BIOLOGY OF IMMUNOGLOBULIN G SUBCLASS PRODUCTION
Immunoglobulin Genomic Organization
Antibodies are heterodimeric glycoproteins composed of 2 light chains and 2 heavy chains. Both the light and heavy chains have variable regions that form the contact site for the antigen (paratope) and constant regions that enable protein assembly and mediate effector functions. The isotype and subclass are determined by the constant region of the heavy chain encoded by discrete loci in the immunoglobulin (Ig) complex. The human Ig system is divided into 5 isotypes: IgM, IgD, IgA, IgE, and IgG. IgA and IgG can be further subdivided into several subclasses. The heavy chain constant region genes are ordered μ, δ, γ3, γ1, α1, γ2, γ4, ε, and α2.
The Basics of Class Switching
The molecular mechanisms by which B cells class switch have been extensively studied. IgM is the first subclass to be produced, and only antigen-experienced B cells undergo class switching to other isotypes. The nature of the antigen influences IgG subclass production, with thymus (T)–independent polysaccharide or glycolipid antigens typically inducing IgM and IgG2 and protein antigens canonically stimulating IgG1 and IgG3. Production of most terminal IgG4 subclass is usually seen only with chronic antigen stimulation, such as in the setting of allergy or parasitic infection. T-dependent protein antigens stimulate follicular B cells that typically require CD4+ T cell signaling to generate class-switched Igs and for the establishment of memory B cells. Briefly, formation of a synapse between T and B cells facilitates CD40-CD40L (CD154) interactions that prime the B cell. Cytokines signal the B cell to switch the isotype and subclass of Ig and to secrete Ig (elegantly reviewed in a study by Stavnezer et al1). A B cell that has switched to a downstream isotype or subclass is incapable of switching back to a more upstream isotype.
B cells that express the more terminal subclasses IgG2 and IgG4 often retain remnants of upstream switch regions,2 suggesting that class switch recombination occurs in a stepwise manner. Moreover, the variable regions of IgG2 and IgG4 often exhibit more extensive somatic hypermutation than IgG1 or IgG3 and may be of higher antigen affinity than earlier subclasses.
It is known that the T follicular–associated interleukin (IL)-21 and TH2-associated cytokines such as IL-4, IL-13, and IL-10 influence Ig isotype and subclass production.3,4 But to date, no dedicated switch factor for any given Ig subclass has been identified, and the signals governing specification of which isotype or subclass is used are incompletely defined. Effort is needed to further elucidate the signals controlling subclass specification, particularly in the setting of alloimmunization.
STRUCTURE AND EFFECTOR FUNCTIONS OF IGG SUBCLASSES
Although the constant regions of the different gamma subclasses are more than 90% homologous, their effector functions and affinity for antigen can vary significantly. IgG1 is by far the most abundant subclass in serum, and IgG3 has the shortest half-life of only 7 days. The constant region of IgG3 has the longest hinge region, yielding the most flexibility and accessibility to effector molecules. The hinge regions of IgG2 and IgG4 are much shorter, and these molecules exhibit more rigidity. Variations in the species of N-linked glycan at the heavy chain constant region 2 (CH2) can dramatically affect antibody effector function.5 Contact sites for engaging complement C1q and Fc gamma receptors (FcγRs) overlap in this CH2 domain, and this is where much of the diversity between IgG subclasses is observed.
IgG3 and IgG1 exhibit the most efficient activation of the classical complement cascade. Under conditions of high antibody titer or high antigen density, IgG2 is capable of activating complement as well, but IgG4 is generally incapable (Table 1). The complement C1 complex binds to the Fc region of IgG via C1q-CH2 interactions. Key amino acid residues in IgG that are required for C1q binding have been identified.7 Because host cells express a constellation of complement regulatory proteins antagonizing the complement cascade, only very high titers of antibody and/or the stronger C1q binding subclasses may be able to trigger terminal complement activation leading to formation the membrane attack complex and cell lysis. Complement components C3d and C4d, commonly detected as part of the histological diagnosis of antibody mediated rejection, are products of cleavage by regulatory proteins and are remnants of different stages of complement activation in the vasculature.
Interestingly, IgG4 molecules are prone to dissociation, forming half molecules of 1 light chain and 1 heavy chain. IgG4 may compete with other subclasses for antigen binding and block complement activation.8 Given that IgG4 often has the highest affinity for antigen but the lowest capacity to elicit effector functions, it has been proposed that chronic antigen exposure stimulates IgG4 as a mechanism of limiting the humoral immune response.
Antibodies enlist adaptive and innate immune cells to carry out effector functions such as phagocytosis, degranulation and antibody-dependent cell-mediated cytotoxicity. Receptors for the Fc regions of IgA, IgG, IgE, and IgM are expressed on a variety of lymphoid and myeloid cells, including B cells, monocytes, natural killer (NK) cells, neutrophils, mast cells, and occasionally, T cells. The human FcγR system is composed of 3 major classes, FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRII and FcγRIII each have several isoforms. Only the FcγRIIb exhibits inhibitory functions via an ITIM; the other FcγRs are activating. The unique FcγR molecules have discrete affinities for different repertoires of IgG subclasses and patterns of expression6 (Table 1). In humans, FcγRIIA, FcγRIIIA, and FcγRIIIB are polymorphic, with allelic variants exhibiting differences in affinity for IgG subclasses relevant in infectious disease, autoimmunity, and response to therapeutic antibodies as well as transplant outcomes.9 Association of a coding polymorphism in FcγR IIA has also been associated with graft survival in retransplant candidates (reviewed in study by Bournazos et al10). We observed that in vitro recruitment of monocytes by HLA antibody–activated endothelial cells varies depending on the IgG subclass of HLA antibody and the monocyte FcγR repertoire.11 In brief, IgG3 and IgG1 bind well to monocyte FcγRI, neutrophil FcγRIIA, and FcγRIIIB, and NK cell FcγRIIIA. IgG2 can only be effectively recognized by 1 allele of FcγRIIA, whereas IgG4 is only bound by FcγRI (see Table 1 for more detail).
In addition to the discussed Fc-mediated effector functions, antibodies are capable of activating agonistic signaling on dimerization of target antigens. Many groups have described that the intracellular signaling cascades downstream of HLA cross-linking by antibodies in antigen-presenting cells and vascular cells. Of relevance to transplantation, antibodies to HLA class I have been shown to trigger endothelial and smooth muscle cell activation of proliferative and inflammatory pathways via calcium signaling, mammalian target of rapamycin, and ERK12-14 that can also be detected in allografts during rejection.15 It is hypothesized that such agonistic signaling promotes endothelial dysfunction and transplant vasculopathy. In theory, all IgG subclasses should be capable of cross-linking HLA molecules and triggering activation of endothelial cells in the allograft vasculature, although this remains to be determined experimentally.
MEASUREMENT OF HLA-SPECIFIC IGG SUBCLASSES
An accurate assay to measure HLA-specific IgG subclasses requires targets carrying stable amounts of HLA molecules and IgG subclass–specific reporter antibodies with a high sensitivity and specificity. The first studies investigating HLA-specific IgG subclasses used cells as the HLA-molecule source and various reporter antibodies with often not-well-characterized specificities.16,17 The introduction of single HLA-antigen beads (SA) revolutionized the ability to detect HLA antibodies. Using this test, system with IgG subclass–specific reporter antibodies has emerged as the preferred assay to detect HLA-specific IgG subclasses. Based on the current literature and our own experience (Hönger et al18), we recommend using the following reporter antibodies because they have a very low cross-reactivity: IgG1, clone HP6001; IgG2, clone HP6002; IgG3, clone HP6050; and IgG4, clone HP6025.
The readout of the SA assay is mostly given as mean fluorescence intensity (MFI), which reflects the number of bound HLA antibodies to the beads. It is important to notice that IgG subclass–specific SA assays produce different MFI values compared with the generic SA assay using a polyspecific reporter antibody binding to all IgG subclasses. In a very clean experimental system, we observed 60% lower MFI for IgG1, 76% lower MFI for IgG2, 37% lower MFI for IgG3, and 50% higher MFI for IgG4 compared with the generic SA assay (Hönger et al18). Furthermore, the background signal in the IgG subclass–specific assay is often substantially lower than in the generic SA assay. Therefore, it seems reasonable to use for each IgG subclass, and each serum as well as each bead, a different MFI cutoff to assign a positive result.19,20 On the other hand, an arbitrary MFI cutoff for all IgG subclass–specific assays might be easier to use and allows for better comparison among different studies.21,22
Although not absolutely correct from a scientific point of view, we favor a pragmatic approach to standardize the IgG subclass–specific assay for clinical studies. We suggest using the previously mentioned IgG subclass–specific reporter antibodies all at the same concentration and to apply 1 arbitrary MFI cutoff for all IgG subclass–specific assays, which is 50% lower than the one used for the generic SA assay.
IGG SUBCLASS PATTERN OF HLA ANTIBODIES
Given the 4 IgG subclasses, 15 different combinations with at least 1 detectable IgG subclass are possible. Based on the complement-activating capability and the class switch evolution of IgG, 3 “biological” groups can be defined: (i) “complement-binding subclasses only” group (ie, IgG1 and/or IgG3), (ii) “expansion to noncomplement-binding subclasses” group (ie, IgG1 and/or IgG3 and IgG2 and/or IgG4), and (iii) “switch to noncomplement-binding subclasses” group (ie, IgG2 and/or IgG4).
Three studies provided details regarding IgG subclass patterns, which are summarized in Figure 1.19,20,22 Despite using different assay conditions and investigating patients at different clinical stages, the IgG subclass patterns are remarkably similar. IgG1 was the most frequently observed isolated subclass (25%-30%), followed by IgG3 (5%), and IgG2 (2%) and IgG4 (2%). With respect to the “biological” groups, the “complement-binding subclasses only” group accounted for 37% to 48% and the “expansion to noncomplement-binding subclasses” group for 47% to 62% of all IgG subclass pattern. The “switch to noncomplement-binding subclasses” group was rarely observed (1%-5%). Importantly, a single, restricted subclass response is also quite rare.
Lowe et al20 found that IgG subclasses induced by blood transfusions were more often restricted to the “complement-binding subclasses only” group, whereas pregnancies and graft failure more often provoked an expansion to noncomplement-binding subclasses. Interestingly, Arnold et al23 reported that an expansion to noncomplement-binding subclasses in patients with de novo donor-specific antibodies to HLA (HLA-DSA) after renal transplantation was associated with a higher frequency of ABMR. Altogether, these data suggest that an expansion to noncomplement-binding subclasses indicates an advanced immune response stimulated by a longer and more intense antigen exposure. It is still unknown, under which conditions a complete switch to noncomplement-binding subclasses with loss of IgG1/3-producing plasma cells will occur.
CLINICAL STUDIES INVESTIGATING THE UTILITY OF IGG SUBCLASS ASSAYS
The presence of HLA-DSA pretransplant or posttransplant is associated with an increased risk for ABMR and allograft loss. However, individual patients with HLA-DSA demonstrate a wide spectrum ranging from uneventful clinical courses to severe ABMR with subsequent allograft loss. Several studies investigated whether an IgG subclass analysis of HLA-DSA helps to improve prediction of ABMR and allograft loss.
Table 2 summarizes those 10 studies that analyzed all 4 IgG subclasses. Because of the different settings (eg, pretransplant vs posttransplant), different IgG subclass assay protocols and cutoffs, and different definitions of outcomes, it is currently impossible to draw any firm conclusions. It seems that the pretransplant IgG subclass pattern is not very predictive for ABMR and allograft loss. By contrast, the posttransplant presence of high IgG3 levels or an expansion to noncomplement-binding subclasses likely provide diagnostic and prognostic value beyond the generic SA assay regarding prediction of the ABMR phenotype and the clinical course of ABMR. Whether this information is clinically helpful to guide treatment strategies is still unknown.
There are several other studies that investigated only 1 single IgG subclass regarding prediction of allograft failure. Such a study design might introduce a bias because the effect of other individual IgG subclasses and specific IgG subclass mixtures cannot be fully investigated. We only highlight 2 studies in this context. Everly et al28 found that de novo posttransplant HLA-DSA being IgG3 and IgM positive are associated with a high risk of renal allograft failure. O'Leary et al29 observed a trend toward inferior survival of liver allograft recipients with pretransplant IgG3 positive HLA-DSA.
A very interesting topic is the dynamic evolution of HLA-DSA IgG subclasses from pretransplant to posttransplant. So far, this has only been systematically investigated by Khovanova et al25 using sera obtained within the first 30 days posttransplant. They observed changes in the IgG subclass profiles, but the correlation with clinical outcomes was rather weak.
Another interesting question is the biological significance of IgG2/IgG4 HLA-DSA when present together with IgG1/IgG3 HLA-DSA. As previously mentioned, some clinical studies suggest that an expansion to these noncomplement-binding subclasses indicates an advanced immune response enabled by ongoing T cell help. These IgG2/IgG4 antibodies might induce complement activation in synergy with IgG1/IgG3 antibodies if they target different epitopes on the same HLA molecule.30 On the other hand, if the IgG2/IgG4 antibodies are directed against the same epitope as the IgG1/IgG3 antibodies, they could potentially block IgG1/IgG3-dependent complement activation. We found in in vitro experiments that this blocking effect starts when IgG2/IgG4 are present in 2-fold higher concentrations than IgG1/IgG3 and it is almost complete at a 10-fold excess (Hönger et al18).
The humoral immune response against HLA antigens is very complex, because the target is highly polymorphic; the process is dynamic and has different effector functions largely depending on the IgG subclasses. Therefore, deciphering the precise role of IgG subclasses requires progress in several areas.
First, the IgG subclass assay needs to be standardized to such an extent that comparison between studies is possible.
Second, because patients typically mount a mixed subclass alloimmune response, the individual contribution of each subclass can be difficult to dissect in these studies. It is unclear whether the predominance of IgG4 late in transplant and its association with chronic rejection reflects the particular pathogenic effect of this subclass or rather is indicative of sustained alloimmune activation.
Third, the clinical relevance of each subclass is beginning to be revealed, but the mechanisms of graft injury elicited by different donor specific IgG subclasses are mostly uncharacterized. Certainly, complement activation is a key pathological mechanism of graft injury, but it is not absolutely required for rejection in humans or in animal models.16,31 NK cells and macrophages also seem to play an important role during ABMR.31-33 However, the contributions of FcγR-mediated functions and differential capacity of IgG subclasses to elicit antibody-dependent cell-mediated cytotoxicity and phagocytosis have yet to be fully elucidated in the context of antibody mediated rejection.
Fourth, the factors regulating isotype and subclass specification are incompletely understood. Different routes of allosensitization seem to stimulate distinct subclass repertoires,20 but the milieu of B cell activation during different alloantigen exposures is uncharacterized. That de novo donor-specific antibody production is highly associated with medication nonadherence34,35 implies that current T cell–directed immunosuppression does have some effect on B cell activation and antibody production. In the studies discussed earlier, each center used different induction therapies. Little is known about how class switching might differ after induction therapies or under immunosuppressive regimens that are used in transplant patients. More studies are needed to describe the “natural history” of anti-HLA IgG subclasses in the pretransplant and posttransplant periods and to understand how they correlate with clinical outcome.
Finally, the measurement of all 4 IgG subclasses in addition to the generic SA assay is quite expensive. These costs seem only be justified if the IgG subclass information provides substantial diagnostic and/or prognostic value beyond the generic SA assay.
1. Stavnezer J, Guikema JE, Schrader CE. Mechanism and regulation of class switch recombination. Annu Rev Immunol
2. Jackson KJ, Wang Y, Collins AM. Human immunoglobulin classes and subclasses show variability in VDJ gene mutation levels. Immunol Cell Biol
3. Avery DT, Bryant VL, Ma CS, et al. IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4. J Immunol
4. Jumper MD, Splawski JB, Lipsky PE, et al. Ligation of CD40 induces sterile transcripts of multiple Ig H chain isotypes in human B cells. J Immunol
5. Allhorn M, Briceno JG, Baudino L, et al. The IgG-specific endoglycosidase EndoS inhibits both cellular and complement-mediated autoimmune hemolysis. Blood
6. Bruhns P, Iannascoli B, England P, et al. Specificity and affinity of human Fc gamma receptors and their polymorphic variants for human IgG subclasses. Blood
7. Sondermann P, Szymkowski DE. Harnessing Fc receptor biology in the design of therapeutic antibodies. Curr Opin Immunol
8. van der Zee JS, van Swieten P, Aalberse RC. Inhibition of complement activation by IgG4 antibodies. Clin Exp Immunol
9. Arnold ML, Fuernrohr BG, Weiss KM, et al. Association of a coding polymorphism in Fc gamma receptor 2A and graft survival in re-transplant candidates. Hum Immunol
10. Bournazos S, Woof JM, Hart SP, et al. Functional and clinical consequences of Fc receptor polymorphic and copy number variants. Clin Exp Immunol
11. Valenzuela NM, Trinh KR, Mulder A, et al. Monocyte recruitment by HLA IgG-activated endothelium: the relationship between IgG subclass and FcγRIIa polymorphisms. Am J Transplant
12. Jin YP, Valenzuela NM, Ziegler ME, et al. Everolimus inhibits anti-HLA I antibody-mediated endothelial cell signaling, migration and proliferation more potently than sirolimus. Am J Transplant
13. Li F, Zhang X, Jin YP, et al. Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner. Hum Immunol
14. Valenzuela NM, Hong L, Shen XD, et al. Blockade of p-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo. Am J Transplant
15. Li F, Wei J, Valenzuela NM, et al. Phosphorylated S6 kinase and S6 ribosomal protein are diagnostic markers of antibody-mediated rejection in heart allografts. J Heart Lung Transplant
16. Gao ZH, McAlister VC, Wright JR Jr, et al. Immunoglobulin-G subclass antidonor reactivity in transplant recipients. Liver Transpl
17. Griffiths EJ, Nelson RE, Dupont PJ, et al. Skewing of pretransplant anti-HLA class I antibodies of immunoglobulin G isotype solely toward immunoglobulin G1 subclass is associated with poorer renal allograft survival. Transplantation
18. Hönger G, Amico P, Arnold ML, Dupont PJ, Spriewald BM, Schaub S. Effects of weak/non-complement-binding HLA antibodies on C1q-binding. HLA
19. Honger G, Hopfer H, Arnold ML, et al. Pretransplant IgG subclasses of donor-specific human leukocyte antigen antibodies and development of antibody-mediated rejection. Transplantation
20. Lowe D, Higgins R, Zehnder D, et al. Significant IgG subclass heterogeneity in HLA-specific antibodies: implications for pathogenicity, prognosis, and the rejection response. Hum Immunol
21. Kaneku H, O'Leary JG, Taniguchi M, et al. Donor-specific human leukocyte antigen antibodies of the immunoglobulin G3 subclass are associated with chronic rejection and graft loss after liver transplantation. Liver Transpl
22. Lefaucheur C, Viglietti D, Bentlejewski C, et al. IgG donor-specific anti-human HLA antibody subclasses and kidney allograft antibody-mediated injury. J Am Soc Nephrol
23. Arnold ML, Ntokou IS, Doxiadis II, et al. Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies. Transpl Int
24. Cicciarelli JC, Kasahara N, Lemp NA, et al. Immunoglobulin G subclass analysis of HLA donor specific antibodies in heart and renal transplant recipients. Clin Transpl
25. Khovanova N, Daga S, Shaikhina T, et al. Subclass analysis of donor HLA-specific IgG in antibody-incompatible renal transplantation reveals a significant association of IgG4 with rejection and graft failure. Transpl Int
26. Viglietti D, Loupy A, Vernerey D, et al. Value of donor-specific anti-HLA antibody monitoring and characterization for risk stratification of kidney allograft loss. J Am Soc Nephrol
27. Kannabhiran D, Everly MJ, Walker-McDermott JK, et al. Changes in IgG subclasses of donor specific anti-HLA antibodies following bortezomib-based therapy for antibody mediated rejection. Clin Transpl
28. Everly JM, Rebellato LM, Haisch CE, et al. Impact of IgM and IgG3 anti-HLA alloantibodies in primary renal allograft recipients. Transplantation
29. O'Leary JG, Kaneku H, Banuelos N, et al. Impact of IgG3 subclass and C1q-fixing donor-specific HLA alloantibodies on rejection and survival in liver transplantation. Am J Transplant
30. Kushihata F, Watanabe J, Mulder A, et al. Human leukocyte antigen antibodies and human complement activation: role of IgG subclass, specificity, and cytotoxic potential. Transplantation
31. Hirohashi T, Chase CM, Della Pelle P, et al. A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody. Am J Transplant
32. Kobashigawa J, Crespo-Leiro MG, Ensminger SM, et al. Report from a consensus conference on antibody-mediated rejection in heart transplantation. J Heart Lung Transplant
33. Hidalgo LG, Sellares J, Sis B, et al. Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies. Am J Transplant
34. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Rates and determinants of progression to graft failure in kidney allograft recipients with de novo donor-specific antibody. Am J Transplant
35. Pizzo HP, Ettenger RB, Gjertson DW, et al. Sirolimus and tacrolimus coefficient of variation is associated with rejection, donor-specific antibodies, and nonadherence. Pediatr Nephrol