage associated” (huSTIgMA, also referred to as PRO362). Subsequently a splice variant of huSTIgMA was cloned, which lacks membrane proximal IgC domain and is 50 amino acids shorter, designated as huSTIgMAshort.

The extracellular IgV domain of CRIg (CRIg-ECD) selectively inhibits the AP pathway by binding to C3b and inhibiting proteolytic activation of C3 and C5. (Bing, “improving therapeutic efficacy of a complement receptor by structure-based affinity maturation” J. biol. chemistry, 284(51), 2009)

Mouse and human CRIg proteins share 83% sequence homology within the IgV domain and they superimpose with a root mean square deviation of 0.37 A for 100C- atoms. Sturctural requirements for C3b binding are also similar in that alanine substitution of residues M86 and D87 or H57 and Q59 showed a greater than 100 fold loss of binding affinity to C3b(Katschke “a novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis” JEM, 204(6) (2007).

Functions: CRIgis a macrophage associated receptor with homology to A33 antigen and JAM1 that is required for the clearance of pathogens form the blood. Next to functioning as a complement receptor for C3 proteins, the extracellular IgV domain of CRIg selectively inhibits the AP by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binding affinity for the covnertase subunit C3b is low requiring a relativley high concentraiton of protein to reach near complete complement inhibition. See regulation of the complement system

CRIg is exclusively expressed on tissue resident macrophages and binds to multimers of C3b and iC3b that are covalently attached to particle surfaces. Next to functionaing as an important clearance receptor, CRIg’s extracellular domain inhibits complement activation through the AP, but not the classical pathway. (He “a role of macrophage complement receptor CRIg in immune clearance and inflammation”, Molecular Immunology, 2008, 45(16), pp. 4041-4047.

CRIg variants with enhanced binding affinity:

Ashkenazi (WO 2006/042329 A2) discloses identification of CRIg and its use for the treatment of complement associated diseases. Also disclosed are CRIg variants having at least about 80% amino acid sequence identiy to a native sequence of CRIg. 

Sidhu (12/387794) disclose CRIg variants with combined amino acid substitutions Q64R and M86Y which showed enhanced binding affinity to C3b compared to native wild type sequence. The mutatns were generated by looking at the crystal structure of CRIg in complex with C3b and using phage display to improve binding affinity for CRIg-ECD for C3b. Phage displayd libraries were generated in which codons encoding residues of CRIg-ECD that contact C3b were subjected to limited randomization. Variants with improved binding affinity for C3b were selected and sequenced and recombinatn mutatn CRIg-ECD proteins were expressed and purified. (Bing, “improving therapeutic efficacy of a complement receptor by structure-based affinity maturation” J. biol. chemistry, 284(51), 2009)

Complement has a variety of important functions which includes:

(1) opsonization of antigens: including bacteria by phagocytosis. The complement component, C3b is the major opsonin of the complement system. Phagocytic cells, as well as some other cells, expresscomplement receptors that bind C3b, enhancing phagocytosis by these cells. C3b may also act as an adjuvant when coupled with protein antigens. C3b targets the antigen directly to the phagocyte, enhancing the initiation of antigen processing and accelerating specific antibody production. The coating of soluble immunogen complexes with C3b is thought to facilitate their binding to complement receptors on erythrocytes which carry these complexes to the liver and spleen. In these organs the complexes are stripped away from the red blood cells and phagocytosed. The terminal sequence of complement activation involves a macromolecular structure called MAC which lyses cells. Gram positive bacteria are generally resistant to complement mediated lysis because the thick peptidoglycan layer in their cell wall prevents insertion of the MAC into the inner membrane.

(2) activation of inflammation: The significance of complement activation is not limited to membrane damage resulting from the attack complex. The active complement peptides contribute to the immune response by increasing vascular permeability and contraction of smooth muscle, promoting immune adherence, granulocyte and platelet aggregation, enhancing phagocytosis, and directing the migration of neutrophils (PMN) and macropahges to the site of inflammation.

The cleavage of C3 and C5 results in the release of two small biologically active peptides, C3a and C5a which act as anaphylatoxins. They amplify the immune response by causing the release of histamine, slow releasing substance of anaphylaxis (SRS-A), and heparin from basophils and mast cells. These substances increase capillary permeability and contraction of smooth muscle resulting in edema and inflammation. In addition to its role as an anaphylatoxin, C5a is a potent chemotactic factor which causes the directed migration of leukocytes including DCs and monocytes to the site of inflammation so these leukocytes will phagocytize and clear immune complexes, bacteria and viruses form the system.  C5a increases the epxression of complement receptors CR1 and CR3 on both PMN and monocytes and is chemotactic for both types of cells.

Complement has a variety of important functions which includes:

(1) opsonization of antigens: including bacteria by phagocytosis. The complement component, C3b is the major opsonin of the complement system. Phagocytic cells, as well as some other cells, expresscomplement receptors that bind C3b, enhancing phagocytosis by these cells. C3b may also act as an adjuvant when coupled with protein antigens. C3b targets the antigen directly to the phagocyte, enhancing the initiation of antigen processing and accelerating specific antibody production. The coating of soluble immunogen complexes with C3b is thought to facilitate their binding to complement receptors on erythrocytes which carry these complexes to the liver and spleen. In these organs the complexes are stripped away from the red blood cells and phagocytosed. The terminal sequence of complement activation involves a macromolecular structure called MAC which lyses cells. Gram positive bacteria are generally resistant to complement mediated lysis because the thick peptidoglycan layer in their cell wall prevents insertion of the MAC into the inner membrane.

(2) activation of inflammation: The significance of complement activation is not limited to membrane damage resulting from the attack complex. The active complement peptides contribute to the immune response by increasing vascular permeability and contraction of smooth muscle, promoting immune adherence, granulocyte and platelet aggregation, enhancing phagocytosis, and directing the migration of neutrophils (PMN) and macropahges to the site of inflammation.

The cleavage of C3 and C5 results in the release of two small biologically active peptides, C3a and C5a which act as anaphylatoxins. They amplify the immune response by causing the release of histamine, slow releasing substance of anaphylaxis (SRS-A), and heparin from basophils and mast cells. These substances increase capillary permeability and contraction of smooth muscle resulting in edema and inflammation. In addition to its role as an anaphylatoxin, C5a is a potent chemotactic factor which causes the directed migration of leukocytes including DCs and monocytes to the site of inflammation so these leukocytes will phagocytize and clear immune complexes, bacteria and viruses form the system.  C5a increases the epxression of complement receptors CR1 and CR3 on both PMN and monocytes and is chemotactic for both types of cells.

Graft-versus-host-disease (GVHD): GVHD (graft-versus-host-disease) occurs when donor derived T cells recognize and react to histo-incompatible recipient antigens leading to a variety of host tissue injuries. GVHD is the major cause of morbidity and mortabiliy after allogeneic BM transplantation, even when siblings are matched at the human luekocyte antigen (HLA) locus. GVHD occurs in both acute and chronic forms, each with different kinetics and distinctive pathology. The skin is the organ the most affected by GVHD and clinical symptoms range form a simple rash to a dramatic epidermolysis. Other affected organs are the gut, the liver, the lung and lymphoid organs. Chronic GVHD occurs less than 100 days after transplantation and affects the same tissues, in addition to the joints and the mucosal surfaces with an incidence of 40-60% in trnasplant recipients surviving more than 100 days.

Strategies to Mitigate GVHD:

Regulatory T cells:

–T-reglatory cells Engineered to express CARs:

Chen, (WO/2021/034689) discloses regulatory T cells that are engineered to expressed chimeric antigen receptors (CARS) that target CD83 on antigen-presenting cells to prevent GVHD. The CARS is made up of three domains: an ectodomain, transmembrane domain and an endodomain. The ectodomain includes the CD83 binding region and is responsible for antigen recognition. The antigen recognition domain is typically an scFv. It also optionally contains a signal peptide so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain transmit an activation signal to the immune effector cell after antigen recognition. For example, the endodomain can contain an intracellular signaling domain and optionally a co-stimulatory signaling region. The signaling domain generaly contains immunoreceptor tyrosine-based activation motifs that activate a signaling cascade when the ITAM is phosphorylated. The “co-stimulatory signaling region” refers to intracellular signaling domains fom costimulatory protein receptors such as CD28 that are able to enhance T cell activaiton by T cell receptors. For example, the CAR can be defined by the formula signal peptide -CD83–hinge domain –transmembrane domain –co-stimulatoyr signaling regions –signaling domain. The T cells which is gnetically modified to express the CAR targeting CD83 is administered to the subject receiving transplanted domor hematopoietic cells or solid organ allografts. CD83 is differentially epxressed on alloreactive T cells. The, the anti-CD83 CAR-T cells will target T cells that cause GVHDand spare graft-versus-leukemia (GVL). Even when donors are fully HLA matched, the minor HLA disparity or the presence of H-Y antigens are sufficient to cause GVHD. 

Partial Liver Transplantation

Massive hepatic resection is the only option for some patients with primary or secondary liver tumors. With regard to small-for-size (SFS) liver transplantation, the use of partial liver grafts has the potential to substantially reduce the donor shortage by allowing the donor organ to be split between 2 recipients.

Xenotransplantation

Due to the shortage of available human organs the pig has been chosesn as a source for xenotransplantation organs. The first major hurdle in carrying out a cross species xenotransplantation is the occurance of hyperacute rejection triggers by complement activation. (Knoell, EP1336618).

Risk associated with Liver Transplantation

Liver resection has bewcome an increasinly safe procedure, but certain procedures remain high risk, such as massive liver resection and small-for-size (SFS) liver transplantation.

Ischaemic-reperfusion injury (IRI):

IRI is an inevitable phenomenon that results following major liver surgery, including partial hepatecotomy and liver transplantation (Gomez, World J Gastroenterol 2007, February 7: 13(5): 657-670).

Tang teaches that partial grafts form split livers have been introduced to expand the donor pool, briding the gap between the increasing number of potential recipients and the inferior number of eligible liver donors but that there are mroe risks in performing partial or small for size liver transplantation, not only due to technical obstances, but also early graft loss resulting from ischemia reperfusion injury (Tan, Transplantation Proceedings, 39, 1338-1344 (2007)).

–Mechanisms:

(i) complement system: Studies using rat models indicate a central role for complement in hepatic IRI. However, in addition to its role in hepatic IRI, evidence indicates that complement activation is required for normal liver regeneration, following either resection or toxic injury. Data indicates that the complement activaiton products C3a and C5a play an important role in the proliferative response and hepatocyte regeneration via an effect on TNF alpha and IL-6 expression. (he, J. Clinical Investigation, 119(8), 2009).

United Network for Organ Sharing

See also Kidney Diseases  See also MHC

In 1901, Landseiner discovered ABO blood group antigens. In 1954, Murray performed the first successful kidney transplant between identical twin brothers. In 1958, Dausset discovered the major histocompatibility complex in human, the human leukocyte antigens (HLA). In 1963, the first liver allograft transplant by starzl and the first lung transplant was performed by Hardy. In 1967 the first heart allograft transplant was performed by Barnard. In 1972, Borel discovered immunosupressive properties of Cyclosporine (isolated from fungus in Norway). In 1983, Baby Fay received the first baboon heart and survived for 21 days. In 1999, Rapamycin was approvied for clinical kindey transplantaion. These are all milestones in transplantation therapy, improving graft transplantation.

Today, a 80% 1 year survival rate for kidney, liver, heart and pancreas transplantation is obtained. However, chronic rejection of transplants remains a large problem. Another major problem is organ shortage. The other problems in translantation are infections due to overimmunosupression. The solution to all of these problem would be to make organs accepted to reduce the need to make transplantations.

Definitions

Autologous graft (“autograft”): is a graft transplanted between two genetically identical individuals.

Allograft (allogeneic graft): is a graft transplanted between two genetically different inidividuas of the same species. In conventional transplantation for allegeneic recipients, multiple HLA class I and class II proteins must be matched for histocompatbility. 

Zenograft: is a graft transplanted between inidividuals of different species. 

Tolerance means the absence of a destructive response to the allograft in an immunocompetant host. , although easy to acheive in small animal models, has been extremely difficult in large animal models and humans. There is also no assay to measure tolerance (no simple assay). There is also a problem of compatbility of tolerance induction strategies with conventional immunosuppression. So any new treatments have to be compatible with immunosupressive drugs people are already taking.

Acute versus Chronic rejection: 

Chronic GVHD usually appears 100 days post transplantation and sevral factors are thought to be involved including upregulation of TGF-beta which casues fibrosis and upregulation of OX40 ligand (OX40L), a TNF family member. In the acute form of the disease, mature T cells present in the bone marrow recognize the donor tissue as foreign. which via APCs casue the activation and proliferation of the donor T cells. Onset for the acute form is usually within 100 days of transplantation (Campbell, US 2017/0327587)

Chronic rejection is poorly defined that involves immune and non-immune components. It is a slow process that occurs months to years after transplantation and characterized by arteriole thickening and interstitial fibrosis. Current immunosuppressive are ineffective at treating chronic rejection.

Mechanisms/Pathways of graft Rejection

Activation of T cells: Allografts are rejected in part by the activation of T cells. The transplant recipient mount a rejections response following CD4+ T cell recognition of foreing antigens in the allograft. These antigens are encoded by the major histocompatibility complex (MHC). There are both class I and Class II MHC molecuels. In human the class I MHC molecules are HLA-A, B and C. The class II MHC molecules are called HLA-DR, DQ and DP. (Rother, WO2005/110481). 

There are important differences in HLA expression between T and B cells, which influence the interpretation of a crossmatch. T cells do not sonstitutively express HLA class II; so the result of a T cell crossmatch generally reflects antibodies to HLA class I only. B cells express both HLA class I and II. Becasue of this, a positive B cell crossmatch is more difficult to interpret thatn a positive T cell crossmatch. It may be due to antibodies directed agaisnt HLA class I, II or both. (Frey, US Patent Applicaiton No: 16/340,453, published as US 2019/0276524).

–Role of Costimulatory molecules and T cell activation: For example, CD40 on APC and CD154 is an important interaction. Antibodies against CD154 to block this interaction has been used in animal studies. Blockage of CD28/B7 and CD40/Cd40L pathways shows synergy in prolonging graft survival.

OX40L is not constituitively expressed but can be induced on professional APCs such as DCs and macrophages. Other types of cells such as Langerhans and NK cells can be induced to expressed OX40L. The OX40L receptor, OX40, is expressed on activted T cells (CD4 and CD8, TH1, TH2 and TH17), which may provide essential signals for the generation of memory T cells, the enhcnacement of the TH2 response and the prolongation of the inflammatory response. OX40 signals into responder T cells renders them resistant to Treg meidated suppression. (Campbell, US 2017/0327587)

Antibody-mediated rejection (AMR): 

AMR, also called “accelerated humoral rejection” is characterized by markedly elevated circulating donor reactive antibodies, microvascular thrombosis, and C4d deposition in the graft.  (Wang “Inhibition of terminal complement components in presensitized transplant recipients prevents antibody-mediated rejection leading to Long-Term graft survival and accomodation” J Immunol 2007, 179: 4451-4463).

In AMR, complement is suggested to be activated by the classical pathway and to play a key role in the pathogenesis. Rother (WO2005/110481) dicloses using a mose model for AMR and showing that anti-C5 mAb in combination with CsA and CyP achieved indefinite heart graft survival over 100 days. 

–In Kidney Transplantation:

Frey, (US Patent Applicaiton No: 16/340,453, published as US 2019/0276524) dicloses a method of treating/reducing antibody mediated rejeciton (AMR) in a human kidney transplant recipient that includes administering an anti-C5 antibody such as ecullizumab at a doese of 1200 mg for 3 hours prior to kidney allograft reperfusion, 900 mg 18-30 hours after reperfusion of the kidney allograt and 7, 14, 21 and 28 post transplantatin and 1200 mg dose adminsitered at week 5, 7 and 9 post transplantation. wherein the recipient is sensitive to a human living donor, received desensitization therapy prior to transplantation for 11 days or more but not post-transplation for at least 9 week. 

Rother (US Patent Application No: 15/243,290, issued as US 9,771,418) discloses a method of treating AMD in a patient having a kidney transpant which includes intravenously adminsitering to the pateint 1200 mg of eculizumab less than 24 hours beofre or during the transplant operation and one post-operative dose within 24 hours of the transplant, 900 mg once a week for four weks and then 1200 mg of eculizumab on week five and bi-weekly therafter. The eculizumab is adminsitered in an amount and with a frequency to maintin at least 50 ug of eculizumab per milliliter in the patient’s blood. 

Stegall (American J of Transplantation, 2011, 11: 2405-2413) discloses an eculizumab dosing region from PNH for AMR which includes 1200 mg immediately prior to transplantation, 600 mg on posoperative day 1 and 600 mg weekly therafter for 4 weeks. At week 4, assessment of DSA levels are performed. Eculizumab was dicontinued in patients whose DSA had signifcantly decreased and eclizumab treatment contined (1200 mg week 5 and then every 2 weks) in patients with persistently high DSA. 

–In Heart Transplantation:

All individuals who reeive a heart tranpslant are at risk for developing antibody-mediated rejeciton (AMR). The growing proportion of sensitized cardiac recipients presents an additional challenge to the transplant practitioner attempting to minimize the occurrence of AMR. Pateints pre-exposed or “sensitived” to antigen exposing events (i.e: lgood transfusions, multiple pregnancies, prior organ transplantations, ventricular support devices) are more likely to both possess preformed and develop antibodies. NCT02013037 by Alexion Pharmaceuticals discloses a non-randomized, open-label efficacy trial investigating use of eculizumab alongside conventional therapy to prevent antibody mediated rejection. Eculizumab as admisntiered 1200 mg at the time of transplantation, 900 mg day 1 post-transplant, on day 5 post-transplant, 1 g/Kg for 2 consecutive days, on days 7, 14 and 21 900 mg and then days 28, 42 and 56 1200 mg. 

Acute/Classical pathway: This is cell mediated (predominantly CD4 and CD8 T cells) directed at donor MHC antigens. CD4 T cells recognize these classes (allosensitization) which help develop effector functions (B cells, CD8T cells, Macrophages) which destroy the graft. Current immunosupression is targeted at acute rejection.

In allorecognition the TCR is working as the antigen. This means the precursor frequency is different. 1-% of T cells will become activated rather then with nominal antigen where 1 in about 10000 T cells are active to respond. The frequency of antigens presented on a cell is much greater then in the traditional sense. In direct recognition, the T cell recognizes an allo APC presenting a foreign pathway. The direct pathway is predominant (greater than 90%). It activates both CD4 and CD8 T cells and is sufficient to cause rejection. There is also an indirect recognition (lower frequency) where peptides are presented by self (host) APC. This is probably important in chronic rejection and may have a role in tolerance induction. This pathway involves epitope spreading.

Hyperacute rejection (HAR): occurs minutes to hours after transplantation due to antibody mediated and complement dependent pathways. HAR is rapid and severe and represents one of the largest obstacles to the success of xenotransplantation techniques. HAR is for the most part mediated by antibodies and complement, there being natural human antibodies, predominantly IgG and IgM subclasses which react with nuermous molecuels on xenotransplant cells, particularly endothelial cells, in vascularized transplants. These are preformed antibodies specific for MHC antigens (eg.., graft before, etc). It is now generally accepted that all or most of the HAR reaction is due to the presence of human antibodies directed against the carbohydrate epitope Gal-alpha (1,3) Gal.

The target of the response is vacular endothelium. This can be avoided by prescreening (taking serum from patient and reacting it against donor cells). Attempts to eradicate HAR have included removal or neutralization of complement and antibody.  

Dendritic Cells in Alloreactivity: DCs are specialized, exptremely potent APC that stimulate both CD4 and CD8 T cells in mixed luekocyte response. 30 years ago it was noted if depleted DCs before transplantation, found greater survival rate. Both donor and host CD contribute to alloactivation (direct and indirect pathways above).

Trying to take advantage of idea that certain DCs can promote tolerogenicity, BM DCs were derived in low dose GM-CSF and shown that they are poor stimulators of T cells. Low dose gave rise to what looked like immature DCs. High does gave rise to mature and immature DC. The low dose, immature DCs transplanted at day 3 prolonged graft survival.

Role of autoantigens in alloreactivity: In one report, gave rats allograft and then challenge rate with specific antigen to see whether ear swelling occurred which indicates T cell response.

Cross-Matching Prior to Transplantation:

Cross-matching was developed in an attempt to identify recipients hwo are likley to develop acute vacular rejection as a graft form a given donor. This phenomenon, HAR, is a result of preformed antiobdies against the donor; referred to as donor-specific antibodies (DSA). Such antibodies are usually formed as the result of previous expsure to HLA, generally through pregnancy, blood transfusion or previous transplantation. Preformed antibodies cause rejection by binding to HLA antigens expressed on the endothelium of vessels in the transplanted kidney, resulting in activaton of the complement cascade with resultant thrombosis and infarction of the graft. (Frey, US Patent Applicaiton No: 16/340,453, published as US 2019/0276524).

Complement Dependent Cytotoxicity Crossmatch: A CDC crossmatch involves placing recipient serum (potentially containing donor-specific anti-HLA antibodies) onto donor lymphocytes (containing HLA antigens). A cytotoxic reaction suggests the presence of preformed donor specific antibodies (DSA).  (Frey, US Patent Applicaiton No: 16/340,453, published as US 2019/0276524).

Treatment Strategies:

The principal stragegies to prevent GVHD center around the depletion of donor T cells. However, this may lead to the loss of GVHD (graft verus tumor effect) and to an increased risk of infections and graft failure. Recently focus has been on host DCs as key stimulators of donor T cells, inducing GVHD. 

Desensitization: refers to donor specific human antigen (HLA or DSAs) reduction techniques used to facilitate kidney transplantation for recipients who are sensitized to their donor organs by lowering the amount of circulating DSA. DSA techniques include for example direct antibody removal by plasmapheresis (PP), immune modulation using intravenous immunoglobulins (IVIg) and attempts to deplete B cells using a variety of immunosuppressive agents. (Frey, US Patent Applicaiton No: 16/340,453, published as US 2019/0276524).

 Antibodies against OX40L: Campbell, (US 2017/0327587) disclsoes antibodies against OX40L which are useful for treatment graft versus host disease. 

See also non-immune cells (for discussion of osteoblasts and osteoclasts)

Osteoporosis:

Osteoprosis is characterised by a progressive loss of bone mass and microarchitecture which leads to incrased fracture risk. The World Helath Organization (wHO) defines osteoporsis as a systemic skeletal disease characterised by low bone mass and microarhitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. with the ageing of the population, the complications of osteoporsis, fractures, represent a growing medical and socio-economic threat in industrialised countires. Switzerland belogs to the countries with the highest and still fastest growing life expectancy at birth worldwide (84.6 years for women and 80.2 for men in 2010). As the incidence of osteoporotic fractures increases exponentially with age, the implementation of meassures aimed at reducing fracture risk is needed to preserve quality of life and to ensure adqueate control of health care costs. “Kurt Lippuner, “the future of osteoporsis treatment –a research update” Current opinion, July 2012). 

Types of Osteoporsis:

The goal of steoporsis treatment is to reduce fractures . this can be acheived either by decreasing bone resorption and/or by increasing bone formation. 

–Primary Osteoporsis: is linked to the normal aging process. There is a link between two hormones, estrogen and progesteron and teh rate at hwih bone is lost. Estrogen regulates osteoclasts that break down bone and progesterone controls osteoblasts, which help in making new bone. Other hromones are also important. Primary osteoporsis can be divided into “primary type I and “primary type II” osteoporsis.

—-Primary Type I osteoporsis: is generally referred to as postmenopausal osteoporsis, as it is seen in women six times more frequently who have gone through menopause, resulting in a drop in levels of estrogen. It occurs in women about 10-15 years after menopause, usually between age 50 and 70. The loss of bone structure because of the increase in bone resorption is connected to estrogen deficiency in women and the lack of testosterone in men. People who suffer from primary type I osteoporosis are at high risk of spinal and wrist factures. 

—-Primary Type II osteoporosis: is caused by a long term calcium deficiency. Women are twice as more likely than men to suffer form Type II osteoporosis. It resutls in loss of outer bone structure and also the inner trabecular bone to wear down and become thin. Studies have linked deficiency in dietary calcium and vitam D decle due to age, or the hyper activity of the parathyroid glands (secondary hyperparathyroidism). It is also called low-turnover osteoporsis becasue the rate of bone turnover is much lower in this type of osteoporsis. 

Secondary Osteoporisis: develop swhen certain medical conditions and medications incrase bone remodeling leading to disruption of bone reformation. The loss of bone mass occurs due to the imbalance beween the production of new bone and the loss of old bone, leading to lower bone turnover rate. An imbalance in hormones from the increased activity of the parathyroid glands or hyperparathyroidism can result in secondary osteoporsis. Hormonal imbalance can also occur form hyperthyroidism, which is an excessive secretion from thyroid glands. Seondary osteoporsis is common amon patients fufering form diabetwes, which can often lead to hyperglycemia or increasing level sof glycosuria. The long term use of oral corticosteroids can cause hypercotisolisms, which increases the chance of developing secondary osteoporsis. 

Treatments:

–Antibodies against endogenous inhibitors of bone formation scelerostin, dickkkofp-1, PTH and PTHrp analogues.

–inhibitors of bone resorption: This includes cathepsin K inhibitors: which may suppress osteoclase function without impairing osteoclast viability and thus maintain bone formation by preserving the osteoclast-osteoblast corsstalk.

—-Bisphophonates: are today’s mainstay of osteoporosis treatment. They act as inhibitors of bone resorption with a high affinity for bone and were shwon to increase BMD and reduce fracture risk in patients with postmenopausal, male, and glucocorticosteroid-induced osteoporsis. Due to their long half-life in bone, they can be adminsitered either orally or intravenously.

–antibody against RNAKL: such as denosumab which inhibits osteoclast formation, function and survival.