British [ournal of Haematology. 1992, 82. 94-98
Enhancement of human T cell responses to allogeneic stimuli by factor VIII concentrates ALISONBATCHELOR,C. MICHAEL STEELA N D CHRISTOPHERA. LUDLAM* Medical Research Council, Human Genetics Unit, Edinburgh, Lothian. and *Department of Haematology, Royal Infirmary of Edinburgh, Edinburgh, Lothian Received 10 Decernber 199 1: accepted for publication 14 April 1992
Summary. The effect of factor VIII concentrate, from commercial and National Health Services manufacturers. on in vitro lymphocyte proliferative response to allogeneic stimulator cells was investigated. Factor VIII preparations 'purified' by ion exchange or by monoclonal antibody affinity had no effect in this assay but lymphocyte proliferation in response to allogeneic cells was markedly and consistently enhanced by some intermediate purity factor VIII concentrates and. to a lesser extent. by a
factor IX preparation. These preparations did not stimulate lymphocyte proliferation in the absence of other mitogens. The co-mitogenic factor(s) present in intermediate purity factor VIII was not identified. However, enhanced proliferation was not due to factor VIII itself, nor to albumin or fibronectin. The clinical relevance of the immunomodulatory activity of intermediate purity factor VIII concentrates i n vitro is discussed.
It has been clear for a number of years that patients receiving factor VIII concentrate for the treatment of haemophilia have subclinical abnormalities of the immune system. These include abnormalities of T cell subsets, irrespective of HIV infection (Berntorp et al. 1989: Moffat et nl, 1985: Carr et al. 1984) and impaired cell mediated immunity. as assessed by the intradermal injection of recall antigens or of dinitrochlorobenzene (DNCR) (Mclvor et al. 1987: Madhok et al. 1986: Cuthbert et al. 1992). It has been suggested that intensive treatment with intermediate purity factor VIII preparations may be responsible for immune modulation in haemophiliacs (Ludlam et aI. 1985; Madhok et al. 1986). Factor VIII concentrates have been shown to impair lymphocyte proliferation in vitro in response to mitogen, recall antigen and monoclonal antibody stimulation (Froebel t't al. 1983; McDonald et al, 1985: Wang et al. 1985). The secretion of interleukin 2 (IL2) and the expression of cell surface activation markers following stimulation in vitro are also reduced in the presence of factor VIII concentrates (Lederman et al. 1986: Thorpe rt a], 1989: Hay rt a]. 1990). However, the relationship between immune abnormalities observed in vivo and the effects of factor VIII concentrate on immune function iri vitro i s controversial. In the present study we have investigated the effects of factor VIII concentrate on lymphocyte proliferation following stimulation with irradiated allogeneic cells. We have com-
pared a number of commercial and National Health Service factor VIII concentrates ranging from intermediate to high purity formulations.
Correspondence: Dr Alison Batchelor. Medical Research Council. Human Genetics Unit. Edinburgh EH.1 2XU. Lothian. Scotland.
MATERIALS AND METHODS Cells. Venous blood was collected into lithium heparin from healthy human volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll/ Hypaque (SG 1.077) (Pharmacia, Uppsala, Sweden) at 900g for 1 5 min. The interface band of mononuclear cells was removed and washed four times in phosphate buffered saline (PBS).An Epstein Barr virus transformed human B lymphoid cell line. RAB12, (HLA type A2A3. B27BW22, DR3,4 W 52W53) was used as the allogeneic stimulator in one-way mixed lymphocyte reactions. RAB12cells were maintained on RPMI 1 6 4 0 (Gibco, Grand Island. N.Y.) supplemented with 0.03% fresh glutamine and 5% heat inactivated fetal calf serum (FCS) in 5% COz at 37°C. The cell line was screened regularly and found consistently negative for mycoplasma contamination. The phenotype of responding PBMC was checked by two-colour immunofluorescence (CD3IFITC + CD19/Phycoerythrin, Becton Dickinson), scoring the proportions of positive cells by flow cytopherometry (FACScan, Becton Dickinson). Prolijieration assays. PBMC were stimulated with irradiated allogeneic lymphoblastoid cells and cultured in RPMI I640 medium containing 0.03% fresh glutamine, 5% heat inactivated FCS and 1 u/ml heparin (Evans Biologicals Ltd.
Factor VIII and T Cell Proliferation Liverpool,U.K.). RAB12cells were exposed to an X-ray dose of 1000 rads prior to inclusion in proliferation assays. 150 pl aliquots containing 5 x lo4 PBMC and 2 x lo4 irradiated RABlz cells were cultured together in 96 V-well plates. Replicate cultures were set up by dispensing 150 p1 aliquots from a 4 ml volume of culture medium containing PBMC, irradiated RAB12 cells and various concentrations of factor VIII, factor IX, fibronectin or control buffers. The factor VIII preparations investigated were SNBTS 28, SNBTS S8 (Scottish National Blood Transfusion Service, Protein Fractionation Centre, Edinburgh, U.K.), high potency Bio-transfusion (Bio-transfusion.Lille, France), Monoclate-P (Armour Pharmaceutical Company, Kankakee, Ill.) and Hemofil M (Baxter Healthcare Corporation, Glendale, Calif.). SNBTS S8 factor VIII is a prototype higher purity factor VIII concentrate which has a lower fibrinogen, but higher fibronectin content compared to SNBTS 28 factor VIII concentrate. The factor IX preparation was SNBTS concentrate (DEFIX).All factor VIII and factor IX preparations were used at a final concentration of 0.1-2 iu/ml. They had been stored and reconstituted according to manufacturers’ instructions. Control buffer included was identical to the buffer solution in which SNBTS 28 factor VIII concentrate is administered. Buffer was present at a final dilution range of 1/ 100 to 1/10. Purified human fibronectin (Chemicon, tissue culture grade) was included at a concentration of 21-425 pg/ml. 18 h prior to cell harvesting, cultures were labelled with 3 7 kBq 3H-thymidine(Amersham, Amersham, Bucks., U.K.) in a 50 pl aliquot of culture medium. Replica cultures were harvested at 24 h intervals for up to 12 d. Cell cultures were harvested onto glass fibre papers, dried and 3H-thymidine incorporation measured using a Matrix 96 direct beta counter (Packard, Ulgersmaweg, Groningen, The Netherlands). Mean values for 3H-thymidine incorporation in counts per minute were calculated from duplicate wells. RESULTS Lymphocyte proliferation in response to irradiated allogeneic lymphoblastoidcells was markedly enhanced in the presence of intermediate purity SNBTS 28 factor VIII concentrate (Fig 1). All seven batches of SNBTS 28 tested behaved in this manner. At the end of the culture 93% of the cells were CD3 positive and 7% expressed CD19; thus the vast majority ofthe proliferating cells were T cells. SNBTS S8 factor VIII concentrate enhanced lymphocyte proliferation in this assay but apparently to a lesser extent (Fig 2). Peripheral blood mononuclear cells did not respond to SNBTS blood products in the absence of irradiated stimulator cells. Enhanced proliferation was not observed when SNBTS control buffer was substituted for concentrate in the lymphocyte proliferation assay (Fig 3). High purity factor VIII concentrate (Bio-transfusion) and monoclonal antibody-purified preparations, Monoclate-P and Hemofil-M had no effect on lymphocyte proliferation in response to allogeneic stimulator cells over a range of concentrations tested (Fig 4). However, SNBTS factor IX concentrate enhanced lymphocyte proliferation in response
95
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Fig 1. Enhanced lymphocyte proliferation in the presence of SNBTS 28 factor VIII concentrate. PBMC were stimulated were irradiated allogeneic lymphoblastoid cells (LCL) in the presence of 0.1-1 iu/ml factor VIII. Cell proliferation was assessed by incorporation of H-
thymidine (counts per minute) at 24 h intervals.
20000
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Fig 2. Effect of prototype high purity SNBTS S8 factor VIII concentrate on lymphocyte proliferation in response to irradiated allogeneic stimulator cells.
96
A. Batchelor. C. M . Steel and C. A. Ludlam PBMC LCL PBMC LCL -0- PBMC'LCL + PBMC LCL -0- PBMC LCL + PBMC LCL -0-
PBnC -t LCL -0 PBNC LSL -0- PBnC LCL BUFFER 1/10 + PBMC LCL BUFFER 1 / 2 0 u PBnC Li:L BUFFER 1 / 1 0 0
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Fig 3 . The proliferativeresponse to allogeneic stimulator cells was not affected by the presence of SNBTS control buffer. Dilutions of buffer included were equivalent to the dilutions of SNBTS factor VIII concentrate investigated
Fig 5. Enhanced lymphocyte proliferation in the presence of SNBTS
factor IX concentrate. PBMC were stimulated with irradiated allogeneic lymphoblastoid cells in the presence of 0.1-2 iu/ml factor IX concentrate.
8000
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Fig 4. Effect of high puritv factor VIII concentrate Monoclate-P on lymphocyte proliferation in response to allogeneic stimulator cells. Similar results were obtained when high purity concentrates Biotransfusion and Hemofil-M were included in this assay system.
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Fig 6. Proliferation in the one-way mixed lymphocyte reaction was not enhanced by the presence of fibronectin. Purified human fibronectin was included at a range of concentrations representative of those found in SNBTS blood products.
Factor VIZI and T Cell Proli,feration to irradiated allogeneic stimulator cells in vitro when present at 2 iu/ml (Fig 5). Purified human fibronectin had no effect on proliferation in response to irradiated allogeneic lymphoblastoid cells when included at a range of concentrations representative of those found in SNBTS blood products (Fig 6). All experiments illustrated in the figures are representative of reproducible data obtained on several occasions. DISCUSSION A range of factor VIII preparations was assayed in a weak one-way mixed lymphocyte reaction in vitro. SNBTS 28, SNBTS S8 and (to a lesser extent) SNBTS factor IX preparations enhanced lymphocyte proliferation in response to irradiated allogeneic lymphoblastoid cells. High potency ion exchange and monoclonal antibody-purifiedproducts had no such effect. This outcome appears to contrast with the commonly recorded inhibition, by factor VIII, of lymphocyte responses to strong mitogens. We did, in fact, confirm that intermediate purity factor VIII preparations cause dosedependent reduction of thymidine-incorporation by PHAstimulated PBMC (data not shown). While we cannot account for the striking differences between the two situations, the weak one-way MLR may provide a better model for immunological stimuli encountered by lymphocytes in vivo than the massive proliferative drive induced by PHA. The monoclonal antibody-purifiedconcentrates Monoclate-P and Hemofil-M contain considerable amounts of human albumin added as a stabilizer.Enhanced lymphocyte proliferation was not therefore due to factor VIII itself nor to albumin. The comitogenic component of Scottish blood products has not been identified. Fibronectin is present at considerable concentrations in both SNBTS 28 and SNBTS S8 factor VIII preparations. Klingemann et al(1986)demonstrated that fibronectin restored defective lymphocyte proliferation in vitro in the mixed lymphocyte reaction of immunocompromised patients. Fibronectin and monoclonal antibodies specific for CD3 have also been shown to be costimulants of human CD4 positive lymphocyte proliferation under appropriate culture conditions (Matsuyama et al, 1989: Shimizu et al, 1990). It therefore seemed plausible that fibronectin might be responsible for enhancing the proliferative response to allogeneic stimulator cells in vitro. However, purified human fibronectin, at a range of concentrations, was found to be completely devoid of co-mitogenic activity in the system employed in this study. SNBTS factor IX concentrate contains very little fibronectin or fibrinogen, proteins which are the main contaminants of intermediate purity factor VIII concentrate. This preparation did, however, enhance proliferation in response to irradiated allogeneic stimulator cells. It is unclear whether the co-mitogenic factor(s) present in SNBTS factor IX is the same as that in SNBTS 28 and S8 concentrates. No blood product assayed was mitogenic in the absence of stimulator cells. Matheson et al (1986) observed a positive blastogenic response to low concentrations of lyophilized factor VIII concentrate in a 7 d thymidine uptake assay. However, responses were fairly weak and only occurred in
97
haemophilia patients who had previously been exposed to factor VIE preparations. PBMC from healthy control subjects did not respond to factor VIII concentrate in vitro. In the present study responder lymphocytes from healthy nonhaemophiliacs had not previously been exposed to factor VIII concentrate and could not therefore be considered antigenically primed to some constituent of the preparation. Our results contrast with those of Lederman et a1 (1986) who reported reduced lymphocyte proliferation in one-way mixed lymphocyte reactions in the presence of lyophilized factor VIII concentrate in vitro. The two sets of results cannot be compared directly as no technical details of culture conditions used by the previous authors are available. We have demonstrated that intermediate purity factor VIII preparations modulate immune reactivity to allogeneic cells in vitro and that they do so at concentrations achievable in the body during therapeutic administration. Such concentrates could therefore potentially affect the immune system in vivo. Infusion of factor VIII may, for example, enhance lymphocyte activation in response to either naturally occurring immune stimuli or ones present in factor concentrates. Enhancement of lymphocyte activation and proliferation is. paradoxically, quite compatible with inhibition of specific immune function, including delayed-type skin reactions (Ascher & Sheppard, 1990).Furthermore, in an HIV-infected haemophiliac, increased T cell activation could lead to increased virus replication and accelerated disease progression (Cuthbert et al, 1990;Simmonds et a!, 1991).Two recent studies record precisely this outcome in comparisons of HIVinfected haemophiliacs treated with intermediate purity or with high purity factor VIII concentrates (Goldsmith et al, 1991; de Biasi et al, 1991). However, relevant data are available from only small numbers of patients to date and further monitoring of the clinical relevance of our findings will be essential. ACKNOWLEDGMENTS We would like to thank Dr Ron McIntosh and the SNBTS Protein Fractionation Centre for kindly supplying the SNBTS concentrates included in this study and for helpful discussions. REFERENCES Ascher, M.S. & Sheppard, H.W. (1990) AIDS as immune system activation. 11. The panergic imnesia hypothesis. Journal of AIDS, 3, 177-190. Berntorp, E.. Jarevi. C., Wedback. A., Bottiger, B., Hansson. B.C.. Nordenfelt, E. & Nilssen. I.M. (1989) Natural history of HIV infection in Swedish haemophiliacs. European Journal of Haernatology, 42, 254-258. deBiasi, R., Rocino, A.. Miraglia, E.. Mastrullo, L. & Quimino. A.A. (199 1)The impact of a very high purity factor VIII concentrate on the immune system of Human Immunodeficiency Virus-infected haemophiliacs: a randomised, prospective, two year comparison with an intermediate priority concentrate. Blood, 78. 19 19-1 922. Carr. R.. Veitch, S.E., Edmond, E.. Peutherer. J.F., Prescott. R.J., Steel, C.M. & Ludlam. C.A. (1984) Abnormalities of circulating lymphocyte subsets in haemophiliacs in an AIDS-free population. Lanret, i. 1431-1434.
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Cuthbert, R.J.G., Ludlam. C.A.. Steel, C.M.. Beatson. D. & Peutherer, J.F. (1992) Immunological studies in HIV seronegative haemophiliacs: relationship to blood product therapy. British journal of Haematology. in press. Cuthbert. R.J.G., Ludlam, C.A.. Steel, C.M.. Tucker, J.. Beatson. D., Rebus, S. & Peutherer. J.F. (1990) Five year prospective study of HIV infection in the Edinburgh haemophiliac cohort. British Medical Journal. 361, 956-961. Froebel. K.S.. Madhok. R., Forbes. C.D.. Lennie. S.E.. Lowe. G.D.O. & Sturrock, R.D. ( 198 3 ) Immunological abnormalities in haemophilia: are they caused by American factor VIII? British Medical Journal. 287. 1091-7093. Goldsmith. J.M.. Deutsche. J.. Tang, M. & Green. I). (1991)CD4 cells in HIV-I infected haemophiliacs: effect of factor VIII concentrates. Thrombosis and Huemostasis. 66, 41 5-419. Hay. C.R.M.. McEvoy. P. & Duggan-Keen. M. (1990) Inhibition of lymphocyte IL-2 receptor expression by factor VIII concentrate: a possible cause of immunosuppression in haemophiliacs. British Journal ofHaemato1ogy. 75, 278-281. Klingemann. H-G.. Tsoi. M.-S. & Storb. R. [ 1986)Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting. Transplantation, 42, 41 2-4 1 7. Iederman. M.M.. Saunders. C.. Toossi. 2.. Lemon, N.. Everson. B. & Ratnoff, O.D. (1986) Anthihemophilic factor (factor VIII) preparations inhibit lymphocyte proliferation and production of interleukin-2. Journal of Laboratory and Clinical Medicine. 197, 471428. Ludlam. C.A., Tucker, I.. Steel. C.M.. Tedder. R.S.. CheingsongPopov, R.,Weiss. R.A., McClelland. D.B.L., Philp. I. & Prescott. R.J. (1985) Human T-lymphotropic virus type 111 (HTLV-111) infection in seronegative haemophiliacs after transfusion of factor VIII. Lancet. ii, 233-236. Madhok. R.. Gracie. A,. Lowe. G.D.O.. Burnett. A,. Froebel. K. & Follett. E. ( 1986) Impaired cell mediated immunity in haemophilia in the absence of infection with the human immunodeficiency virus. British Medical Journal. 293, 978-980.
Matheson. D.S.. Green. B.J., Poon, M.-C., Bowen, T.J., Fritzler. M.J. & Hoar. D.I. ( I 986) T lymphocytes from hemophiliacs proliferate after exposure to factor VIII product. Vox Sanguinis, 51, 92-95. Matsuyama.T.. Yamada, A.. Kay.J., Yamada. K.M.,Akiyama, S.K. & Schlossman. S.F. (1989) Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex. Journal of Experimental Medicine, 170, 1133-1148. McDonald. C.. Jackson, V.. Kilpatrick. D.C., Yap, P.-L. & Prowse, C. (1985) A diffusable factor in Scottish coagulation factor concentrates can inhibit lymphocyte transformation in vitro. Vox Sunguinis, 49, 187-194. McIvor. R.A.. McCluskey, D.R., Mayne, E.E. & McKirgan. J. (1987) Immune response to multiple skin test antigens in haemophiliacs. Ulster Medical Journal, 56, 95-99. Moffat. E.H.. Bloom, A.L., Jones. J . , Matthews. N. & Newcombe. R.G. (1985) A study of cell mediated and humoral immunity in haemophilia and related disorders. British Journal of Haematology, 61. 157-167. Shimizu, Y.. Van Seventer, G.A., Horgan. K.J. & Shaw, S. (1990) Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin. Journal of Immunology, 145, 59-67. Simmonds. P.. Beatson. D., Cuthbert. R.J.G., Watson, H., Reynolds, B.. Peutherer, J.F., Parry. J.V.. Ludlam C.A. & Steel, C.M. (1991) Determinants of HIV disease progression: a 6 year longitudinal study in the Edinburgh haemophilia/HIV cohort. Lancet, 338, 1159-1163. Thorpe. R.. Dilger. P.. Dawson. N.J. & Barrowcliffe, T.W. (1989) Inhibition of interleukin-2 secretion by factor VIII concentrates; a possible cause of immunosuppression in haemophiliacs. British Journal of Haematology, 71, 387-391. Wang.Y.. Beck.E.A..Furlan,M.&deWeck.A.L. (1985)Inhibitionof human T lymphocyte proliferation in vitro by commercial factor VIII concentrates. Vox Sanguinis. 48, 343-349.
Enhancement of human T cell responses to allogeneic stimuli by factor VIII concentrates ALISONBATCHELOR,C. MICHAEL STEELA N D CHRISTOPHERA. LUDLAM* Medical Research Council, Human Genetics Unit, Edinburgh, Lothian. and *Department of Haematology, Royal Infirmary of Edinburgh, Edinburgh, Lothian Received 10 Decernber 199 1: accepted for publication 14 April 1992
Summary. The effect of factor VIII concentrate, from commercial and National Health Services manufacturers. on in vitro lymphocyte proliferative response to allogeneic stimulator cells was investigated. Factor VIII preparations 'purified' by ion exchange or by monoclonal antibody affinity had no effect in this assay but lymphocyte proliferation in response to allogeneic cells was markedly and consistently enhanced by some intermediate purity factor VIII concentrates and. to a lesser extent. by a
factor IX preparation. These preparations did not stimulate lymphocyte proliferation in the absence of other mitogens. The co-mitogenic factor(s) present in intermediate purity factor VIII was not identified. However, enhanced proliferation was not due to factor VIII itself, nor to albumin or fibronectin. The clinical relevance of the immunomodulatory activity of intermediate purity factor VIII concentrates i n vitro is discussed.
It has been clear for a number of years that patients receiving factor VIII concentrate for the treatment of haemophilia have subclinical abnormalities of the immune system. These include abnormalities of T cell subsets, irrespective of HIV infection (Berntorp et al. 1989: Moffat et nl, 1985: Carr et al. 1984) and impaired cell mediated immunity. as assessed by the intradermal injection of recall antigens or of dinitrochlorobenzene (DNCR) (Mclvor et al. 1987: Madhok et al. 1986: Cuthbert et al. 1992). It has been suggested that intensive treatment with intermediate purity factor VIII preparations may be responsible for immune modulation in haemophiliacs (Ludlam et aI. 1985; Madhok et al. 1986). Factor VIII concentrates have been shown to impair lymphocyte proliferation in vitro in response to mitogen, recall antigen and monoclonal antibody stimulation (Froebel t't al. 1983; McDonald et al, 1985: Wang et al. 1985). The secretion of interleukin 2 (IL2) and the expression of cell surface activation markers following stimulation in vitro are also reduced in the presence of factor VIII concentrates (Lederman et al. 1986: Thorpe rt a], 1989: Hay rt a]. 1990). However, the relationship between immune abnormalities observed in vivo and the effects of factor VIII concentrate on immune function iri vitro i s controversial. In the present study we have investigated the effects of factor VIII concentrate on lymphocyte proliferation following stimulation with irradiated allogeneic cells. We have com-
pared a number of commercial and National Health Service factor VIII concentrates ranging from intermediate to high purity formulations.
Correspondence: Dr Alison Batchelor. Medical Research Council. Human Genetics Unit. Edinburgh EH.1 2XU. Lothian. Scotland.
MATERIALS AND METHODS Cells. Venous blood was collected into lithium heparin from healthy human volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll/ Hypaque (SG 1.077) (Pharmacia, Uppsala, Sweden) at 900g for 1 5 min. The interface band of mononuclear cells was removed and washed four times in phosphate buffered saline (PBS).An Epstein Barr virus transformed human B lymphoid cell line. RAB12, (HLA type A2A3. B27BW22, DR3,4 W 52W53) was used as the allogeneic stimulator in one-way mixed lymphocyte reactions. RAB12cells were maintained on RPMI 1 6 4 0 (Gibco, Grand Island. N.Y.) supplemented with 0.03% fresh glutamine and 5% heat inactivated fetal calf serum (FCS) in 5% COz at 37°C. The cell line was screened regularly and found consistently negative for mycoplasma contamination. The phenotype of responding PBMC was checked by two-colour immunofluorescence (CD3IFITC + CD19/Phycoerythrin, Becton Dickinson), scoring the proportions of positive cells by flow cytopherometry (FACScan, Becton Dickinson). Prolijieration assays. PBMC were stimulated with irradiated allogeneic lymphoblastoid cells and cultured in RPMI I640 medium containing 0.03% fresh glutamine, 5% heat inactivated FCS and 1 u/ml heparin (Evans Biologicals Ltd.
Factor VIII and T Cell Proliferation Liverpool,U.K.). RAB12cells were exposed to an X-ray dose of 1000 rads prior to inclusion in proliferation assays. 150 pl aliquots containing 5 x lo4 PBMC and 2 x lo4 irradiated RABlz cells were cultured together in 96 V-well plates. Replicate cultures were set up by dispensing 150 p1 aliquots from a 4 ml volume of culture medium containing PBMC, irradiated RAB12 cells and various concentrations of factor VIII, factor IX, fibronectin or control buffers. The factor VIII preparations investigated were SNBTS 28, SNBTS S8 (Scottish National Blood Transfusion Service, Protein Fractionation Centre, Edinburgh, U.K.), high potency Bio-transfusion (Bio-transfusion.Lille, France), Monoclate-P (Armour Pharmaceutical Company, Kankakee, Ill.) and Hemofil M (Baxter Healthcare Corporation, Glendale, Calif.). SNBTS S8 factor VIII is a prototype higher purity factor VIII concentrate which has a lower fibrinogen, but higher fibronectin content compared to SNBTS 28 factor VIII concentrate. The factor IX preparation was SNBTS concentrate (DEFIX).All factor VIII and factor IX preparations were used at a final concentration of 0.1-2 iu/ml. They had been stored and reconstituted according to manufacturers’ instructions. Control buffer included was identical to the buffer solution in which SNBTS 28 factor VIII concentrate is administered. Buffer was present at a final dilution range of 1/ 100 to 1/10. Purified human fibronectin (Chemicon, tissue culture grade) was included at a concentration of 21-425 pg/ml. 18 h prior to cell harvesting, cultures were labelled with 3 7 kBq 3H-thymidine(Amersham, Amersham, Bucks., U.K.) in a 50 pl aliquot of culture medium. Replica cultures were harvested at 24 h intervals for up to 12 d. Cell cultures were harvested onto glass fibre papers, dried and 3H-thymidine incorporation measured using a Matrix 96 direct beta counter (Packard, Ulgersmaweg, Groningen, The Netherlands). Mean values for 3H-thymidine incorporation in counts per minute were calculated from duplicate wells. RESULTS Lymphocyte proliferation in response to irradiated allogeneic lymphoblastoidcells was markedly enhanced in the presence of intermediate purity SNBTS 28 factor VIII concentrate (Fig 1). All seven batches of SNBTS 28 tested behaved in this manner. At the end of the culture 93% of the cells were CD3 positive and 7% expressed CD19; thus the vast majority ofthe proliferating cells were T cells. SNBTS S8 factor VIII concentrate enhanced lymphocyte proliferation in this assay but apparently to a lesser extent (Fig 2). Peripheral blood mononuclear cells did not respond to SNBTS blood products in the absence of irradiated stimulator cells. Enhanced proliferation was not observed when SNBTS control buffer was substituted for concentrate in the lymphocyte proliferation assay (Fig 3). High purity factor VIII concentrate (Bio-transfusion) and monoclonal antibody-purified preparations, Monoclate-P and Hemofil-M had no effect on lymphocyte proliferation in response to allogeneic stimulator cells over a range of concentrations tested (Fig 4). However, SNBTS factor IX concentrate enhanced lymphocyte proliferation in response
95
20000 PBMC
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Fig 1. Enhanced lymphocyte proliferation in the presence of SNBTS 28 factor VIII concentrate. PBMC were stimulated were irradiated allogeneic lymphoblastoid cells (LCL) in the presence of 0.1-1 iu/ml factor VIII. Cell proliferation was assessed by incorporation of H-
thymidine (counts per minute) at 24 h intervals.
20000
PBMC
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PBMC.LCL Factor V l l l 2iu/ml -e. PBMC LCL Factor V l l l l i u / m l U PBMC-LCL Faclor V l l i 0 5iu/mi 9
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Fig 2. Effect of prototype high purity SNBTS S8 factor VIII concentrate on lymphocyte proliferation in response to irradiated allogeneic stimulator cells.
96
A. Batchelor. C. M . Steel and C. A. Ludlam PBMC LCL PBMC LCL -0- PBMC'LCL + PBMC LCL -0- PBMC LCL + PBMC LCL -0-
PBnC -t LCL -0 PBNC LSL -0- PBnC LCL BUFFER 1/10 + PBMC LCL BUFFER 1 / 2 0 u PBnC Li:L BUFFER 1 / 1 0 0
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Fig 3 . The proliferativeresponse to allogeneic stimulator cells was not affected by the presence of SNBTS control buffer. Dilutions of buffer included were equivalent to the dilutions of SNBTS factor VIII concentrate investigated
Fig 5. Enhanced lymphocyte proliferation in the presence of SNBTS
factor IX concentrate. PBMC were stimulated with irradiated allogeneic lymphoblastoid cells in the presence of 0.1-2 iu/ml factor IX concentrate.
8000
201100
I is- PBMC
+- > C L
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PEMC L C I 0 PBflC L C L F i i l o r Vlll Ilu/ml -a- P a m L C L Faclor VIII 0 S i d m l u-PBfiC L C L F a c l o r V l l l 0 l i u / m l
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Fibronectin 425ug1ml Fibronectin 212pglrni PBMC.LCL Fibronectln 42pg1ml
* P6MC:LCL
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W Y S INZUBATIOII
Fig 4. Effect of high puritv factor VIII concentrate Monoclate-P on lymphocyte proliferation in response to allogeneic stimulator cells. Similar results were obtained when high purity concentrates Biotransfusion and Hemofil-M were included in this assay system.
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Fig 6. Proliferation in the one-way mixed lymphocyte reaction was not enhanced by the presence of fibronectin. Purified human fibronectin was included at a range of concentrations representative of those found in SNBTS blood products.
Factor VIZI and T Cell Proli,feration to irradiated allogeneic stimulator cells in vitro when present at 2 iu/ml (Fig 5). Purified human fibronectin had no effect on proliferation in response to irradiated allogeneic lymphoblastoid cells when included at a range of concentrations representative of those found in SNBTS blood products (Fig 6). All experiments illustrated in the figures are representative of reproducible data obtained on several occasions. DISCUSSION A range of factor VIII preparations was assayed in a weak one-way mixed lymphocyte reaction in vitro. SNBTS 28, SNBTS S8 and (to a lesser extent) SNBTS factor IX preparations enhanced lymphocyte proliferation in response to irradiated allogeneic lymphoblastoid cells. High potency ion exchange and monoclonal antibody-purifiedproducts had no such effect. This outcome appears to contrast with the commonly recorded inhibition, by factor VIII, of lymphocyte responses to strong mitogens. We did, in fact, confirm that intermediate purity factor VIII preparations cause dosedependent reduction of thymidine-incorporation by PHAstimulated PBMC (data not shown). While we cannot account for the striking differences between the two situations, the weak one-way MLR may provide a better model for immunological stimuli encountered by lymphocytes in vivo than the massive proliferative drive induced by PHA. The monoclonal antibody-purifiedconcentrates Monoclate-P and Hemofil-M contain considerable amounts of human albumin added as a stabilizer.Enhanced lymphocyte proliferation was not therefore due to factor VIII itself nor to albumin. The comitogenic component of Scottish blood products has not been identified. Fibronectin is present at considerable concentrations in both SNBTS 28 and SNBTS S8 factor VIII preparations. Klingemann et al(1986)demonstrated that fibronectin restored defective lymphocyte proliferation in vitro in the mixed lymphocyte reaction of immunocompromised patients. Fibronectin and monoclonal antibodies specific for CD3 have also been shown to be costimulants of human CD4 positive lymphocyte proliferation under appropriate culture conditions (Matsuyama et al, 1989: Shimizu et al, 1990). It therefore seemed plausible that fibronectin might be responsible for enhancing the proliferative response to allogeneic stimulator cells in vitro. However, purified human fibronectin, at a range of concentrations, was found to be completely devoid of co-mitogenic activity in the system employed in this study. SNBTS factor IX concentrate contains very little fibronectin or fibrinogen, proteins which are the main contaminants of intermediate purity factor VIII concentrate. This preparation did, however, enhance proliferation in response to irradiated allogeneic stimulator cells. It is unclear whether the co-mitogenic factor(s) present in SNBTS factor IX is the same as that in SNBTS 28 and S8 concentrates. No blood product assayed was mitogenic in the absence of stimulator cells. Matheson et al (1986) observed a positive blastogenic response to low concentrations of lyophilized factor VIII concentrate in a 7 d thymidine uptake assay. However, responses were fairly weak and only occurred in
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haemophilia patients who had previously been exposed to factor VIE preparations. PBMC from healthy control subjects did not respond to factor VIII concentrate in vitro. In the present study responder lymphocytes from healthy nonhaemophiliacs had not previously been exposed to factor VIII concentrate and could not therefore be considered antigenically primed to some constituent of the preparation. Our results contrast with those of Lederman et a1 (1986) who reported reduced lymphocyte proliferation in one-way mixed lymphocyte reactions in the presence of lyophilized factor VIII concentrate in vitro. The two sets of results cannot be compared directly as no technical details of culture conditions used by the previous authors are available. We have demonstrated that intermediate purity factor VIII preparations modulate immune reactivity to allogeneic cells in vitro and that they do so at concentrations achievable in the body during therapeutic administration. Such concentrates could therefore potentially affect the immune system in vivo. Infusion of factor VIII may, for example, enhance lymphocyte activation in response to either naturally occurring immune stimuli or ones present in factor concentrates. Enhancement of lymphocyte activation and proliferation is. paradoxically, quite compatible with inhibition of specific immune function, including delayed-type skin reactions (Ascher & Sheppard, 1990).Furthermore, in an HIV-infected haemophiliac, increased T cell activation could lead to increased virus replication and accelerated disease progression (Cuthbert et al, 1990;Simmonds et a!, 1991).Two recent studies record precisely this outcome in comparisons of HIVinfected haemophiliacs treated with intermediate purity or with high purity factor VIII concentrates (Goldsmith et al, 1991; de Biasi et al, 1991). However, relevant data are available from only small numbers of patients to date and further monitoring of the clinical relevance of our findings will be essential. ACKNOWLEDGMENTS We would like to thank Dr Ron McIntosh and the SNBTS Protein Fractionation Centre for kindly supplying the SNBTS concentrates included in this study and for helpful discussions. REFERENCES Ascher, M.S. & Sheppard, H.W. (1990) AIDS as immune system activation. 11. The panergic imnesia hypothesis. Journal of AIDS, 3, 177-190. Berntorp, E.. Jarevi. C., Wedback. A., Bottiger, B., Hansson. B.C.. Nordenfelt, E. & Nilssen. I.M. (1989) Natural history of HIV infection in Swedish haemophiliacs. European Journal of Haernatology, 42, 254-258. deBiasi, R., Rocino, A.. Miraglia, E.. Mastrullo, L. & Quimino. A.A. (199 1)The impact of a very high purity factor VIII concentrate on the immune system of Human Immunodeficiency Virus-infected haemophiliacs: a randomised, prospective, two year comparison with an intermediate priority concentrate. Blood, 78. 19 19-1 922. Carr. R.. Veitch, S.E., Edmond, E.. Peutherer. J.F., Prescott. R.J., Steel, C.M. & Ludlam. C.A. (1984) Abnormalities of circulating lymphocyte subsets in haemophiliacs in an AIDS-free population. Lanret, i. 1431-1434.
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Cuthbert, R.J.G., Ludlam. C.A.. Steel, C.M.. Beatson. D. & Peutherer, J.F. (1992) Immunological studies in HIV seronegative haemophiliacs: relationship to blood product therapy. British journal of Haematology. in press. Cuthbert. R.J.G., Ludlam, C.A.. Steel, C.M.. Tucker, J.. Beatson. D., Rebus, S. & Peutherer. J.F. (1990) Five year prospective study of HIV infection in the Edinburgh haemophiliac cohort. British Medical Journal. 361, 956-961. Froebel. K.S.. Madhok. R., Forbes. C.D.. Lennie. S.E.. Lowe. G.D.O. & Sturrock, R.D. ( 198 3 ) Immunological abnormalities in haemophilia: are they caused by American factor VIII? British Medical Journal. 287. 1091-7093. Goldsmith. J.M.. Deutsche. J.. Tang, M. & Green. I). (1991)CD4 cells in HIV-I infected haemophiliacs: effect of factor VIII concentrates. Thrombosis and Huemostasis. 66, 41 5-419. Hay. C.R.M.. McEvoy. P. & Duggan-Keen. M. (1990) Inhibition of lymphocyte IL-2 receptor expression by factor VIII concentrate: a possible cause of immunosuppression in haemophiliacs. British Journal ofHaemato1ogy. 75, 278-281. Klingemann. H-G.. Tsoi. M.-S. & Storb. R. [ 1986)Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting. Transplantation, 42, 41 2-4 1 7. Iederman. M.M.. Saunders. C.. Toossi. 2.. Lemon, N.. Everson. B. & Ratnoff, O.D. (1986) Anthihemophilic factor (factor VIII) preparations inhibit lymphocyte proliferation and production of interleukin-2. Journal of Laboratory and Clinical Medicine. 197, 471428. Ludlam. C.A., Tucker, I.. Steel. C.M.. Tedder. R.S.. CheingsongPopov, R.,Weiss. R.A., McClelland. D.B.L., Philp. I. & Prescott. R.J. (1985) Human T-lymphotropic virus type 111 (HTLV-111) infection in seronegative haemophiliacs after transfusion of factor VIII. Lancet. ii, 233-236. Madhok. R.. Gracie. A,. Lowe. G.D.O.. Burnett. A,. Froebel. K. & Follett. E. ( 1986) Impaired cell mediated immunity in haemophilia in the absence of infection with the human immunodeficiency virus. British Medical Journal. 293, 978-980.
Matheson. D.S.. Green. B.J., Poon, M.-C., Bowen, T.J., Fritzler. M.J. & Hoar. D.I. ( I 986) T lymphocytes from hemophiliacs proliferate after exposure to factor VIII product. Vox Sanguinis, 51, 92-95. Matsuyama.T.. Yamada, A.. Kay.J., Yamada. K.M.,Akiyama, S.K. & Schlossman. S.F. (1989) Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex. Journal of Experimental Medicine, 170, 1133-1148. McDonald. C.. Jackson, V.. Kilpatrick. D.C., Yap, P.-L. & Prowse, C. (1985) A diffusable factor in Scottish coagulation factor concentrates can inhibit lymphocyte transformation in vitro. Vox Sunguinis, 49, 187-194. McIvor. R.A.. McCluskey, D.R., Mayne, E.E. & McKirgan. J. (1987) Immune response to multiple skin test antigens in haemophiliacs. Ulster Medical Journal, 56, 95-99. Moffat. E.H.. Bloom, A.L., Jones. J . , Matthews. N. & Newcombe. R.G. (1985) A study of cell mediated and humoral immunity in haemophilia and related disorders. British Journal of Haematology, 61. 157-167. Shimizu, Y.. Van Seventer, G.A., Horgan. K.J. & Shaw, S. (1990) Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin. Journal of Immunology, 145, 59-67. Simmonds. P.. Beatson. D., Cuthbert. R.J.G., Watson, H., Reynolds, B.. Peutherer, J.F., Parry. J.V.. Ludlam C.A. & Steel, C.M. (1991) Determinants of HIV disease progression: a 6 year longitudinal study in the Edinburgh haemophilia/HIV cohort. Lancet, 338, 1159-1163. Thorpe. R.. Dilger. P.. Dawson. N.J. & Barrowcliffe, T.W. (1989) Inhibition of interleukin-2 secretion by factor VIII concentrates; a possible cause of immunosuppression in haemophiliacs. British Journal of Haematology, 71, 387-391. Wang.Y.. Beck.E.A..Furlan,M.&deWeck.A.L. (1985)Inhibitionof human T lymphocyte proliferation in vitro by commercial factor VIII concentrates. Vox Sanguinis. 48, 343-349.
