Physicochemical Characterization and Partial Purification of Mouse Immune Interferon

773 J. gen. Virol. (I979) , 44, 773-78t Printed in Great Britain Physico-chemical Characterization and Partial Purification of Mouse Immune Interferon By J U A N A W I E T Z E R B I N , S I M O N S T E F A N O S , M I G U E L L U C E R O , ERNESTO FALCOFF, Institut Curie, Section de Biologie, 75z3I Paris, Cedex o5, France J U D I T H A. O ' M A L L E Y AND E U G E N E S U L K O W S K I Department o f Viral Oncology, Roswell Park Memorial Institute, Buffalo, N. Y. 14263, U.S.A. (Accepted 20 March I979) SUMMARY Mouse immune (type T) interferon was produced from suspensions of spleen cells (i × Io 7 cells/ml) treated with 3/~g/ml of phytohaemagglutinin. The crude interferon was chromatographed on four sorbents with varying affinities, namely concanavalin A-Sepharose, Affi-Gel 202, Blue Sepharose CL-6B and PhenylSepharose CL-4B. With each of these the interferon activity was observed to have considerable heterogeneity. By means of affinity chromatography, mouse immune interferon was purified IOO to 2o0 times with concomitant complete recovery of activity. INTRODUCTION Specific antigens and stimulants of T and B lymphocytes are able to trigger interferon synthesis in immunocompetent cells (Wheelock, I965; Falcoff, I972; Wallen et al. t973; Stobo et al. I974; Valle et al. 1975a). Together with immune interferon, many other lymphokines are also produced in the course of a cell-mediated immune response (Youngner & Salvin, i973; Bartfeld & Vilc~k, i975; Johnson & Baron, r976a). We have recently shown (Wietzerbin et al. I977; Barot-Ciorbaru et aL I978), in accordance with findings of other laboratories (Youngner & Salvin, I973; Valle et al. 1975b; Johnson & Baron, I976b), that the properties of the interferon induced by mitogens depend on the nature of the cell population which is induced. For example, the mouse immune interferon induced by B-cell mitogens is antigenically related to virus-induced interferon and is stable on treatment at pH 2. However, interferons induced by T-cell mitogens, such as phytohaemagglutinin or concanavalin A (Con A) (Johnson & Baron, I976b; Wietzerbin et al. 1977; Wietzerbin et al. 1978a) are antigenically different from virus-induced interferon and are unstable at pH 2. This T-type interferon induced by a non-specific stimulant such as phytohaemagglutinin, has properties like those of the type II interferon of Youngner & Salvin (x973) which is induced by injecting tuberculin (a T-dependent specific antigen) into BCG-sensitized mice. During the last few years, many studies have been concerned with the physico-chemical properties, purification, mechanism of action and immunological properties of virusinduced interferons (Metz, 1975; Johnson & Baron, I976a; Lewis et al. I977; De MaeyerGuignard et al. I978), but none with immune interferons. Interest in these latter interferons has recently increased because the findings of Johnson & Baron 0976b) and Sonnenfeld et al. (I977) indicate that T-type (type 1I) interferons have exceptionally potent immunosuppressive activity by comparison with virus-induced interferon. In this report, we have Downloaded from www.microbiologyresearch.org by oo22-I317/79/oooo-3567 $o2.oo~ 1979 SGMIP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 774 J. W I E T Z E R B I N A N D OTHERS developed a method for producing relatively large amounts of phytohaemagglutinininduced interferon from mouse spleen cell cultures. Furthermore, in order to evaluate some biological properties of this type of interferon and its immunosuppressive activity in particular, it has been necessary to develop simple and efficient means for its partial purification. To this end, we have explored affinity chromatography of mouse immune interferon on several sorbents with different chromatographic characteristics. METHODS Animals. Nude heterozygous mice (nu/+) were supplied by Centre de S61ection et d'Elevage d'Animaux de Laboratoire, CSEL, Orleans, France. Media. RPNI 164o and foetal calf serum were purchased from Flow Laboratories; I99 medium was obtained from Institut Pasteur. Chromatographic sorbents and chemicals. Con A-Sepharose, CH-Sepharose 4 B, Blue Sepharose CL-6B and Phenyl-Sepharose CL-4B were purchased from Pharmacia; Affi-Gel 202 from Bio-Rad Laboratories; Methyl C~-D-mannopyranoside (c~-MM) from Sigma; fluorescamine from Roche Diagnostics; phytohaemagglutinin (PHA) from Wellcome; gentamicin from UNILABO, France. All other reagents were of analytical grade. Preparation of mouse PH,4-interferon. Nude heterozygous mice (nu/q-) were killed by cervical dislocation. The spleens were rapidly removed and placed into 199 medium in Petri dishes kept on ice. Spleen cells were obtained by teasing the spleen tissue through a nylon filter and repeated washes with 199 medium. After centrifugation, the cells were resuspended at I x IO7 cells/ml in RPMI I64O medium supplemented with 5~o foetal calf serum, 2 mM-glutamine and o-oo4~ gentamicin. The cell suspensions were incubated in Petri dishes (Nunclon, 9o mm) with purified PHA at a concentration of 3 #g/ml for 24 h at 37 °C in a humidified (5 ~ CO2) incubator. The cells were then spun off and the medium was treated with ammonium sulphate to a final saturation of 40 ~ at 4 °C. The precipitate was removed by centrifugation and the supernatant was concentrated (Io to t 5 times) under vacuum and dialysed against phosphate-buffered physiological saline (PBS). The titres of the resulting interferon preparations were in the range of t × Io4 to 1.5 × lO4 units/ml and specific activity (units per mg protein) of interferon was about 5 × lO~. Interferon assay. Interferon preparations were assayed either by the colorimetric procedure of Finter 0969) or by a cytopathogenic inhibition test (Havelt & Vilc~k, 1972) in Lcell monolayers using vesicular stomatitis virus as a challenge virus. All interferon titres are expressed in reference units. Protein determination. Protein concentration was measured by a fluorometric assay (B~Shlen et al. 1973) with bovine serum albumin as the standard. Chromatographic procedure. All interferon preparations were dialysed against appropriate buffers at 4 °C for 24 h and the column was equilibrated initially with the same buffer. The equilibration and development of the columns were performed at 4 °C. The flow rate from the column was 2o to 40 ml/cm2/h and was maintained by means of a peristaltic pump. Fractions of I ml were collected in plastic test tubes. Even-numbered fractions were used for protein determination; odd-numbered fractions, collected into test tubes containing o'5 ml of a 1 ~o solution of bovine serum albumin in PBS, were used for interferon assay. RESULTS Chromatography on concanavalin A-agarose The chromatographic behaviour of mouse immune interferon applied to a Con A-agarose column in a buffer with I M-NaCI is illustrated in Fig. I. The interferon activity was distributed among two fractions: (i) the breakthrough fraction containing about 4o ~ of the total Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 Purification of mouse immune interferon iI 4 I E1 tl I ! I I E2 Ea 775 ! 11 1000 l e- I' .o 2 i, ~. .-- 0 10 20 30 40 50 /'/'/] 0"10 ~__. ,% 60 Fractionnumber 500 o-o 70 80 90 Fig. i. Chromatography of mouse immune interferon on Con A-Sepharose. Five ml of crude interferon, containing a total of 12500 units of activity and 26"5mg protein, were dialysed against o.o2 M-phosphate buffer, pH 7"4 (PB) containing I M-NaC! and applied to a Con A-Sepharose column (0"9× 8 cm). The column was washed with 30 ml PB (I M-NaC1)and then a linear concentration gradient of ~-MM (El) was developed by mixing 20 ml PB (I M-NaC1) and 20 ml o'I M-~-MM in PB (I M-NaCI). The column was then washed briefly with 5 ml oq M-~-MM in PB (i M-NaCi) (E2) and finally with I5 ml 50 ~ (v/v) ethylene glycol in E~ (Ea). The overall recovery of interferon activity was 95 ~, with 38 % in the breakthrough fraction and 62 ~ eluted with the ~-MM gradient. O - - - (3, Protein; • • , interferon; - - - --, ~x-MMgradient. applied activity and (ii) a fraction eluted with ~ - M M which constitutes about 60 ~o of the activity. The true chromatographic character of both fractions was established by rechromatographing them individually on freshly prepared lectin columns; they were recovered in their original positions. Thus, it may be concluded that the lack of binding of the first fraction ('breakthrough fraction') was not due to a limited capacity of the column employed in the experiment; also that the binding of the second fraction ('c~-MM fraction') might be caused by adventitious complexing to other glycoproteins present in the preparation. However, the bulk of glycoproteins was eluted after interferon, and this position was unchanged when the pooled fractions were subjected to re-chromatography. The purification of the ' ~ - M M fraction' is about Ioo-fold under conditions such as in the experiment described in Fig. I. When the chromatography was performed with an equilibrating solvent of lower ionic strength, namely PBS (o. 15 u), a considerable amount of protein was retained on the column and was subsequently eluted with ~ - M M ; this resulted in a substantially lower purification factor for the ' ~ - M M fraction' of interferon (not shown). Chromatography on Aft-Gel 202 Apparent heterogeneity of mouse immune interferon, as observed on lectin chromatography, prompted us to evaluate further several other sorbents with differing chromatographic characteristics. Among other sorbents, mouse immune interferon was therefore chromatographed on Affi-Gel 2o2 as shown in Fig. 2. When mouse immune interferon was applied at p H 5 and at a low ionic strength of the solvent, the majority of interferon activity - - about 9o ~o - - was retained on the sorbent. However, interferon activity could be readily recovered by an increase in p H (El) and the ionic strength (E2) of the solvent. Both of these fractions were shown to re-chromatograph in their original positions (not shown) indicating again some heterogeneity in the population of interferon molecules. In view of the nature of the l i g a n d - which clearly has the potential for both ionic (carboxyl group) and hydrophobic (two runs of three methylene units along the arm) interactions d this heterogeneity may be ascribed to differences in charge and hydrophobicity of both subpopulations of Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 776 J. W l E T Z E R B I N AND OTHERS 1000 I I I I I l I ' I , I ' I ~ I ' I • I ' t • I Ez ;~ El 750 o~ lOOO = 2 I / I II / / ! I/ t 500 o.s 500 ~ 1 -= 250 t- 10 2b 3o 4o so 6o 7o 8o 9 Fraction number Fig. 2 7 5 3 pH Fig. 3 Fig. 2. Chromatography of mouse immune interferon on Atfi--Gel 202. Eight ml of concentrated interferon containing a total of 20000 units of activity and 80 mg protein were dialysed against o'05 M-sodium acetate, pH 5, and applied to a column (0'9 × 8 cm) of Affi-Gel 202 equilibrated with the same buffer. The breakthrough fraction from the column contained about I2 70 of the applied interferon activity and the majority of the protein. Elution of the column with PB (El) resulted in the displacement of 38 ~o of the interferon activity. Finally, the column was developed with a linear concentration gradient of NaCI (E~) formed by mixing 20 ml PB and 20 ml 0'5 M-NaCI in PB; this resulted in the recovery of the remaining 50 ~ of interferon activity. © - - - ©, Protein; • • , interferon; - - - - - , NaCI gradient. Fig. 3. pH stability of mouse immune interferon. One ml samples of an interferon preparation, containing 75o units of activity were dialysed for z4 h at 4 °C against 20o ml of the following buffers: HCI-KC1, pH 2 (0-05 M); glycine-HCl, pH 3 (0'05 M); sodium acetate, pH 4 and 5 (0"05 M); sodium phosphate buffer pH 6, 7 and 8 (0"05 M) and tris-HCl, pH 9 (0"05 M). The samples were then dialysed against PBS (pH 7"4) for an additional 24 h. All were titrated for the interferon activity in a single assay. interferon molecules. W h e n m o u s e i m m u n e interferon was a p p l i e d on C H - S e p h a r o s e 4 B u n d e r identical solvent conditions, all o f the p r o t e i n a n d the i n t e r f e r o n activity were recovered in the b r e a k t h r o u g h fraction (not shown). It is relevant to observe t h a t a l t h o u g h C H - S e p h a r o s e 4 B carries terminal c a r b o x y l h e a d groups, as A M - G e l does, its a r m is less hydrophobic. C h r o m a t o g r a p h y o f b o t h interferon s u b p o p u l a t i o n s f r o m the A f f i - G e l 202 c o l u m n , i.e., E1 a n d E~ p o o l e d f r a c t i o n s ; o n C o n A - S e p h a r o s e revealed t h a t they c o u l d be in t u r n subdivided into n o n - b i n d i n g a n d binding c o m p o n e n t s . Thus, the heterogeneity o f m o u s e i m m u n e interferon, as revealed on Affi-Gel 202, c a n n o t be simply ascribed to the v a r i e d extent o f glycosylation which p r e s u m a b l y w o u l d result in v a r y i n g a m o u n t s o f c h a r g e d sugar residues (sialic acid). Significantly, the c h r o m a t o g r a p h y o f i m m u n e interferon o n A f f i - G e l 2o2 (Fig. 2) can serve well as a purification step as the retention o f interferon activity is quite selective (most p r o t e i n s pass t h r o u g h the c o l u m n unretained). T h e specific activity o f an interferon p r e p a r a t i o n can be increased f r o m 2"5 × I0 ~ (crude p r e p a r a t i o n ) to 4 × [04 (El-pool) a n d to ] × IO5 (E2-pool). Stability of immune interferon at low p H The need to use low p H conditions during the a d s o r p t i o n phase o f A m - G e l 2o2 c h r o m a t o g r a p h y p r o m p t e d us to examine the p H stability o f o u r i m m u n e interferon p r e p a r a t i o n (Fig. 3). A s can be seen f r o m the p H - s t a b i l i t y curve, the exposure o f the interferon Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 Purification o f mouse immune interferon 18 I I I I I | E11 6f I 3000 14 ,~ /! 6 4 I!~k 2000 ~ // 9 ./J k~rk 5 J 2 1 I 10 20 30 40 50 60 70 80 Fraction number Fig. 4 i i i I 5000 i 4000 '3 1 [ i' /I I i 10 3000 /----75 ! 0 0 I IE2 ,!! T~ 3 I , ? 6 4 1000 i E11 7 5, ,J II 8 I 8 ~ 1o = m I IE2 777 / ! fl ] l, sot-t ~ 25t 70 80 20 ,oJ 30 40 50 60 Fraction number Fig. 5 Fig. 4. Chromatography of mouse immune interferon on Blue Sepharose CL-6B. Eight ml of an interferon preparation, containing 32000 units of activity and 88 mg protein, were dialysed against PBS and applied on a Blue Sepharose CL-6B column (0'9 × 5 cm). The column was washed wtih t5 ml PBS and then a linear concentration gradient of NaCI (Ex) was developed by mixing I7 ml PBS and 17 ml 1"35 M-NaC1 in PBS. The column was finally Outed with 20 ml 50 ~o (v/v) ethylene glycol in PB (I M-NaC1) (E2). There was complete recovery of interferon activity from the column. (3 - - - (3, Protein; • • , interferon; . . . . , NaCI gradient. Fig. 5. Chromatography of mouse immune interferon on Phenyl-Sepharose CL-4B. Seven ml of interferon containing 24 500 units of activity and 77 mg protein, were dialysed against PBS and applied to a Phenyl-Sepharose CL-4B column (o'9 × 5 cm). The column was washed with Io ml PBS and then with 20 ml PB (E0. A linear concentration gradient of ethylene glycol (E~) was developed by mixing 15 ml PB and I5 ml 75 ~ (v/v) ethylene glycol in PB. The recovery of interferon activity was complete. O - - - ©, Protein; • • , interferon; - - - - - , ethylene glycol gradient. preparation to pH 5 for almost 24 h did not result in any decrease in its activity; however, this did occur at still lower pH values. It is also of interest to observe here that the interferon activity was stable at pH values as high as pH 9The survival of some of the activity at pH 3 and pH 2 could indicate the presence of a small subpopulation of immune interferon with pH-stability characteristics of virus induced mouse interferon. In order to test this notion, a 5 ml sample of a mouse immune interferon preparation having 2500 units per ml, was exposed to pH 2 for 24 h; its titre decreased to 300 units per ml. Chromatography of this remaining interferon activity on an AN-Gel 202 column revealed the presence of all the fractions (not shown) observed on chromatography of an untreated interferon preparation (Fig. 2). Thus, no selective survival of a particular chromatographic component can be postulated. Moreover, the remaining activity after pH 2 treatment could not be neutralized by an antiserum raised against interferon induced by Newcastle disease virus (titres of 40000 against ~o units of interferon) even at a dilution as low as I:2o. Therefore, both the chromatographic and immunological evidence is consistent with the notion that the residual interferon activity has the properties of immune interferon. Chromatography on Blue Sepharose CL-6B To advance the physico-chemical characterization of mouse immune interferon by means of chromatography, an additional sorbent was employed, Blue Sepharose CL-6B. The chromophore of this sorbent, Cibacron Blue F3GA, is known to interact with several interferons and, in particular, with mouse L-cell interferon (De Maeyer-Guignard & De Maeyer, Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 2000 1000 E 778 J. W I E T Z E R B I N AND OTHERS I976; Jankowski et al. t976; Cesario et al. I976). The chromatographic behaviour of mouse immune interferon on Blue Sepharose CL-6B is shown in Fig. 4. Again, a conspicuous heterogeneity of the interferon preparation can be observed. This is an expected result in view of the apparent heterogeneity of mouse immune interferon on Con A-Sepharose and Affi-Gel 202 columns. Unexpectedly, however, a significant portion of activity was not retained on this column. A study of interaction of this interferon fraction with immobilized polyribonucleotides may prove to be of a particular interest; it has been postulated (De Maeyer-Guignard et al. I977) that the binding sites for Cibacron Blue F3GA and polyribonucleotides are identical. Chromatography on Phenyl-Sepharose CL-4B The potential of mouse immune interferon to enter into hydrophobic interactions was indicated by its chromatographic behaviour on Affi-Gel 2o2 and Blue Sepharose CL-6B. Nevertheless, one could argue that electrostatic interactions rather than hydrophobic ones, between the interferon molecule and those tigands, are of primary significance. In order then to establish unequivocally that the immune interferon has a potential for hydrophobic interactions as well, it was necessary to select a hydrophobic sorbent which does not carry any charged groups. To this end, a mouse immune interferon preparation was chromatographed on a Phenyl-Sepharose CL-4B column. The results of this experiment are shown in Fig. 5. It is immediately clear that the mouse immune interferon is strongly and quite selectively retained on this sorbent. Thus, it appears that mouse immune interferon displays a significanl apparent hydrophobicity just as other mammalian interferons (Davey et al. I976a). Partial purification by chromatographic procedures Figure 6 illustrates the partial purification of mouse immune interferon by sequential chromatography on Affi-Gel 2o2 followed by Phenyl-Sepharose CL-4B. An interferon preparation was applied on an Affi-Gel 202 column in o'o5 M-sodium acetate, pH 5"0. The column was briefly washed with PB (o.o2 M-sodium phosphate, pH 7"4; Ex). The interferon activity was then eluted and transferred directly on to a Phenyl-Sepharose CL-4B column with o'25 M-sodium chloride in PB (E2). After the transfer, the columns were disconnected and the Phenyl-Sepharose CL-4B column was equilibrated with PB (E3). The displacement of interferon activity was then effected with 50 ~o (v/v) ethylene glycol in PB (E4). Recovery of activity from both columns was about 93 ~ . A significant portion of activity, about 34 of the applied amount, was recovered (in this particular experiment) in the breakthrough fractions from Affi-Gel 2o2 column. The leakage of activity from this column varies from preparation to preparation. The remaining 66Yo of activity, transferred on PhenylSepharose CL-4B column, was recovered with a significant, IOO-to 2oo-fold purification. DISCUSSION The rigorous physico-chemical characterization of mouse immune interferon must await its complete purification. The recent interest in immune (Type II) interferon and, in particular, its immunosuppressive effects on antibody production, has prompted several laboratories to attempt its partial purification (Sonnenfeld et al. 1977; Mizrahy et aL 1978). In our own purification attempt, we have chosen to probe first some physico-chemical properties of this interferon before an advanced purification procedure could be elaborated. As a result of the use of several sorbents of diverse chromatographic behaviour, some structural features of mouse immune interferon have been revealed. The results of chromatography of this interferon on Con A-agarose (Fig. l) suggest that a significant portion of interferon molecules is glycosylated. To what extent the remainder Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 Purification of mouse immune interferon 1-0 I A I I I 779 I 12ooo Et~,~,E2 1000 0-5 i 6' i 0 0 E E g BI I I I l I -~ 3000 "~ 1.0 0-5 /1 0 60 70 80 90 100 Fraction number 1'°°° 110 120 Fig. 6. Chromatography of mouse immune interferon on Affi-Gel 2oz followed by PhenylSepharose CL-4B. O n e m l of an interferon preparation containing I5OOO units of activity and 30 m g protein were applied on an Afli-Gel 202 c o l u m n (5 ml bed volume). The column was washed with o'o5 M-Na acetate, p H 5, then with PB ( E 0 and linked up with a Phenyl-Sepharose CL-4B column 0 " 5 ml bed volume). The interferon was transferred from the first column to the second one with 0'25 M-NaCI in PB (E0. The columns were then disconnected and the Phenyl-Sepharose CL-4B c o l u m n was washed with PB (E3). Finally, the interferon activity was displaced with 5o % ethylene glycol in PB (E4). A, Affi-Gel 2o2 c o l u m n ; B, Phenyl-Sepharose CL-4B column. © - - - ©, Protein; • • , interferon. of interferon molecules not recognized by this tectin are glycosylated, remains an open question. It has been reported that human lymphocytes stimulated in the presence of tunicamycin led to the synthesis of aglycosylated interferon (Mizrahy et al. I978). In mouse spleen cell cultures similar doses of antibiotic completely inhibited interferon production. At lower doses, some interferon was obtained but it behaved as standard preparations. The results of chromatography of interferon on Affi-Gel 202 (Fig. 2) again underscore its molecular heterogeneity. The selective retention of interferon on this ligand may be a result of both electrostatic and hydrophobic interactions as was previously suggested for mouse L-cell interferon (Davey et al. I976b). The affinity of mouse immune interferon (Fig. 4) for Cibacron Blue F3GA (the chromophore of Blue Sepharose CL-6B) may indicate the presence of a polynucleotide binding site on this interferon. Similar observations made for mouse L-cell interferon in the past have led to such a conclusion (De Maeyer-Guignard et al. ~977). This facet of the structure of the immune interferon molecule has been investigated (Wietzerbin et al. ~978 b). Downloaded from www.microbiologyresearch.org by IP: 54.198.184.77 On: Mon, 18 Jul 2016 18:22:25 780 J. W I E T Z E R B I N AND OTHERS From its chromatographic behaviour on Phenyl-Sepharose CL-4B (Fig. 5), mouse immune interferon appears to be hydrophobic like other mammalian interferons (Davey et al. I976a). The purification of mouse immune interferon on individual columns of Affi-Gel 202 and Phenyl-Sepharose CL-4B is considerable. Moreover, these sorbents can be linked in sequential chromatography (Fig. 6) due to the compatibility of solvent elution conditions on Affi-Gel 202 with the solvent adsorption conditions on Phenyl-Sepharose CL-4B. As a result, a significant purification, about 2oo-fold, has been achieved. A salient feature of the chromatography of mouse immune interferon on all these affinity sorbents is the nearly complete or complete recovery of interferon activity. This is not a readily accomplished feat by more traditional means. The loss of activity during a purification procedure, if significant, can preclude any reliable statements about the heterogeneity of the purified preparation; it is always possible that a particular component is lost. Therefore, the purification by more classical means, which results in low purification factors and poor overall recovery of activity, may be found unsatisfactory. This is especially important in the case of immune interferons since their crude preparations have relatively low titres of activity and apparently display significant molecular heterogeneity. The resolving power of the affinity sorbents utilized in this investigation must still await a more complete evaluation until the chromatographic behaviour of some other lymphokines (MIF, lymphotoxin, etc.) known to be present in crude immune interferon preparations, is fully explored. One of us (J. W.) wishes to express her gratitude to Dr W.A. Carter, Director of Medical Viral Oncology, Roswell Park Memorial Institute, for hospitality during her visit (July I977). This work was supported by Direction G6n6rale de la Recherche Scientifique et Technique research grant 77.7. I375 and INSERM grant CRL 78.4.o82I. Note added in proof. Since this paper was submitted, a paper by L. C. Osborne, J. A. Georgiades & H. M. Johnson, concerning the production and partial purification of mouse interferon, has appeared in Infection and lmmunity (I979) 23, 80-86. REFERENCES BAROT-CIORBARU, R., WIETZERBIN, J., PETIT, J. F., CHEDID, L., FALCOFF, E. & LEDERER, E. (I978). Induction of interferon synthesis in mice by fractions from Nocardia. Infection and Immunity x9, 353-356. BARTFELI), H. & VILCglC, J. (I975). Immunologically specific production of interferon in cultures of rabbit blood lymphocytes: association with in vitro tests for cell-mediated immunity. Infection and Immunity x2, 1112-Iii 5. B6HLEN, V., STEIN, S., DAIRMAN,W. & UDENERIENO,S. 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