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A. Varthaman et al.
transfected cells or with immature human dendritic cells failed to trigger Toll-like receptor signaling28 or to induce dendritic cell maturation.29
The engagement of FVIII in the coagulation cascade induces a burst of thrombin production that leads to the generation of fibrin and to the consolidation of the platelet clot. Thrombin not only cleaves fibrinogen into fibrin but also cleaves proteinase-associated receptors, which in turn induce the release of pro-inflammatory cytokines and chemokines.30 Initial experiments elegantly suggested that FVIII-mediated thrombin-dependent activation of pro- teinase-associated receptors provides adjuvant signals for an anti-ovalbumin immune response in FVIII-deficient mice.31 However, later work using non-active FVIII with the V634M mutation in FVIII-deficient mice or normal FVIII in wild-type mice with impaired coagulation ruled out the possibility of FVIII being not only the target but also the trigger of the anti-FVIII immune response.1,32
The inability of FVIII to activate the immune system directly suggests that FVIII is not involved in the genera- tion of danger signals required to mount efficient primary immune responses.
Factor VIII inhibitor development: a case for inflammatory assaults?
Major bleeds, surgery, infections or vaccination at the time of FVIII administration have been proposed as poten- tial inflammatory assaults that could predispose hemo- philia A patients to develop allo-antibodies to exogenous FVIII.22 Counter-intuitively, experiments in FVIII-deficient mice recently demonstrated that exposure to the live- attenuated measles-mumps-rubella vaccine at the time of FVIII administration has no influence on the incidence or intensity of immune responses to therapeutic FVIII.33 The experimental data were confirmed by retrospective clini- cal observations from the PEDNET registry showing that pediatric vaccination given in close proximity to the administration of FVIII is not associated with an increased risk of FVIII inhibitor development.34 Interestingly, influen- za vaccination concurrent to FVIII treatment significantly reduced the incidence of anti-FVIII immune responses in mice.33
Chronic inflammation associated with recurrent bleed- ing35 as well as acute hemarthrosis following knee injury36 in FVIII-deficient mice failed to increase the immune response to exogenous FVIII. In fact, the induction of heme-oxygenase 1 (HO-1), a stress-inducible enzyme with potent anti-inflammatory activity, following injec- tion of heme,35 hemolyzed blood or following knee punc- ture-associated hemolysis (unpublished data), was shown to reduce the immune response to therapeutic FVIII. Interestingly, these observations find resonance in hemo- philia A patients. Indeed, a clinical trial was initiated in which the exposure of previously untreated patients to immunological danger signals was avoided during treat- ment with FVIII:37,38 the first treatment with FVIII was avoided if the patient was bleeding or had an infection; surgery was avoided during the first 20 days of exposure to FVIII; vaccinations were not given on the same day as FVIII treatment; and bleeds were treated as early as possi- ble to shorten the time of potential tissue damage. Such a drastic protocol led to an inhibitor incidence of 40% pro- voking the early termination of the EPIC study (ClinicalTrials.gov Identifier: NCT01376700).
Taken together, the large majority of the investigations
performed over the last 10 years failed to identify candi- date danger signals the control of which would unequivo- cally reduce the immunogenicity of therapeutic FVIII, reflecting the possibility that danger signals may not be as important as first thought for the initiation of the anti- FVIII immune response. Alternatively, the background level of activation of the innate/adaptive immune system may be sufficient to allow activation of naïve FVIII-specif- ic T cells without the need for overt danger signals, pro- vided that a sufficient amount of FVIII is internalized, processed by antigen-presenting cells and presented to T cells. In line with this, we recently observed that the phys- iological baseline activation of complement C3 (i.e., spon- taneous complement C3 tick-over)39,40 increases endocyto- sis of FVIII by immature dendritic cells without inducing their maturation. The mere in vivo elimination of C3 allowed a drastic reduction of the immunogenicity of ther- apeutic FVIII in FVIII-deficient mice.41
Self-recognition: the original sin of the immune system
The recognition of self, and in particular FVIII, is part of normal immune homeostasis.42 It is clear today that both B and T lymphocytes undergo positive and negative selec- tion processes, in the bone marrow and thymus, respec- tively, wherein highly autoreactive lymphocytes are elim- inated and poorly autoreactive ones are retained. In the case of T lymphocytes, thymic T cells with intermediate affinities for self-antigens may differentiate into natural regulatory T cells that suppress autoreactive T-cell responses.43 Regulatory T cells may also develop in the periphery upon encounter of antigens under non-inflam- matory conditions.44 Self-recognition may thus be consid- ered as the original sin of the adaptive immune system: the functionality of the emerging lymphocytes and of their antigen-specific receptors is in essence based on the recognition of self-proteins, which lays the ground for the selection of potential harmful autoreactivity. In this con- text, physiological immune homeostasis relies on recogni- tion of self, balanced by a tight control of the autoreactive lymphocytes. This is particularly true for the recognition of FVIII under physiological conditions (Figure 1).
Recognition of endogenous factor VIII by T cells under physiolog- ical conditions
The existence of FVIII-reactive T cells in healthy sub- jects was first suggested by the seminal work of the group led by B Conti-Fine. In vitro assays revealed proliferation of CD4+ T cells in the presence of the FVIII protein and over- lapping peptides spanning the different domains of FVIII in more than 50% of healthy donors.45,46 Recently, preex- isting FVIII-specific CD4+ T cells in healthy individuals were quantified accurately following cycles of stimulation of CD4+ T cells by FVIII-loaded mature autologous mono- cyte-derived dendritic cells.47 The frequency of preexisting FVIII-specific T cells was evaluated to be about 2 cells per million CD4+ T cells.47 This value is in the same range as that of CD4+ T cells specific for foreign proteins such as ovalbumin or keyhole limpet hemocyanin, i.e., 1.3 and 20 CD4+ T cells per million, respectively.48 In marked con- trast, it is up to one log higher than that of preexisting T cells specific for immunogenic therapeutic proteins such as adalimumab, rituximab, infliximab and erythropoietin (0.1 to 1 cell/million CD4+ T cells),48,49 and two logs greater than that of preexisting T cells specific for the non- immunogenic humanized therapeutic antibody
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haematologica | 2019; 104(2)