SummaryAntibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH–HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10−4 and 20% in those with MRD ⩾10−4 (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5–32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ⩾10−5 was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH–HTS method may facilitate routine MRD quantification in clinical trials.

Eliminating alloreactive cells from T-cell populations would enable the transfer of immune function to patients who receive stem cell transplants. However, highefficiency depletion has proved difficult to achieve. We sought to develop ex vivo approaches for the maximal depletion of alloreactive CD4 ؉ T cells. Using a flow cytometric cell sorting approach after mixed lymphocyte reaction (MLR) culture, we have found that sorted CFSE bright (5-(and-6)-carboxyfluorescein diacetate succinmidyl ester) (nondivided) and activation antigen-negative cells are markedly depleted of alloreactivity. With HLA-mismatched peripheral blood mononuclear cell (PBMC) stimulators we have consistently attained (90%-95%) depletion of alloreactivity. Importantly, when purified matured monocyte-derived dendritic cells (DCs) are used as stimulators, a 100-fold (99%) reduction in alloreactivity was attained, resulting in abrogation of the secondary MLR. Significantly, the CFSE bright CD25 ؊ cells recovered from these cultures retained general immunoreactivity, including responses to Candida and cytomegalovirus (CMV) antigens. In addition, a CFSE-based approach was tested and found to be sufficient for graft-versus-host disease (GVHD) prevention in vivo, in a major histocompatibility complex (MHC) class II disparate murine model. This efficient approach to selectively deplete mature alloantigen-specific T cells may permit enhanced immune reconstitution without GVHD. (Blood.

IntroductionIn spite of the significant advances in the treatment of hemophilia over the last 30 years, the development of antibodies that neutralize the procoagulant activity of factor VIII (factor VIII inhibitors) remains a serious complication of treatment with factor VIII products. Recent prospective studies of previously untreated patients with severe hemophilia have reported the incidence of inhibitor development to be approximately 20% to 25%.1-11 Factor VIII inhibitors may also develop in individuals without congenital factor VIII deficiency. Acquired autoimmune hemophilia occurs with an incidence of 0.2 to 1.0 per 1,000,000 per year in the general population.12,13 In both congenital and acquired hemophilia, anti-factor VIII antibody inhibitors are significant contributors to the morbidity and mortality of those affected. As such, these cases present difficult therapeutic challenges.Much work over the last decade has focused on defining the epitopes on factor VIII to which inhibitor antibodies bind. While this work has clarified some of the mechanisms by which inhibitors neutralize the procoagulant function of factor VIII, our knowledge of the factor VIII-specific CD4+ T helper cells, whose activation is crucial for the synthesis of factor VIII inhibitors, remains quite limited. This review will, first, discuss how a better understanding of the cellular mechanisms involved in the immune response to factor VIII may lead to improved methods of long-term inhibitor suppression and, perhaps, even prevention of inhibitor development. Lastly, this review will describe the work in our laboratory that has begun to characterize the CD4+ T cell response to factor VIII in patients with hemophilia and in normal subjects.