The discovery of natural regulatory T cell epitopes (Tregitopes [i]) in the sequence of therapeutic mAbs represents a paradigm shift for protein therapeutics, allergy, autoimmunity and transplantation. These natural Treg epitopes (also known as Tregitopes) are promiscuous MHC Class II T cell epitopes located in the Fc and framework regions of Fab from IgG. For several years now, we have hypothesized that the important anti-inflammatory effect of IVIG is due to Tregitopes that are present in the Fc and conserved Fab regions of IgG.
Does IVIG treatment activate nTregs through Tregitopes? Evidence is accumulating.
A number of ideas have been put forth about IVIG, such as: IVIG acts through anti-idiotypic networks, it contains antibodies to specific autoimmune antigens, and/or it blocks FcRn, leading to accelerated degradation of IgG. We’ve based the discussion below on mechanisms of action for IVIG proposed by Kaveri et al. [ii]. The data reviewed here lends support to our hypothesis that Tregitopes are responsible for the immunomodulatory effect of IVIG:
(1) IVIG is only anti-inflammatory at high doses. This points to a role for FcRn, and, of course, for the role of a subfraction (or peptide derived from a small fraction) of IVIG, such as a Tregitope.
What’s the mechanism? The high does of IVIG is said to overwhelm FcRn [iii]. Blockage of FcRn would increase processing of IgG in the proteolytic pathway, enhancing the presentation of Tregitopes (derived from IgG Fc and Fab) to natural regulatory T cells; leading to nTreg suppression of effector immune responses.
The case of FcRn-deficient mice supports the hypothesis. In these mice, loss of FcRn (like overwhelming FcRn with IVIG) may lead to increased Tregitope presentation (from any antibody)[iv].
Furthermore, blocking FcRn leads to improved immunomodulatory effect of IVIG; reducing FcRn activity also ameliorates disease in an autoimmune model [v]. And blocking FcRn during pregnancy inhibits the development of tolerance to allergens [vi].
(2) IVIG modulates the production of cytokines and cytokine antagonists associated with nTregs such as IL10 [vii]. This effect could be due to induction of circulating, Tregitope specific Tregs by Tregitopes present in IVIG.
(3) Treatment with IVIG directly expands the number of circulating Tregs; the expansion is associated with amelioration of autoimmune disease; nTregs are required for this improvement [viii]. Activation of natural regulatory T cells by APC presenting Tregitope could lead to the rapid decrease in titer of circulating autoantibodies that is often observed within hours following the infusion of IVIG.
(4) The long-term modification of immune repertoires in IVIG-treated individuals could be attributed to antigen-specific tolerance by nTregitopes activated by Tregitope.
The Tregitope hypothesis for IVIG mechanism of action does not exclude other possible mechanisms of action of IVIG. Perhaps the effect in some diseases is due, in part, to the anti-idiotypic network. Nor does the Tregitope hypothesis discount the potential for interactions of IVIG with immune cell surface receptors that may be relevant to IVIG effects in T cell- as well as B cell-mediated diseases.
Sugar and everything nice
Anthony and Ravetch have described a link between the Fc region sialylation, binding to DC-sign and tolerance in their papers published in Science in 2008 and PNAS 2009. What does DC-sign do? Its role is to traffic antigens (and Tregitope) to the proteolytic pathway [ix].
Isn’t that interesting? DC-sign directs IVIG (and its Tregitopes) to the proteolytic pathway. Over the past year or so, we have been pondering why sialylation and trafficking of immunoglobulin to the proteolytic pathway might be critically important for the effect of IVIG. Here are some of our recent thoughts:
(1) Does Fc region sialylation perhaps serve to target Tregitopes to the proteolytic pathway? The linkage between Fc (which contains Tregitope 289) and immunosuppression has been known for a long time (Baxevani, 1986 [x]). In fact, targeting IVIG to the proteolytic pathway leads to induction of tolerance in a class II dependent manner [xi].
Sialic acids are often linked to complex carbohydrates in a wide range of glycoproteins, which allows them to be involved in immunomodulation.
Why would a sialylation be the key to immunomodulation? Perhaps glycosylation is controlled during the course of immune response, and thus saialylation (we postulate) may be an important off-switch for inflammation. When immune response is completed (or antigen perists), sialyation of the immunoglobulin by an aging plasma cell may lead to the induction of regulatory T cells to the antigen, shutting down immune response. The interplay between regulatory and effector T cell responses therefore may be controlled by antibody glycosylation.
(2) Link between sialylation and autoimmune disease. There is also clearly a link between sialylation and increased processing of IgG. It follows that a lack of sialylation might lead to increased disease, in some cases, and that appears to be true. In Rheumatoid arthritis (RA), there is a marked increase in the percentage of serum IgG glycans lacking sialic acid and galactose residues; the level of these forms of IgG is more than two fold above age-matched healthy controls – and they correlate with disease activity [xii]. Levels of IgG-G0 fall during remission of the disease and also during pregnancy, when patients with the disease experience amelioration of symptoms.
(3) Some people dispute the role of DC-Sign. DC-sign mediated degradation of IVIG and presentation of Tregitopes is not the only means of delivering Tregitopes to nTregs – in fact the role of DC-Sign in the induction of tolerance by Fc IVIG is disputed [xiii]. How might one explain the induction of tolerance in the absence of trafficking by DC-sign? In unpublished work, we have shown that the Tregitope system is redundant; there are multiple (as many as eight) Tregitopes in IgG; some are in conserved regions in the framework of the variable region, others are in the CH1 and variable light chain domains.
Sugar and Splice
For best effect, IVIG combines sialylation (trafficking to the proteolytic pathway so as to better present Tregitopes) and linkage to the antigen to which tolerance is required. That’s why delivery of antigen with sialylated Fc would be tolerogenic – a parallel to work on antiDec205 published by Nussensweig and Steinman, in which co-delivery of antigen and antibody to dendritic cells results in the induction of tolerance. Of course, other explanations for that phenomenon abound, but we think that the effect is due to Tregitopes.
What’s the published evidence? In published work, we have shown that tetramer staining confirms that Tregitopes specifically activate CD4+CD25+FoxP3+ natural regulatory T cells (nTregs). In vitro, co-incubation of antigens with “Tregitopes” in vitro leads to suppression of effector cytokine and chemokine secretion, reduced proliferation of effector T cells, and expansion of antigen-specific adaptive Tregs (aTregs).
What’s new? In vivo, co-administration of Tregitopes with a range of proteins (such as FVIII, thyroid stimulating hormone receptor, ovalbumin, and autoantigens) leads to suppression of T cell and antibody responses to the test antigens. Furthermore, we have demonstrated that differences in the Tregitope content of monoclonal therapeutics such as ofatumumab and rituximab, humira and infliximab, explain differences observed in the clinical setting.
What is emerging from our work is the concept that Tregitopes play an important role in immune regulation, and that close attention to their effect may be critically important for the development of monoclonals and protein replacement therapies.
Regulatory T cell induction in the context of protein therapy and inflammation may contribute to the design of improved biologic therapeutics for a wide range of clinical conditions. It is likely that consideration of Tregitopes in protein design will lead to the design of safer, more effective protein therapeutics, whether these proteins are monoclonal antibodies, novel scaffolds, replacement therapies or biosimilars. Furthermore, Tregitopes are likely to be responsible for the anti-inflammatory activity of IVIG.
Those who wish to further explain the myriad properties of IVIG would do well to bear Tregitopes in mind.
[i] De Groot AS, Moise L, McMurry JA, Wambre E, Van Overtvelt L, Moingeon P, Scott DW, Martin W. Activation of natural regulatory T cells by IgG Fc-derived peptide “Tregitopes”. De Groot AS, Moise L, McMurry JA, Wambre E, Van Overtvelt L, Moingeon P, Scott DW, Martin W. Blood 2008 Oct 15;112(8):3303-11. Epub 2008 Jul 2
[ii] Sibéril S, Elluru S, Graff-Dubois S, Negi VS, Delignat S, Mouthon L, Lacroix-Desmazes S, Kazatchkine MD, Bayary J, Kaveri SV. Intravenous immunoglobulins in autoimmune and inflammatory diseases: a mechanistic. Ann N Y Acad Sci. 2007 Sep;1110:497-506.
[iii] Hansen RJ, Balthasar JP Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia Blood. 2002 Sep 15;100(6):2087-93.
[iv] Roopenian DC, Christianson GJ, Sproule TJ, Brown AC, Akilesh S, Jung N, Petkova S, Avanessian L, Choi EY, Shaffer DJ, Eden PA, Anderson CL. The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs. J Immunol. 2003 Apr 1;170(7):3528-33.
[v] Petkova SB, Akilesh S, Sproule TJ, Christianson GJ, Al Khabbaz H, Brown AC, Presta LG, Meng YG, Roopenian DC. Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease. Int Immunol. 2006 Dec;18(12):1759-69. Epub 2006 Oct 31.
[vi] Polte T, Hennig C, Hansen G. Allergy prevention starts before conception: maternofetal transfer of tolerance protects against the development of asthma J Allergy Clin Immunol. 2008 Nov;122(5):1022-1030.e5.
[vii] Reske D, Thomas AV, Petereit HF, Fink GR, Schroeter M. Impact of immunomodulatory treatment on leukocyte cytokine production in multiple sclerosis patients and healthy donors. Neuroimmunomodulation. 2009;16(6):385-91. Epub 2009 Jul 17.
[viii] Ephrem A, Chamat S, Miquel C, Fisson S, Mouthon L, Caligiuri G, Delignat S, Elluru S, Bayry J, Lacroix-Desmazes S, Cohen JL, Salomon BL, Kazatchkine MD, Kaveri SV, Misra N. Expansion of CD4+CD25+ regulatory T cells by intravenous immunoglobulin: a critical factor in controlling experimental autoimmune encephalomyelitis. Blood. 2008 Jan 15;111(2):715-22. Epub 2007 Oct 11.
[ix] Dakappagari N, Maruyama T, Renshaw M, Tacken P, Figdor C, Torensma R, Wild MA, Wu D, Bowdish K, Kretz-Rommel A. Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses. J Immunol. 2006 Jan 1;176(1):426-40.
[x] Baxevanis CN, Ioannides CD, Reclos GJ, Papamichail M. Evidence for distinct epitopes on human IgG with T cell proliferative and suppressor function. Eur J Immunol. 1986 Aug;16(8):1013-6.
[xi] el-Amine M, Hinshaw JA, Scott DW.In vivo induction of tolerance by an Ig peptide is not affected by the deletion of FcR or a mutated IgG Fc fragment. Int Immunol. 2002 Jul;14(7):761-6.
[xii] Przybysz M, Maszczak D, Borysewicz K, Szechiński J, Katnik-Prastowska I. Relative sialylation and fucosylation of synovial and plasma fibronectins in relation to the progression and activity of rheumatoid arthritis. Glycoconj J. 2007 Dec;24(9):543-50. Epub 2007 Jul 3.
[xiii] Bayry J, Bansal K, Kazatchkine MD, Kaveri SV. DC-SIGN and alpha2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):E24;