- Email: [email protected]
[43] Methods and procedures for experiments with mixed cell populations: Transfer of the antiviral state induced by interferon
[43]
TP,ANSFEg OF IF-INDUCED ANTIVIRALSTATE
353
[43] M e t h o d s a n d P r o c e d u r e s f o r E x p e r i m e n t s w i t h M i x e d Cell Populations: T r a n s f e r of t h e Antiviral State Induced by Interferon B y J. E D W I N B L A L O C K
We have shown that induction of the antiviral state is probably mediated by secondary messenger molecules that are induced at the cell membrane by interferon, signal the nucleus to produce antiviral protein(s), and by cell-to-cell transfer induce viral resistance in adjacent cells. ~ These findings resulted from coupling two previous observations. First, many cell types communicate among themselves by gap junctional transfer of metabolites and small control molecules2 Second, interferon action shows species preference. 4 We reasoned that, if induction of the antiviral state was mediated by small secondary molecules, these by gap junctional transfer might influence adjacent cells. This was tested by coculturing two different cell species in the presence of interferon to which only one cell species was sensitive and determining whether the other became resistant to virus infection. It was found that under these conditions the cell species not directly sensitive to interferon became resistant to virus infection. The cell-to-cell transfer of viral resistance was initiated by interferon, was rapid, and required ongoing RNA synthesis in the recipient cellY This process represents a major amplification system for interferon action? Assay of Interferon-Induced Transfer of Viral Resistance Freshly trypsinized mouse L cells and human amnion (WISH) cells in Eagle's minimal essential medium supplemented with 2% fetal calf serum were cocultured in a 1 : 1 ratio in Micro Test II tissue culture plates (Falcon Plastics, Oxnard, California). The total number of cells in each well (about 28 mm 2) was 1.5 × 105 in 0.15 ml, which is comprised of 7.5 × 104 cells/weU of each of the two cell species. Controls consisted of an equivalent number of either cell species alone. Various concentrations of mouse virus-type interferon in 0.1 ml or an equivalent volume of medium were J. E. Blalock and S. Baron, Nature (London) 269, 422 (1977). 2 j. E. Blalock and S. Baron, J. Gen ViroL 42, 363 (1979).
3 W. R. Lowensteinin "Cell Membranes: Biochemistry,Cell Biologyand Pathology"(G. Weissmannand R. Claiborne, eds.), p. 105. HP Publ., New York, 1975. 4 R. Z. Lockhart, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), p. 11. North-Holland, Amsterdam, 1973. J. E. Blalock,Proc. Soc. Exp. Biol. Med. 162, 80 (1979).
METHODS IN ENZYMOLOGY,VOL. 79
Copyright © 1981by Academic Press. Inc. All rights of reproductionin any form reserved. ISBN 0-12-181979-5
354
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
[43]
TABLE I TRANSFER OF INTERFERON-INDUCED VIRAL RESISTANCE BETWEEN CELLS OF VARIOUS SPECIES
Cells~ Donor
Recipient
Interferon (300 units/ml)
Loglo inhibition of VSV yield from recipient cellsb
WISH RK-13
BHK-21 WISH CE Vero WISH Vero MDCK
Human Rabbit Mouse Mouse Mouse Rabbit Human
0.7 0.8
L
L L RK- 13 WISH
0.8
0.8 1.2 None None
WISH, human amnion cells; RK-13, rabbit kidney cells; L, mouse fibroblasts; BHK-21, baby hamster kidney cells; CE, secondary chick embryo cells; Vero, African green monkey kidney cells; MDCK, canine kidney cells. b Log10 inhibition was calculated according to the formula in the text. added, and cultures were incubated overnight at 37 ° in a 4% CO2 atmosphere. Units o f interferon are expressed in terms o f the N I H reference m o u s e interferon. Supernatant fluids were decanted, and each well was infected with 103 plaque-forming units (PFU) o f poliovirus in 0.1 ml. Virus yields f r o m pooled triplicate cultures were d e t e r m i n e d a p p r o x i m a t e l y 24 hr later by a slightly modified microplaque assay in which 0.5% methylcellulose (1500 centipoise) was substituted for c a r b o x y m e t h y l cellulose. ~ Results o f a typical experiment are shown in Fig. 1. Poliovirus did not replicate in m o u s e L cells, and m o u s e interferon did not alter the yield of poliovirus from the h u m a n W I S H cells. Coculturing m o u s e L cells and h u m a n W I S H cells in the absence of interferon did not affect the yield of poliovirus from the W I S H cells, whereas m o u s e interferon caused a dose-dependent reduction in poliovirus yields from h u m a n W I S H cells in the cocultures. The log10 inhibition of virus yields in the cocultures in the presence of m o u s e interferon is taken as a m e a s u r e of the transfer of interferon-induced viral resistance. Choice o f Cell Species
The p r o t o t y p e transfer s y s t e m consists of m o u s e interferon, m o u s e L cells, and h u m a n W I S H cells, but other cell combinations and their respective interferons m a y be substituted. T Table I shows a compilation of 6 j. B. Campbell, J. Grunberger, M. A. Kochman, and S. L. White, Can. J, Microbiol. 21, 1247 (1975). 7 T. K. Hughes, J. E. Blalock, and S. Baron, Arch. Virol. 58, 77 (1978).
[43]
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
355
6.0
0 "r"
K
I",,,
"v5,0
.o >,.
q~ I,,..
._o 0
n
o
÷
4.~
< I.C
J,
Nine
L Cells
1.6 Lo(JIoMouse
zo
•
3.0
Interferon (U/ml)
FIG. 1. Interferon-induced transfer of viral resistance from mouse L cells to human amnion (WISH) cells. *Poliovirus does not replicate in mouse cells. Each point represents the mean yield and the 95% confidence limits.
356
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
[43]
the cell combinations we have tested and the results obtained. The conditions of the assay for these combinations are the same as mentioned above except that vesicular stomatitis virus (VSV) was substituted for poliovirus. The only apparent prerequisites to choosing the species of cells employed are that the donor cell species be sensitive to its respective interferon while the recipient cell species is insensitive to that same interferon. Additionally one must have a recipient cell species that is permissive for an interferon-sensitive virus. By the appropriate choice of cell types transfer between cells within a species may be also observed. For instance, human amnion WISH and human amnion U cells differ in their kinetic response to human fibroblast interferon. WISH cells respond about 1-2 hr faster than U cells. When U cells and WISH cells are cocultured, the U cells now respond at the same rate as the WISH cells, s Also, mouse L cell clones can be obtained that differ 10-fold in their sensitivity and 100-fold in the maximum protection afforded by mouse fibroblast interferon. By cocultivation of a highresponder and a low-responder clone, the response of the low-responder clone approaches that of the high response) These results are interpreted to result from transfer of viral resistance within cells of the same species. In general, transfer within a species is more efficient than between species. 8
Choice of Virus Theoretically any interferon-sensitive virus may be employed as the challenge so long as it replicates in the recipient cells. In practice we have used poliovirus, vesicular stomatitis virus, Sindbis virus, and vaccinia virus. All these viruses under the proper conditions can be used to demonstrate transferred resistance. Care must be taken when virus types are used that at higher multiplicities of infection (MOI) can overcome directly induced viral resistance, since transferred resistance is also overwhelmed. 2 For example, at an input MOI of 0.1 PFU/cell vaccinia virus yield was inhibited by mouse interferon (7500 units/ml) in L cells alone (98.5% reduction) as well as in a 1 : 1 L cell and WISH cell mixture (93.3% reduction). At an input MOI of 1 PFU/celI, vaccinia virus was not sensitive to mouse interferon in L cells alone and transfer of resistance to WISH cells was not observed. ~ The MOI effect is one of many characteristics that show that transferred resistance is very similar if not identical to the directly induced antiviral state. Transferred viral resistance seems to parallel virus sensitivity to the antiviral state, which is directly induced by interferon. In most instances, 8 j. E. Blalock and G. J. Stanton,J. Gen. Virol, 41,325 (1978).
[43]
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
357
one would want to work with the most sensitive virus type possible. Poliovirus is convenient to use since it is relatively sensitive to interferon and has the added advantage that it grows only in human cells. In the mouse interferon, mouse L cell, and human WISH cell system, this allows for the direct demonstration that resistance develops in the human cells. However, we commonly employ VSV as a challenge, since in our experience it is more sensitive to interferon than is poliovirus. When VSV is used as a challenge there is in theory the complication that it grows in both mouse and human cells. In practice, this does not affect the results, since for all intents and purposes VSV does not replicate in mouse L cells at the concentrations of mouse interferon employed. The VSV yield from mouse interferon-treated L cells is usually 2-3 log10 below the WISH cell yield when 10-100 units of mouse interferon are used. When VSV is the challenge virus, the amount of viral resistance that is transferred to WISH cells is based on an expected virus yield. This yield usually gives equivalent results whether it is calculated from the percentage of WISH cells in a L cell-WISH cell mix without interferon or the virus yield from an equivalent number of WISH cells with mouse interferon. Virus yields from interferon-treated or nontreated human WISH cells are very similar to the yield from non-interferon-treated L cells, and the virus yield from mouse interferon-treated L cells is negligible in comparison. The log10 inhibition of VSV yield from a transfer experiment that employed a 1 : 1 ratio of L cells to WISH cells would employ the following formula: -
log10 virus yield from cell mix with interferon 1/2 virus from cell mix without interferon
A comparison of the results obtained when VSV or poliovirus are used as a challenge in a transfer experiment are shown in Fig. 2. The conditions are as described in the assay procedure section. Interferon-induced transfer of resistance from L cells to WISH cells is demonstrable with both virus types. While a similar degree of protection of WISH cells is eventually achieved with both poliovirus and VSV, viral resistance is detected with less interferon when VSV is the challenge. Therefore, VSV is a more sensitive virus for the detection of transferred viral resistance. As expected, VSV was more sensitive than poliovirus to human interferon treatment of WISH cells alone (data not shown).
Choice of Interferon In the mouse system we have found that both virus type, and immune type interferon9 will effect the transfer of viral resistance from mouse L J. E. Blalock, J. Georgiades, and H. M. Johnson, J. Immunol. 122, 1018 (1979).
358
[43]
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
2.0
• L * WISH Cells o WISH Cells
~O (/)
2
"* e " ~. ~"
/
=v*
?-/ VSV / o
/
o _J
/ /
/ /
/ Poliovirus or VSV p='L':"
None
0
0
1.0 2.0 LogloMouse Interferon (U/ml)
0
3.0
FIG. 2. Interferon-induced transfer of viral resistance: comparison of vesicular stomatitis virus (VSV) and poliovirus yield reduction. Log10 inhibition represents reduction in virus yield from WISH cells.
cells to human WISH cells. On a per unit basis, mouse immune-type interferon is about 2.5-10 times more efficient than virus type interferon at causing the transfer of viral resistance (Table II). All interferons used in Table I were virus type. Since both virus-type and immune-type inter-
[43]
359
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
TABLE II COMPARISON OF TRANSFER OF ANTIVIRAL RESISTANCE WITH IMMUNE-TYPE AND VIRus-TYPE INTERFERONS
Ration of viral inhibition transferred to WISH cells: viral inhibition on L cells Interferon~' (units/ml) 3 10 33 100
Immune-type interferon 0.56 0.29 0.10 0.10
Virus-type interferon
Ratio of interferon transfer activity, immune-type:virus-type
0.056 0.063 0.040 0.025
10 4.6 2.5 4
Interferons were produced as described by Blalock et al, 9 and assayed by VSV yield reduction as described in the text. b Ratio = antilogl0 (logt0 viral inhibition transferred to WISH cells-logt0 viral inhibition on L cells). Log10inhibition transferred to WISH cells was calculated according to the formula in the section on choice of virus. Log~0 inhibition on L cells was calculated by substituting the virus yields from L cells alone into the same formula. ferons seem to elicit the same or similar sequences o f events in the recipient cell during transfer, 9 the choice o f interferon type does not seem crucial. We do not, however, have information on h u m a n leukocyte-type interferon. Comments The transfer o f interferon-induced viral resistance is very dependent on the cell density. As expected the cell-to-cell transfer process does not o c c u r when cells are not in c o n t a c t ) Thus it is imperative that transfer experiments be performed at cell confluency or above. In microtiter wells (28 mm 2) confluency occurs at about 1 × l05 cells/well. Cell concentrations a b o v e confluency show greater transferred resistance. We usually employ 1.5 x 105 cells/well, but also get very good results at 2.25 × 10~ cells/well. Transfer o f viral resistance also depends on the ratio o f donor to recipient cells at a given cell density. For transfer from L to W I S H cells, the minimum ratio that shows transfer is about 1 L cell to 2 W I S H cells. For simplicity we usually employ a ratio o f 1 to 1. Greater degrees of transfer, if desired, can be obtained by increasing the ratio o f L to W I S H cells. Based on the n u m b e r o f plaques that we usually c o u n t in transfer experiments a 0.5 log10 reduction in virus yield is significant at a p < 0.05.
TP,ANSFEg OF IF-INDUCED ANTIVIRALSTATE
353
[43] M e t h o d s a n d P r o c e d u r e s f o r E x p e r i m e n t s w i t h M i x e d Cell Populations: T r a n s f e r of t h e Antiviral State Induced by Interferon B y J. E D W I N B L A L O C K
We have shown that induction of the antiviral state is probably mediated by secondary messenger molecules that are induced at the cell membrane by interferon, signal the nucleus to produce antiviral protein(s), and by cell-to-cell transfer induce viral resistance in adjacent cells. ~ These findings resulted from coupling two previous observations. First, many cell types communicate among themselves by gap junctional transfer of metabolites and small control molecules2 Second, interferon action shows species preference. 4 We reasoned that, if induction of the antiviral state was mediated by small secondary molecules, these by gap junctional transfer might influence adjacent cells. This was tested by coculturing two different cell species in the presence of interferon to which only one cell species was sensitive and determining whether the other became resistant to virus infection. It was found that under these conditions the cell species not directly sensitive to interferon became resistant to virus infection. The cell-to-cell transfer of viral resistance was initiated by interferon, was rapid, and required ongoing RNA synthesis in the recipient cellY This process represents a major amplification system for interferon action? Assay of Interferon-Induced Transfer of Viral Resistance Freshly trypsinized mouse L cells and human amnion (WISH) cells in Eagle's minimal essential medium supplemented with 2% fetal calf serum were cocultured in a 1 : 1 ratio in Micro Test II tissue culture plates (Falcon Plastics, Oxnard, California). The total number of cells in each well (about 28 mm 2) was 1.5 × 105 in 0.15 ml, which is comprised of 7.5 × 104 cells/weU of each of the two cell species. Controls consisted of an equivalent number of either cell species alone. Various concentrations of mouse virus-type interferon in 0.1 ml or an equivalent volume of medium were J. E. Blalock and S. Baron, Nature (London) 269, 422 (1977). 2 j. E. Blalock and S. Baron, J. Gen ViroL 42, 363 (1979).
3 W. R. Lowensteinin "Cell Membranes: Biochemistry,Cell Biologyand Pathology"(G. Weissmannand R. Claiborne, eds.), p. 105. HP Publ., New York, 1975. 4 R. Z. Lockhart, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), p. 11. North-Holland, Amsterdam, 1973. J. E. Blalock,Proc. Soc. Exp. Biol. Med. 162, 80 (1979).
METHODS IN ENZYMOLOGY,VOL. 79
Copyright © 1981by Academic Press. Inc. All rights of reproductionin any form reserved. ISBN 0-12-181979-5
354
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
[43]
TABLE I TRANSFER OF INTERFERON-INDUCED VIRAL RESISTANCE BETWEEN CELLS OF VARIOUS SPECIES
Cells~ Donor
Recipient
Interferon (300 units/ml)
Loglo inhibition of VSV yield from recipient cellsb
WISH RK-13
BHK-21 WISH CE Vero WISH Vero MDCK
Human Rabbit Mouse Mouse Mouse Rabbit Human
0.7 0.8
L
L L RK- 13 WISH
0.8
0.8 1.2 None None
WISH, human amnion cells; RK-13, rabbit kidney cells; L, mouse fibroblasts; BHK-21, baby hamster kidney cells; CE, secondary chick embryo cells; Vero, African green monkey kidney cells; MDCK, canine kidney cells. b Log10 inhibition was calculated according to the formula in the text. added, and cultures were incubated overnight at 37 ° in a 4% CO2 atmosphere. Units o f interferon are expressed in terms o f the N I H reference m o u s e interferon. Supernatant fluids were decanted, and each well was infected with 103 plaque-forming units (PFU) o f poliovirus in 0.1 ml. Virus yields f r o m pooled triplicate cultures were d e t e r m i n e d a p p r o x i m a t e l y 24 hr later by a slightly modified microplaque assay in which 0.5% methylcellulose (1500 centipoise) was substituted for c a r b o x y m e t h y l cellulose. ~ Results o f a typical experiment are shown in Fig. 1. Poliovirus did not replicate in m o u s e L cells, and m o u s e interferon did not alter the yield of poliovirus from the h u m a n W I S H cells. Coculturing m o u s e L cells and h u m a n W I S H cells in the absence of interferon did not affect the yield of poliovirus from the W I S H cells, whereas m o u s e interferon caused a dose-dependent reduction in poliovirus yields from h u m a n W I S H cells in the cocultures. The log10 inhibition of virus yields in the cocultures in the presence of m o u s e interferon is taken as a m e a s u r e of the transfer of interferon-induced viral resistance. Choice o f Cell Species
The p r o t o t y p e transfer s y s t e m consists of m o u s e interferon, m o u s e L cells, and h u m a n W I S H cells, but other cell combinations and their respective interferons m a y be substituted. T Table I shows a compilation of 6 j. B. Campbell, J. Grunberger, M. A. Kochman, and S. L. White, Can. J, Microbiol. 21, 1247 (1975). 7 T. K. Hughes, J. E. Blalock, and S. Baron, Arch. Virol. 58, 77 (1978).
[43]
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
355
6.0
0 "r"
K
I",,,
"v5,0
.o >,.
q~ I,,..
._o 0
n
o
÷
4.~
< I.C
J,
Nine
L Cells
1.6 Lo(JIoMouse
zo
•
3.0
Interferon (U/ml)
FIG. 1. Interferon-induced transfer of viral resistance from mouse L cells to human amnion (WISH) cells. *Poliovirus does not replicate in mouse cells. Each point represents the mean yield and the 95% confidence limits.
356
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
[43]
the cell combinations we have tested and the results obtained. The conditions of the assay for these combinations are the same as mentioned above except that vesicular stomatitis virus (VSV) was substituted for poliovirus. The only apparent prerequisites to choosing the species of cells employed are that the donor cell species be sensitive to its respective interferon while the recipient cell species is insensitive to that same interferon. Additionally one must have a recipient cell species that is permissive for an interferon-sensitive virus. By the appropriate choice of cell types transfer between cells within a species may be also observed. For instance, human amnion WISH and human amnion U cells differ in their kinetic response to human fibroblast interferon. WISH cells respond about 1-2 hr faster than U cells. When U cells and WISH cells are cocultured, the U cells now respond at the same rate as the WISH cells, s Also, mouse L cell clones can be obtained that differ 10-fold in their sensitivity and 100-fold in the maximum protection afforded by mouse fibroblast interferon. By cocultivation of a highresponder and a low-responder clone, the response of the low-responder clone approaches that of the high response) These results are interpreted to result from transfer of viral resistance within cells of the same species. In general, transfer within a species is more efficient than between species. 8
Choice of Virus Theoretically any interferon-sensitive virus may be employed as the challenge so long as it replicates in the recipient cells. In practice we have used poliovirus, vesicular stomatitis virus, Sindbis virus, and vaccinia virus. All these viruses under the proper conditions can be used to demonstrate transferred resistance. Care must be taken when virus types are used that at higher multiplicities of infection (MOI) can overcome directly induced viral resistance, since transferred resistance is also overwhelmed. 2 For example, at an input MOI of 0.1 PFU/cell vaccinia virus yield was inhibited by mouse interferon (7500 units/ml) in L cells alone (98.5% reduction) as well as in a 1 : 1 L cell and WISH cell mixture (93.3% reduction). At an input MOI of 1 PFU/celI, vaccinia virus was not sensitive to mouse interferon in L cells alone and transfer of resistance to WISH cells was not observed. ~ The MOI effect is one of many characteristics that show that transferred resistance is very similar if not identical to the directly induced antiviral state. Transferred viral resistance seems to parallel virus sensitivity to the antiviral state, which is directly induced by interferon. In most instances, 8 j. E. Blalock and G. J. Stanton,J. Gen. Virol, 41,325 (1978).
[43]
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
357
one would want to work with the most sensitive virus type possible. Poliovirus is convenient to use since it is relatively sensitive to interferon and has the added advantage that it grows only in human cells. In the mouse interferon, mouse L cell, and human WISH cell system, this allows for the direct demonstration that resistance develops in the human cells. However, we commonly employ VSV as a challenge, since in our experience it is more sensitive to interferon than is poliovirus. When VSV is used as a challenge there is in theory the complication that it grows in both mouse and human cells. In practice, this does not affect the results, since for all intents and purposes VSV does not replicate in mouse L cells at the concentrations of mouse interferon employed. The VSV yield from mouse interferon-treated L cells is usually 2-3 log10 below the WISH cell yield when 10-100 units of mouse interferon are used. When VSV is the challenge virus, the amount of viral resistance that is transferred to WISH cells is based on an expected virus yield. This yield usually gives equivalent results whether it is calculated from the percentage of WISH cells in a L cell-WISH cell mix without interferon or the virus yield from an equivalent number of WISH cells with mouse interferon. Virus yields from interferon-treated or nontreated human WISH cells are very similar to the yield from non-interferon-treated L cells, and the virus yield from mouse interferon-treated L cells is negligible in comparison. The log10 inhibition of VSV yield from a transfer experiment that employed a 1 : 1 ratio of L cells to WISH cells would employ the following formula: -
log10 virus yield from cell mix with interferon 1/2 virus from cell mix without interferon
A comparison of the results obtained when VSV or poliovirus are used as a challenge in a transfer experiment are shown in Fig. 2. The conditions are as described in the assay procedure section. Interferon-induced transfer of resistance from L cells to WISH cells is demonstrable with both virus types. While a similar degree of protection of WISH cells is eventually achieved with both poliovirus and VSV, viral resistance is detected with less interferon when VSV is the challenge. Therefore, VSV is a more sensitive virus for the detection of transferred viral resistance. As expected, VSV was more sensitive than poliovirus to human interferon treatment of WISH cells alone (data not shown).
Choice of Interferon In the mouse system we have found that both virus type, and immune type interferon9 will effect the transfer of viral resistance from mouse L J. E. Blalock, J. Georgiades, and H. M. Johnson, J. Immunol. 122, 1018 (1979).
358
[43]
INTERFERON ACTIVITY AT THE CELLULAR LEVEL
2.0
• L * WISH Cells o WISH Cells
~O (/)
2
"* e " ~. ~"
/
=v*
?-/ VSV / o
/
o _J
/ /
/ /
/ Poliovirus or VSV p='L':"
None
0
0
1.0 2.0 LogloMouse Interferon (U/ml)
0
3.0
FIG. 2. Interferon-induced transfer of viral resistance: comparison of vesicular stomatitis virus (VSV) and poliovirus yield reduction. Log10 inhibition represents reduction in virus yield from WISH cells.
cells to human WISH cells. On a per unit basis, mouse immune-type interferon is about 2.5-10 times more efficient than virus type interferon at causing the transfer of viral resistance (Table II). All interferons used in Table I were virus type. Since both virus-type and immune-type inter-
[43]
359
TRANSFER OF IF-INDUCED ANTIVIRAL STATE
TABLE II COMPARISON OF TRANSFER OF ANTIVIRAL RESISTANCE WITH IMMUNE-TYPE AND VIRus-TYPE INTERFERONS
Ration of viral inhibition transferred to WISH cells: viral inhibition on L cells Interferon~' (units/ml) 3 10 33 100
Immune-type interferon 0.56 0.29 0.10 0.10
Virus-type interferon
Ratio of interferon transfer activity, immune-type:virus-type
0.056 0.063 0.040 0.025
10 4.6 2.5 4
Interferons were produced as described by Blalock et al, 9 and assayed by VSV yield reduction as described in the text. b Ratio = antilogl0 (logt0 viral inhibition transferred to WISH cells-logt0 viral inhibition on L cells). Log10inhibition transferred to WISH cells was calculated according to the formula in the section on choice of virus. Log~0 inhibition on L cells was calculated by substituting the virus yields from L cells alone into the same formula. ferons seem to elicit the same or similar sequences o f events in the recipient cell during transfer, 9 the choice o f interferon type does not seem crucial. We do not, however, have information on h u m a n leukocyte-type interferon. Comments The transfer o f interferon-induced viral resistance is very dependent on the cell density. As expected the cell-to-cell transfer process does not o c c u r when cells are not in c o n t a c t ) Thus it is imperative that transfer experiments be performed at cell confluency or above. In microtiter wells (28 mm 2) confluency occurs at about 1 × l05 cells/well. Cell concentrations a b o v e confluency show greater transferred resistance. We usually employ 1.5 x 105 cells/well, but also get very good results at 2.25 × 10~ cells/well. Transfer o f viral resistance also depends on the ratio o f donor to recipient cells at a given cell density. For transfer from L to W I S H cells, the minimum ratio that shows transfer is about 1 L cell to 2 W I S H cells. For simplicity we usually employ a ratio o f 1 to 1. Greater degrees of transfer, if desired, can be obtained by increasing the ratio o f L to W I S H cells. Based on the n u m b e r o f plaques that we usually c o u n t in transfer experiments a 0.5 log10 reduction in virus yield is significant at a p < 0.05.