Page 194 - 2019_03-Haematologica-web

D.P. Hart et al.
ment burden for this group is surprisingly high, with 44% of a large London cohort being reported to have received some hemostatic treatment in a 2-year observation win- dow, 79% of whom received tFVIII concentrate.9 Consequently, inhibitor surveillance in non-severe hemo- philia A requires adult treaters to be ever vigilant.8 In con- trast to the systematic inhibitor screening in patients exposed early to tFVIII for severe hemophilia A,5 inhibitor screening in the setting of non-severe hemophilia A is cur- rently more reactive and sporadic,9 but recognized to be of increasing importance given the aging population of those living with non-severe hemophilia A.10 Inhibitor occur- rence in non-severe hemophilia A can be devastating, with neutralization of infused FVIII concentrate and potential cross-reactivity with endogenous FVIII. This cross-reactiv- ity occurs in at least 50% of identified cases11 and results in loss of a patient’s previous non-severe FVIII activity baseline level (FVIII:C) resulting in a worsening bleeding phenotype, often in later decades of life.8 This, in turn, results in increased bleed rates and an increased risk of premature mortality.12 In this context, the early detection of inhibitor occurrence – or, better still, the ability to reli- ably predict an individual’s risk of developing inhibitors before any have formed – has the potential to influence subsequent clinical decisions in ways that would substan- tially improve the patient’s outcomes.
The T-cell dependency of inhibitor generation is well described, with confirmed tFVIII-specific CD4+ T-cell responses13–15 and immunoglobulin class switching.16 The activation of CD4+ T cells depends on their interaction with “foreign” peptides – in this case, tFVIII-derived pep- tides spanning the location of the endogenous F8 missense mutation – presented by major histocompatibility com- plex (MHC) class II molecules. However, not all such for- eign peptides are perceived to be immunologically differ- ent from self and, if the difference is undetected, there is presumed negligible risk of an immune response. There are two key mechanisms at work here. Firstly, not all pep- tides are capable of binding to an individual’s repertoire of MHC molecules and are therefore never presented to T cells. Secondly, not all binding peptides are distinguishable from self-peptides bound to the same MHC molecules; in such cases, T cells that are capable of binding to these MHC:peptide complexes are expected to have been removed from the T-cell repertoire by self-tolerance mechanisms.17
What is unclear, however, is whether an understanding of MHC presentation and self-tolerance can enable us to make useful predictions about the inhibitor risk of patients with missense mutation hemophilia A – for example, by accu- rately predicting whether individual patients have a negligi- ble risk of developing inhibitors. The aim of this study was to address this point directly. Given that MHC molecules are encoded by genes that are among the most polymor- phic in the human genome, and there are several hundred disease-causing F8 missense mutations, the first aim of this study was to predict inhibitor risk based on an analysis of tFVIII peptide presentation by MHC molecules. Such an investigation poses a huge combinatorial challenge – one that is arguably impractical to address using purely in vitro techniques. Building on the approach developed in an earli- er study that we undertook using a much smaller dataset,18 we analyzed MHC:peptide complexes associated with 25 common human leukocyte antigen (HLA) class II alleles and 956 distinct F8 missense mutations, requiring over four
million peptide-HLA isoform combinations to be evaluated. However, this preliminary analysis did not take into account the possibility that fortuitous cross-matches between tFVIII-derived peptides and peptides at other locations – both within the FVIII protein sequence itself, and more generally to other proteins in the human pro- teome – may play a protective role by ensuring that T cells capable of triggering an immune response have been removed from the repertoire by self-tolerance mecha- nisms. This includes, but is far from limited to, the well- described homology between FVIII and factor V.19 Such human proteome cross-matching is the main focus of this study. Here we demonstrate that cross-matches between tFVIII and other parts of the proteome are commonplace and have a profound impact on the predicted inhibitor risk
for individuals living with non-severe hemophilia A.
Methods
Novel peptide-MHC surfaces
In previous work, we developed a methodology for predicting which patients with non-severe hemophilia A are at risk of devel- oping antibodies against tFVIII.18 Specifically, we predicted which F8 missense mutation/HLA isoform combinations would present novel peptide-MHC (pMHC) surfaces to CD4+ T cells, taking into account the reasonable assumption that T cells capable of binding to pMHC surfaces that are formed by endogenous FVIII peptides and presented by the same MHC molecules would have been removed from the T-cell repertoire by central tolerance mecha- nisms. Novelty arises when a tFVIII-derived peptide is an MHC- binder (most are not) and either (i) the equivalent endogenous FVIII-derived peptide is a non-binder (this may occur if the mis- sense mutation is at an MHC-facing, peptide-anchoring position, as residues at such positions anchor the peptide to the MHC mol- ecule) or (ii) the relevant amino-acid difference is at a T-cell recep- tor (TCR)-facing position (Figure 1A).
Our earlier work focused exclusively on the location of the hemophilia-causing F8 missense mutation and HLA-DR presenta- tion. Here we extend our analysis to include HLA-DP and -DQ presentation and, crucially, to take into account the possibility of fortuitous peptide cross-matches to other locations – both within FVIII itself, and more generally within other proteins in the human proteome (Figure 1B).
Peptide-MHC binding prediction
We used the in silico tool NetMHCII20 to predict the strength with which a given peptide binds to an MHC molecule – specifi- cally, the portable version of NetMHCII 2.2 (Technical University of Denmark, http://www.cbs.dtu.dk/services/NetMHCII/). If a peptide is predicted to bind with a 50% inhibitory concentration (IC50) of <1000 nmol/L – the conventional threshold used to indi- cate that peptide-MHC class II binding is of biological signifi- cance21 – it is a candidate for forming a novel pMHC surface. Deciding whether a given combination of tFVIII peptide and MHC molecule forms a novel pMHC surface in comparison to the corresponding endogenous FVIII-derived peptide requires the considerations outlined in Figure 1 to be made, given: (i) knowl- edge of the positions of the MHC anchoring pockets for the cho- sen HLA isoform (generally these are at positions 1, 4, 6 and 9); and (ii) the binding register of the peptide (predicted by NetMHCII), which specifies the stretch of nine consecutive amino-acid residues that form the preferred binding core within the MHC groove. Predictions were made for 25 common HLA- DR, -DP and -DQ isoforms with estimated worldwide population
600
haematologica | 2019; 104(3)