transforming-growth-factor-beta and Hemophilia-A

Evidence suggests that haemophiliacs treated with factor VIII concentrates show abnormalities in immune functions. The basis of this is not clear, but some factor VIII concentrates down-regulate Fc receptors on monocytes which may explain the impaired function of these cells. Some concentrates inhibit lymphocyte proliferation and interleukin-2 secretion by human T-cell lines and peripheral blood lymphocytes. They can also inhibit activity of other cytokines such as interleukin-4 and interleukin-5 and secretion of cytokines such as interleukin-1 and granulocyte macrophage colony stimulating factor. These effects are product-related and vary from total inhibition to virtually no detectable inhibition. Of particular significance is that the degree of inhibition is not related to the purity or gross protein composition of the products. The inhibitory activity is not due to factor VIII itself as antibody affinity purified factor VIII products are entirely non-inhibitory. The main inhibitory protein components appear to be of approximately 200 kDa and 60 kDa (by gel filtration). Recent evidence suggests that transforming growth factor-beta (TGF-beta), derived from the plasma used for fractionation, is a major contaminant of 'inhibitory' concentrates and is responsible for the effects, observed in vitro, of concentrates on cytokine secretion or activity. The levels of TGF-beta varied between products and correlated with inhibition of interleukin-2 secretion from stimulated T-cells. The presence of TGF-beta in concentrates may therefore explain the immunosuppression observed in recipients of these products. Correlation of the inhibitory effects with clinically important consequences such as increased susceptibility to infections or decreased CD4 counts also remains to be established.

Topics: Factor VIII; Hemophilia A; Humans; Immunosuppressive Agents; In Vitro Techniques; Interleukin-2; Laboratories; T-Lymphocytes; Transforming Growth Factor beta

The clinical use of therapeutic proteins can be complicated by the development of anti-product antibodies. We have previously observed that O-phospho-l-serine (OPLS) reduced antibody response to FVIII in Hemophilia-A (HA) mice. However, the mechanism underlying this observation is not clear. We hypothesize that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells (DCs). We tested this hypothesis using in vivo, in vitro, and ex vivo methods. Naive HA mice that were pre-exposed to FVIII in the presence of OPLS showed substantially lower antibody response following rechallenge with OPLS free FVIII as compared with dexamethasone-pretreated mice. Exposure of OPLS to bone-marrow-derived dendritic cells (BMDCs) in culturing conditions resulted in an increase in the regulatory cytokine TGF-β and a decrease in proinflammatory cytokines TNF-α and IL12p70. This was accompanied by a significant reduction in upregulation of costimulatory marker CD40, as measured by flow cytometry. Furthermore, ex vivo matured BMDCs in the presence of FVIII and OPLS failed to elicit a robust immune response in HA mice compared with FVIII-treated BMDCs. Our data suggest that OPLS modulates the immune response by altering the function and maturation of DCs, resulting in the induction of tolerogenic properties. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3457-3463, 2014.

Topics: Adjuvants, Immunologic; Animals; Antibodies; CD40 Antigens; Cells, Cultured; Coagulants; Dendritic Cells; Disease Models, Animal; Factor VIII; Hemophilia A; Immune Tolerance; Interleukin-12; Mice, Transgenic; Phosphoserine; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Factor VIII; Genetic Therapy; Hemophilia A; Interleukin-2; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Plasmids; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transgenes

The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A.. We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells.. The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII-iDCs) in the presence or absence of the NFkappaB pathway blocking compound Andrographolide (Andro-cFVIII-iDCs). Three weekly intravenous infusions of one million cFVIII pulsed-iDCs were administered to a group of five hemophilic Balb/c mice. Anti-FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII.. We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co-cultured T cells. Following repeated infusion of the cFVIII-iDCs and Andro-cFVIII-iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long-term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T-cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII-iDC infusion and the elaboration of the immunosuppressive cytokines IL-10 and TGF-beta following andrographolide-treated cFVIII-iDCs.. This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.

Topics: Animals; Antigen Presentation; Dendritic Cells; Dogs; Factor VIII; Hemophilia A; Immune System Phenomena; Immune Tolerance; Interleukin-10; Mice; Mice, Inbred BALB C; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Treatment Outcome

Immunological abnormalities have been reported in haemophiliacs. Although infections with HIV, hepatitis and other viruses may contribute to these abnormalities, immune defects are detectable also in HIV seronegative haemophiliacs. It is likely that chronic exposure to extraneous proteins in clotting factor concentrates (CFCs) may play a role in immunomodulation, but the underlying mechanisms remain unclear. The results of the present paper show that: a) soluble HLA class I (sHLA-I), soluble Fas-ligand (sFas-L) and transforming growth factor beta 1 (TGF-beta1) are detectable in plasma derived but not in recombinant CFCs; b) the level of sHLA-I and sFas-L is proportional to the grade of CFCs purity whereas TGF-beta1 showed very variable levels; c) soluble molecules detected in CFCs exert immunomodulatory effects in vitro like apoptosis induction in Jurkat cells and inhibition of mixed lymphocyte reaction response, antigen-specific lymphocyte cytotoxic activity and neutrophil chemotaxis.

Topics: Apoptosis; Blood Coagulation Factors; Chemotaxis, Leukocyte; Cytotoxicity Tests, Immunologic; Drug Contamination; Fas Ligand Protein; Hemophilia A; HLA Antigens; Humans; Immunosuppression Therapy; Jurkat Cells; Lymphocyte Culture Test, Mixed; Lymphocytes; Membrane Glycoproteins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Recombinant Proteins; Respiratory Burst; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma-derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor-beta (TGF-beta) as an immunomodulatory component present in plasma-derived FVIII concentrate. However, the addition of neutralizing antibody to TGF-beta did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen-stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) -stimulated CD4 and CD8 T-cell subsets as determined by Annexin V binding and DNA fragmentation. T-cell subsets showed a pdFVIII dose-dependent inhibition of entry into S-phase and G(1) arrest. Addition of neutralizing anti-TGF-beta reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T-cell subsets as determined by Annexin V staining. TGF-beta has been reported to be a potent inhibitor of T-cell proliferation, arresting the cell cycle in G(1) phase and causing apoptosis. Together, these findings suggest that TGF-beta is a significant immunomodulatory component of pdFVIII concentrates.

Topics: Adult; Apoptosis; Cell Culture Techniques; Cell Cycle; Cell Division; DNA Fragmentation; Factor VIII; Female; Hemophilia A; Humans; Immune Tolerance; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; T-Lymphocyte Subsets; Transforming Growth Factor beta

In Japan, the proportion of hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) is 40%, whereas more than 90% are infected with hepatitis C virus (HCV). To evaluate the immunological status of hemophiliacs infected with HIV-1, we investigated the pattern of cytokine production in peripheral blood mononuclear cells (PBMCs) of HIV-1-seropositive and -seronegative hemophiliacs, HIV-1-seropositive non-hemophiliacs, and healthy individuals. The production of IL-18 and IL-1beta from PBMCs stimulated with Staphylococcus aureus Cowan strain 1 (SAC) in the HIV-1-seropositive hemophiliacs was significantly decreased in comparison with the other groups. On the other hand, IL-12 production in both HIV-1-seropositive groups was significantly lower than in HIV-1-seronegative groups. TNF-alpha and IL-6 production was similar among the four groups. In contrast, plasma levels of TGF-beta1 were increased in HIV-1-seropositive hemophiliacs, HIV-1-seropositive nonhemophiliacs, and HIV-1-seronegative hemophiliacs, with the highest levels being in HIV-1-seropositive hemophiliacs, suggesting that coinfection with HIV-1 and HCV increases the level of plasma TGF-beta in HIV-1-seropositive hemophiliacs. Treatment of PBMCs from healthy individuals with TGF-beta1 inhibited IL-18 and IL-1beta production without affecting IL-6, IL-10, or TNF-alpha production. Suppression of the expression of caspase 1 mRNA, which is known to be an IL-1beta-converting enzyme and which also cleaves the precursor of IL-18, was observed in the SAC-stimulated PBMCs from healthy individuals after treatment with TGF-beta1 and in the SAC-stimulated PBMCs from HIV-1-seropositive hemophiliacs, suggesting that the decreased production of IL-18 and IL-1beta in HIV-1-seropositive hemophiliacs may be related to the downregulation of caspase 1 mRNA induced by high levels of TGF-beta1 in plasma.

Topics: Adolescent; Adult; Caspase 1; Cytokines; Hemophilia A; HIV Infections; HIV-1; Humans; Interleukin-1; Interleukin-18; Leukocytes, Mononuclear; Lymphocyte Activation; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcus aureus; Transforming Growth Factor beta

Topics: Cytokines; Depression, Chemical; Drug Contamination; Factor VIII; Hemophilia A; Humans; Transforming Growth Factor beta

In previous studies, we have shown that some, but not all low-, intermediate-, and high-purity factor VIII concentrates inhibit interleukin-2 (IL-2) secretion from phytohemagglutinin (PHA)-stimulated T lymphocytes. We now present evidence that this inhibitory action of concentrates is, at least in part, due to contamination with transforming growth factor-beta (TGF-beta). Originally identified in platelets, TGF-beta is a 25-kD homodimer that has been shown to be a natural and potent inhibitor of many immunologic responses. Using a specific bioassay, we have measured TGF-beta in various factor VIII concentrates. While some concentrates contained substantial amounts of the cytokine, there was a wide variation in concentrations of TGF-beta in different products. These levels correlated with the degree of inhibition of IL-2 secretion from T cells exhibited by each product (P = .0001). Noninhibitory concentrates contained no detectable TGF-beta. Addition of a specific TGF-beta 1 antibody reversed the inhibitory effect of some concentrates on IL-2 secretion by PHA-stimulated Jurkat T cells and interleukin-5 (IL-5)-induced proliferation of an erythroleukemic cell line. These findings suggest that TGF-beta contamination is a major contributory factor to the inhibitory activity of some factor VIII concentrates on cytokine secretion or activity, and may partially explain the reported immunosuppressive effects in recipients of these blood products.

Topics: Antibodies; Cell Line; Drug Contamination; Factor VIII; Hemophilia A; Humans; Immunologic Deficiency Syndromes; Interleukin-2; Interleukin-5; Phytohemagglutinins; T-Lymphocytes; Transforming Growth Factor beta