The IL-10 polarized cytokine pattern in innate and adaptive immunity cells contribute to the development of FVIII inhibitors
- Amanda CO Silveira†1Email author,
- Marcio AP Santana†2,
- Isabella G Ribeiro†1, 2,
- Daniel G Chaves†2 and
- Olindo A Martins-Filho†1
© Silveira et al.; licensee BioMed Central. 2015
Received: 24 June 2014
Accepted: 8 December 2014
Published: 16 January 2015
Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors.
Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized.
Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4+ and CD8+ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10.
The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.
KeywordsImmune regulation Intracellular cytokine staining Cytokine profile FVIII inhibitors Hemophilia A
Hemophilia A (HA) is an X-linked inherited bleeding disorder resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII) . HA is classified as mild, moderate, or severe based on the degree of FVIII residual activity . Treatment of patients with HA involves replacement therapy with plasma-derived FVIII (pdFVIII) or recombinant FVIII . A major clinical complication observed during replacement therapy is the development of antibodies against FVIII (called inhibitors) that block its procoagulant activity. Approximately 10%–15% patients with HA and 25%–30% patients with severe HA develop inhibitors . Patient’s age at the time of the first exposure to replacement therapy and type and frequency of FVIII exposure are risk factors for inhibitor formation; in addition, mutations in the gene encoding FVIII and variations in the immune system are important risk factors for inhibitor formation . Anti-FVIII antibodies are immunoglobulin G (IgG) antibodies, mainly IgG4; in some cases, IgG1 and IgG2 can also be detected [6,7].
Type 1 cells have been established to play a role in the initial immune response to FVIII, and type 2 cells act in the development of strong inhibitor production. CD4+ T cells are important for the production of inhibitors because they secrete both proinflammatory and anti-inflammatory/regulatory cytokines . Studies have described that polymorphisms in genes encoding cytokines such as TNF-α and IL-10 greatly affect inhibitor production [5,9-11]. Several studies have focused on immune response in patients with HA to elucidate the mechanisms underlying inhibitor production. We observed that the global cytokine profiles of innate and adaptive immune cells showed a major anti-inflammatory/regulatory pattern in patients with HA who were positive for inhibitors [HAα-FVIII(+)] and showed a mixed pattern, with a bias toward an inflammatory cytokine profile, in patients who were negative for inhibitors [HAα-FVIII(−)]. In addition, we proposed that these cytokine profile patterns may be the key elements in the production of distinct subclasses of anti-FVIII antibodies [12-14]. To understand immune response to FVIII, we characterized the cytokine patterns of peripheral blood leukocytes from whole blood samples of healthy blood donors, HAα-FVIII(+) patients, and HAα-FVIII(−) patients. In addition, we examined the differential synthesis of proinflammatory (IFN-γ and TNF-α) and anti-inflammatory/regulatory (IL-4, IL-5, and IL-10) cytokines in innate (neutrophils and monocytes) and adaptive (CD4+ and CD8+ T and B cells) immune cells.
Study population and sample collection
Characterization of patients with HA who were positive for inhibitors
Level of circulating FVIII
Historical peak of Bethesda titer (UB/mL)
Bethesda titer (UB/mL) at the time of blood collection
Immunophenotyping of cell subsets and intracellular cytokines
Peripheral blood cells were immunostained in the dark for 30 min at room temperature with TriColor-labelled [TC-phycoerythrin (PE)-cyanin 5 (Cy5)] monoclonal antibodies (mAbs) (Caltag, Burlingame, CA, USA), including anti-CD4 (clone S3.5), anti-CD8 (clone M-L233), anti-CD14 (clone TüK4), anti-CD16 (clone 3G8), and anti-CD19 (clone 4G7) mAbs. After lysis/fixation, the leukocytes were permeabilized by incubation with phosphate-buffered saline (PBS) permeabilization reagent (PBS supplemented with 0.5% [w/v] saponin [Sigma, St Louis, MO, USA]) for 10 min at room temperature. Fixed/Permeabilized cells were then incubated in the dark for 30 min at room temperature with 20 μL PE-labeled anti-cytokine mAbs (IFN-γ, clone B27; TNF-α, clone MAB11; IL-4, clone MP4-25D2; IL-5, clone TRFK5; and IL-10, JES3-9D7 [e-Bioscience, San Diego, CA, USA]) in PBS permeabilization reagent.
Flow cytometry acquisition and analysis
After immunophenotyping, leukocyte suspensions were run in a FACScalibur® flow cytometer (Becton Dickinson, San Jose, CA, USA) to collect 30,000 ungated events per sample. The acquired data were analyzed using CellQuest software (Franklin Lakes, NJ, USA). Distinct gating strategies were used to analyze cytokine-expressing leukocyte subpopulations from innate and adaptive immune cells. Neutrophils were selected as SSCHighCD16High+ cells and monocytes were selected as CD14High+ cells on FL3/anti-CD16-TC and FL3/anti-CD14-TC versus laser side-scatter (SSC) dot plots, respectively. Lymphocyte populations were first selected on laser forward-scatter (FSC) versus SSC dot plots. The number of gated neutrophils, monocytes, and lymphocytes ranged from 19,500 to 22,500, 1,200 to 2,100, and 6,000 to 9,000, respectively. After the initial gate selection, the frequencies of cytokine-positive cells were quantified using quadrant statistics applied on FL3/anti-cell surface marker-TC versus FL2/anti-cytokine-PE dot plots. PE-Cy5-labeled antibodies were detected on FL3 channel, and PE-labeled antibodies were detected on FL2 channel. Distinct tubes were used to evaluate the percentage of cytokine-positive T cells (CD4+ and CD8+) and B cells (CD19+). Data were expressed as the percentage of cytokine-positive cells among gated neutrophils, monocytes, and total lymphocytes. The results were assembled further to calculate the global cytokine profiles of immune cells, as proposed earlier . Briefly, the median percentage for each cytokine-positive cell population was calculated using the values obtained for each study group [BD, HAα-FVIII(−), and HAα-FVIII(+) groups].
Statistical analyses were performed using GraphPad Prism 5 software (San Diego, CA, USA). Because all data files assumed a non-Gaussian distribution, statistical comparisons were performed using non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test to evaluate cytokine profiles of innate and adaptive immune cells from BDs, HAα-FVIII(−) patients, and HAα-FVIII(+) patients. Differences were considered significant when p-values were <0.05.
Monocytes and neutrophils from HAα-FVIII(+) patients have an anti-inflammatory/regulatory cytokine profile
High synthesis of IL-10 is the hallmark of CD4+ and CD8+ T cells in HAα-FVIII(+) patients
B cells from patients with HA and BDs have similar cytokine profiles
The treatment regimen of FVIII varies among different countries and among centers in the same country. For patients included in this study, on-demand treatment was used to manage clinically evident bleeding. FVIII replacement therapy depends on age, severity, and treatment regimen. Recent studies have shown that median annual on-demand FVIII utilization (IU kg−1 · year−1) varies between 1,100 and 1,429 IU kg−1 · year−1 [13,14]. Our data showed that patients receiving on-demand treatment at Fundação Hemominas used 1,452 IU kg−1 · year−1 of FVIII. Recent reports have shown that replacement therapy with these amounts of FVIII products can cause several changes in the immune profile of patients with HA [15-19].
Production of anti-FVIII inhibitors remains a challenge in the treatment of patients with HA. Therefore, understanding the cellular compartmentalization of immune responses is important. Little is known about the cytokine profiles and cell types implicated in auto- and alloimmune responses to FVIII. Previous studies have shown that patients with HA who are positive for inhibitors have a major anti-inflammatory/regulatory immune cytokine profile while those without inhibitors have a mixed pattern, with a bias toward an inflammatory cytokine profile. These findings support and suggest that proinflammatory-modulated immune response may favor the synthesis of anti-FVIII IgG1 antibodies and prevent the synthesis of anti-FVIII IgG4 inhibitors [12,20,21]. It has been suggested that immunological context along with the intensity of treatment favors the class switching of FVIII-specific antibodies to IgG4 .
The findings of the present study highlight the significantly lower frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, and IL-4-positive neutrophils and higher frequencies of IL-10-positive neutrophils in HAα-FVIII(+) patients. Recent studies have shown that neutrophils not only synthesize cytokines in response to various inflammatory stimuli in chronic inflammatory disorders (such as rheumatoid arthritis, inflammatory bowel diseases, diabetes, and mycobacterial infections) but also regulate immune responses such as those suggested in HA. Some studies have also proposed that the cytokine profile of neutrophils is similar to the cytokine profiles of monocytes and macrophages. Therefore, modulation of cytokines derived from monocytes and neutrophils is a potentially useful strategy for therapeutic immunointervention [23-25].
Stimulation of B cells to produce FVIII inhibitors is dependent on CD4+ T . However, recent reports describe the contribution of CD8+ T cells in this context. A recent study has shown an association between CD8+ T cells and induction of immune tolerance. It was found that human CD8+ T cells also functioned as regulatory T cells to induce immune tolerance by suppressing activated T cells through IL-10 production [27-29]. In addition, CD8+ cytotoxic cells mediate the apoptosis of T cells that contribute to peripheral tolerance . Proliferation of CD8+ suppressor cells and activated CD8+ T cells is observed in patients with HA; moreover, proliferation of suppressor cells seems to be mainly related to FVIII infusion . These regulatory T cells are important for regulating other T cells without the intervention of antigen-presenting cells (APCs). In addition, it is well known that repeated infusions of pdFVIII concentrates skews TCR repertoires for CD8+ T cells [15,19].
Analysis of T cells from HAα-FVIII(−) patients showed elevated levels of almost all cytokines. In contrast, T cells from HAα-FVIII(+) patients showed decreased levels of all the tested cytokines, except IL-10. The immune response against FVIII develops as a classical T cell-dependent antibody-mediated immune response. Infused FVIII is recognized, internalized, and processed by APCs and is presented to antigen-specific CD4+ T cells that provide activation signals to antigen-specific B cells, thus resulting in the synthesis of anti-FVIII antibodies and long-lived FVIII-specific memory B cells [8,32,33]. The data obtained in this study showed that the cytokine microenvironment promoted by T cells is determinant for the presence or absence of FVIII inhibitors. These data are consistent with those of different studies that observed a mixed cytokine pattern, with a bias toward an inflammatory cytokine profile, in HAα-FVIII(−) patients and anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients [7,8,12,34]. The changes observed in CD8+ T cell compartment also seem to be related to the production of inhibitors and the regulatory profiles of these cells. In HAα-FVIII(−) patients, increased cytokine production by lymphocytes contributed to the prevention of inhibitor production by modulation of the immune system; in contrast, in the inhibitor-positive patients, reducing the production of cytokines inducing immune microenvironment modifying the expansion of lymphocytes with a regular profile, increasing the production of IL-10 in order to re-modulate the immune system; However, other changes, which need investigation, prevent this process, thus resulting in the production of inhibitors.
Analysis of B cells showed that HAα-FVIII(−) patients had higher levels of IL-4-positive B cells and HAα-FVIII(+) patients had higher levels of IL-10-positive B cells. Because of their central role in inhibitor production, it is important to elucidate the cytokine profile of B cells in patients with HA. The IL-10-predominant cytokine profile of B cells observed in this study is well characterized as a downregulator of TNF-α, IL-1α, and IL-1β production. However, it stimulates B cell proliferation and differentiation and antibody production . Importantly, B cells are not merely antibody producers but actively contribute by regulating immune response and secreting cytokines that amplify humoral and cellular immune responses [36-39].
Typically, various cytokines and chemokines act as mediators in an immune response. It is important to note that genes encoding IL-10 and TNF-α were the first genes located outside the gene encoding FVIII to be associated with inhibitor production in patients with HA [9-11,40]. However, different risk factors such as ethnicity, type of mutation in the gene encoding FVIII, family history of inhibitor production, type of FVIII (plasmatic or recombinant) therapy, patient age at the time of first exposure to replacement therapy, initial doses of FVIII concentrate, mode of infusion, surgery, intensity of treatment or regular prophylaxis, and inflammatory state or HLA haplotype of patients are associated with inhibitor production and should be investigated further. Other causes of inhibitor production against FVIII include stress, age, malignancy, coinfections, pregnancy, and antibiotics [41-43]. Based on these findings, we hypothesize that anti-inflammatory/regulatory cytokine-dominant microenvironment determined by monocytes, neutrophils, T cells, and B cells may favor the synthesis of anti-FVIII IgG4 with inhibitory activity. In addition, the proinflammatory microenvironment corroborates with the synthesis of anti-FVIII IgG1 non-inhibitory antibody [12,20,21].
The limitations of the study include limited number of patients and absence of information on mutations in the gene encoding FVIII. An important issue to be considered in future investigations is to analyze the kinetic conversion of a proinflammatory immune response to an anti-inflammatory/regulatory immune response against FVIII in HAα-FVIII(+) patients and the risk factors that may be associated with this conversion during HA treatment. The anti-inflammatory/regulatory cytokine profile of innate immune cells in patients with HA could be an important biomarker for the development of high inhibitor titers .
We thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) financial support. O.A.M.F. is thankful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research fellowship (PQ) and D.G.C. acknowledges the FAPEMIG for the BIP fellowship programme.
- Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991;30 Suppl 43:10363–70.PubMedView Article
- Fischer K, Van Den Berg M. Prophylaxis for severe haemophilia: clinical and economical issues. Haemophilia. 2003;9 Suppl 4:376–81.PubMedView Article
- Spiegel PCJ, Stoddard BL. Optimization of factor VIII replacement therapy: Can structural studies help in evading antibody inhibitors? Br J Haematol. 2002;119 Suppl 2:310–22.PubMedView Article
- Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia. 2003;9 Suppl 4:418–35.PubMedView Article
- Agostini D, Rosset C, Botton MR, Kappel DB, Vieira IA, Gorziza RP, et al. Immune system polymorphisms and factor VIII inhibitor formation in Brazilian haemophilia A severe patients. Haemophilia. 2012;18 Suppl 6:416–8.View Article
- Lollar P. Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX. J Thromb Haemost. 2004;2 Suppl 7:1082–95.PubMedView Article
- Reding MT, Lei S, Lei H, Green D, Gill J, Conti-Fine BM. Distribution of Th1- and Th2-induced anti-factor VIII IgG subclasses in congenital and acquired haemophilia patients. Thromb Haemost. 2002;88 Suppl 4:568–75.PubMed
- Hu G, Guo D, Key NS, Conti-Fine BM. Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients. Thrombd Haemost. 2007;97 Suppl 5:788–94.
- Astermark J, Oldenburg J, Carlson J, Pavlova A, Kavakli K, Berntorp E, et al. Polymorphisms in the TNFA gene and the risk of inhibitor development in patients with hemophilia A. Blood. 2006;108 Suppl 12:3739–45.PubMedView Article
- Astermark J, Oldenburg J, Pavlova A, Berntorp E, Lefvert AK, Mibs Study Group. Polymorphisms in the IL10 but not in the IL1beta and IL4 genes are associated with inhibitor development in patients with hemophilia A. Blood. 2006;107 Suppl 8:3167–72.PubMedView Article
- Chaves DG, Belisario AR, Castro G, Santoro MM, Velloso-Rodrigues C. Analysis of cytokine genes polymorphism as markers for inhibitor development in haemophilia A. Int J Immunogenet. 2010;37 Suppl 2:79–82.PubMedView Article
- Chaves DG, Velloso-Rodrigues C, Oliveira CA, Santoro MM, Martins-Filho AO. A shift towards a T cell cytokine deficiency along with an anti-inflammatory/regulatory microenvironment may enable the synthesis of anti-FVIII inhibitors in haemophilia A patients. Clin Exp Immunol. 2010;162 Suppl 3:425–37.PubMed CentralPubMedView Article
- Nerich V, Tissot E, Faradji A, Demesmay K, Bertrand MA, Lorenzini JL, et al. Cost-of-illness study of severe haemophilia A and B in five French haemophilia treatment centres. Pharm World Sci. 2008;30:287–92.PubMedView Article
- Epstein J, Xiong Y, Woo P, Li-Mcleod J, Spotts G. Retrospective analysis of differences in annual factor VIII utilization among haemophilia A patients. Haemophilia. 2012;18:187–92.PubMedView Article
- Matsutani T, Sakurai Y, Yoshioka T, Tsuruta Y, Suzuki R, Shima M, et al. Replacement therapy with plasma-derived factor VIII concentrates induces skew in T-cell receptor usage and clonal expansion of CD8+ T-cell in HIV-seronegative hemophilia patients. Thromb Haemost. 2003;90:279–92.PubMed
- Gouw SC, Van Den Berg HM, Le Cessie S, Van Der Bom JG. Treatment characteristics and the risk of inhibitor development: a multicenter cohort study among previously untreated patients with severe hemophilia A. J Thromb Haemost. 2007;5:1383–90.PubMedView Article
- Gouw SC, Van Der Bom J, Van Den Berg HM. Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study. Blood. 2007;109 Suppl 11:4648–54.PubMedView Article
- Van Helden PMW, Van Haren SD, Fijnvandraat K, Van Den Berg HM, Voorberg J. Factor VIII-specific B cell responses in haemophilia A patients with inhibitors. Haemophilia. 2010;16:35–43.PubMedView Article
- Sakurai Y, Matsutani T, Yoshioka T, Takeda T, Yoshioka A, Shima M. Alterations of T cell receptor Vβ repertoire of CD8 T lymphocytes in immune tolerance induction in two hemophilia A patients with inhibitors. Vojnosanit Pregl. 2011;68 Suppl 12:1047–50.PubMedView Article
- Oliveira CA, Velloso-Rodrigues C, Machado FC, Carvalho BN, Gentz SH, Martins-Filho OA, et al. Cytokine profile and FVIII inhibitors development in haemophilia A. Haemophilia. 2013;19 Suppl 3:139–42.View Article
- Pimentel JP, Chaves DG, Araújo AR, de Araújo EM, da Silva F, Neves WL, et al. Anti-inflammatory/regulatory cytokine microenvironment mediated by IL-4 and IL-10 coordinates the immune response in hemophilia A patients infected chronically with hepatitis C virus. J Med Virol. 2013;85 Suppl 6:1009–18.PubMedView Article
- Van Helden PMW, Van Den Berg HM, Gouw SC, Kaijen PH, Zuurveld MG, Mauser-Bunschoten EP, et al. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A. Br J Haematol. 2008;142 Suppl 4:644–52.PubMedView Article
- Kasama T, Miwa Y, Isozaki T, Odai T, Adachi M, Kunkel SL. Neutrophil-derived cytokines: potential therapeutic targets in inflammation. Curr Drug Targets Inflamm Allergy. 2005;4 Suppl 3:273–9.PubMedView Article
- Hatanaka E, Monteagudo PT, Marrocos MS, Campa A. Neutrophils and monocytes as potentially important sources of proinflammatory cytokines in diabetes. Clin Exp Immunol. 2006;146 Suppl 3:443–7.PubMed CentralPubMedView Article
- Wright HL, Moots RJ, Bucknall RC, Edwards RC. Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford). 2010;49 Suppl 9:1618–31.View Article
- Wroblewska A, Reipert BM, Pratt KP, Voorberg J. Dangerous liaisons: how the immune system deals with factor VIII. J Thromb Haemost. 2013;11:47–55.PubMedView Article
- Le Gal FA, Riteau B, Sedlik C, Khalil-Daher I, Menier C, Dausset J, et al. HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes. Int Immunol. 1999;11:1351–6.PubMedView Article
- Endharti AT, Rifa’I M, Shi Z, Fukuoka Y, Nakahara Y, Kawamoto Y, et al. Cutting Edge: CD8CD122 Regulatory T Cells Produce IL-10 to Suppress IFN Production and Proliferation of CD8 T Cells. J Immunol. 2005;175:7093–7.PubMedView Article
- Shi Z, Okuno Y, Rifa’I M, Endharti AT, Akane K, Isobe K, et al. Human CD8CXCR3 T cells have the same function as murine CD8CD122 Treg. Eur J Immunol. 2009;39:2106–19.PubMedView Article
- Herndon JM, Stuart PM, Ferguson TA. Peripheral Deletion of Antigen-Specific T Cells Leads to Long-Term Tolerance Mediated by CD8 Cytotoxic Cells. J Immunol. 2005;174:4098–104.PubMedView Article
- Dianzani U, Pileri A, Bianchi A, Camponi A, Tamponi G, Massaia M. Biochemical and immunologic abnormalities in peripheral blood T lymphocytes of patients with hemophilia A. Eur J Haematol. 1988;41 Suppl 4:334–40.PubMed
- André S, Meslier Y, Dimitrov DJ, Repessé Y, Kaveri SV, Lacroix-Desmazes S, et al. A cellular viewpoint of anti-FVIII immune response in hemophilia A. Clin Rev Allergy Immunol. 2009;37 Suppl 2:105–13.PubMedView Article
- Pautard B, D’Oiron R, Te Li Thiao V, Lavend’Homme R, Saint-Remy JM, Peerlinck K, et al. Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII-specific T cells. J Thromb Haemost. 2011;9 Suppl 6:1163–70.PubMedView Article
- Qadura M, Othman M, Waters B, Chegeni R, Walker K, Labelle A, et al. Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells. J Thromb Haemost. 2008;6 Suppl 12:2095–104.PubMedView Article
- Rousset F, Garcia E, Defrance T, Péronne C, Vezzio N, Hsu DH, et al. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci U S A. 1992;89 Suppl 5:1890–3.PubMed CentralPubMedView Article
- Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, et al. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol. 2000;1 Suppl 6:475–82.PubMedView Article
- Duddy ME, Alter A, Bar-Or A. Distinct profiles of human B cell effector cytokines: A role in immune regulation? J Immunol. 2004;172 Suppl 6:3422–7.PubMedView Article
- Lund FE. Cytokine-producing B lymphocytes - key regulators of immunity. Curr Opin Immunol. 2008;20 Suppl 3:332–8.PubMed CentralPubMedView Article
- Wojciechowski W, Harris DP, Sprague F, Mousseau B, Makris M, Kusser K, et al. Cytokine-producing effector B cells regulate type 2 immunity to H. polygyrus. Immunity. 2009;30 Suppl 3:421–33.PubMed CentralPubMedView Article
- Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factors VIII and IX. Haemophilia. 2006;12 Suppl 6:15–22.PubMedView Article
- Lacroix-Desmazes S, Navarrete AM, André S, Bayry J, Kaveri SV, Dasgupta S. Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A. Blood. 2008;112 Suppl 2:240–9.PubMedView Article
- Ghosh K, Shety S. Immune response to FVIII in hemophilia A: an overview of risk factors. Clin Rev Allergy Immunol. 2009;37 Suppl 2:58–66.PubMedView Article
- Pinto P, Ghosh K, Shetty S. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients. Haemophilia. 2012;18 Suppl 5:794–7.PubMedView Article
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