THE JOURNAL
OF
ALLERGY AND
CLINICAL
IMMUNOLOGY NUMBER
VOLUME. 63
2
Editorials
‘Transfer of cellular transfer factor Charles H. Kirkpatrick,
immunity
M.D. &dzesdu, Md.
Since the original reports by Lawrence and associates’, 2 that it was possible to transfer delayed cutaneous hypersensitivity from sensitive human donors to insensitive recipients with lysates and dialysates of blood leukocytes, there has been controversy over the specificity, the mechanism, and even the existence of the phenomenon. The arguments against transfer factor derived from several sources. First, there was difficulty in confirming the reports that similar transfers could be achieved in guinea pigs.3* 4 Second, it was difficult to envision mechanisms by which molecules with molecular weights of 10,000 daltons or less could transfer the information required for reactivity against a vast repertory of antigenic determinants. Third, most of the experiments in man studied transfer of reactivity to ubiquitous microbial antigens such as tuberculin, streptococcal proteins, diphtheria toxoid, Can&&, etc., and it could be argued that the apparent transfers were due to enhancing or adjuvant-like activities of the dialysates in previously sensitized or “primed” recipients. A fourth argument reasoned that transfer factor contained minute amounts of “super-antigen” and that the apparently specific transfers were really the results of active immunization All of the arguments against transfer factor cannot be resolved at this time. but with the aid of properly
Reprint requests to: Charles H. Kirkpatrick, I IB- 13, National Institutes of Health,
with
M.D., Bldg. 10, Room Bethesda, MD 20014.
controlled studies in humans and with the recently developed animal models,“-g additional evidence for the phenomenon has accrued. For example, the crucial issue of specificity is unresolved, but recent findings are more compatible with specific than with nonspecific mechanisms. Maurer’O reported that lysates of peripheral blood leukocytes from donors with delayed hypersensitivity to ethylene oxide-treated human serum could transfer hypersensitivity to nonimmune recipients. Zuckerman and co-workers” were able to transfer delayed skin reactivity to keyhole limpet hemocyanin (KLH) from a KLH-sensitive donor to ten of ten nonimmune recipients with dialyzable transfer factor. Their donor was also reactive to purified protein derivative (PPD), and 8 of the 10 recipients also became responsive to this antigen. Two other transfer factor preparations, one from a KLH-negative, PPD-positive donor and the other from a pool of donors with unknown sensitivities, failed to transfer reactivity to KLH, but each of 5 recipients of the PPD-sensitive transfer factor developed reactivity to tuberculin. Recently, Burger and associates’* studied transfer of reactivity to KLH and added an important control. Transfer factor was prepared from the donors both before and after they were sensitized with KLH. The preimmunization transfer factor failed to sensitize any of the six recipients, while the postimmunization transfer factor sensitized 42 of 46 recipients. Other reports provide evidence that a previous “priming” exposure to an antigen is not essential for Vol.
63, No.
2, pp.
71-73
72
Kirkpatrick
passive transfer of delayed cutaneous reactivity to that antigen with transfer factor. Eichberg and co-workersL3 transferred delayed-type reactivity to primates that were housed in germ-free isolators. Kirkpatricki reported that 21 of 23 patients with chronic mucocutaneous candidiasis developed delayed hypersensitivity to Candidu after receiving transfer factor from Candidu-sensitive donors. In contrast, none of 5 candidiasis patients who were treated with transfer factor from Candidu-insensitive donors became reactive to Cundidu. This observation also argues against nonspecific, adjuvant-like activities being the only immunologic effect of transfer factor. In addition, there are no recognized genetic restrictions on this type of passive transfer in humans. Indeed, human transfer factor has been used to sensitize subhuman primates,13 mice,6 and cattle,x, g and murine and bovine transfer factorhas sensitized humans.” Since the original report by Landsteiner and Chase”’ that reactivity to tuberculin or chlorodinitrobenzene (CDNB) could be transferred from sensitive to insensitive guinea pigs with lymphoid cells, contact allergic reactions have been regarded as an expression of the cell-mediated immunity. In contrast to the relative ease of transferrring delayed cutaneous reactivity to intradermal tests with transfer factor, efforts to transfer reactivity to contact allergens have generally been unrewarding. One of the earliest studies was conducted by Brandriss,” who used dialyzable transfer factor from CDNB-sensitive, PPD-sensitive donors. Four of 7 recipients became reactive to PPD, but only 2 recipients developed “weak but definite reactions” to CDNB. Arala-Chaves and PintoI also attempted to transfer sensitivity to CDNB with leukocyte lysates. The experimental design was unique in that the challenge dose of antigen was applied to the skin either 72 hr before or at the same time that the transfer factor was injected. None of the 16 recipients who were challenged with 10 pg of CDNB before injections of transfer factor became reactive. Three of 19 (15.8%) recipients of both 10 and 20 pg challenge tests developed areas of erythema with papules or vesicles; similar reactions did not occur in any of the 21 control subjects challenged with the skin tests alone. Epstein Elsewhere in this issue of the JOURNAL, and Byers report another attempt to transfer contact allergy with dialyzable transfer factor. A central question in their study was the possibility that passive sensitization with transfer factor would occur only in persons who had been “primed” with subsensitizing doses of antigen (beryllium fluoride). The results were compatible with this model. None of the 20 subjects who were repeatedly patch-tested with BeFz
J. ALLERGY
CLIN. IMMUNOL. FEBRUARY 1979
and injected with Be0 became reactive. None of the 14 subjects who received transfer factor but did not have “priming” exposures of antigen became reactive. But 14 of 38 recipients (37%) who were both repeatedly exposed to beryllium salts and given transfer factor from beryllium-sensitive donors developed patch test reactivity to BeF,. Even then the sensitive state appeared to be transitory; 12 recipients were reactive one week after receiving transfer factor but only 4 persons were still reactive one week later. The finding that testing with BeFz at the same time that the transfer factor was injected did not produce positive responses confirms previously reported work in mice.7 In these experiments positive responses could not be obtained for at least 24 hr after injection of the transfer factor. It is also curious that only 3 recipients developed granulamatous responses to intradermal BeO, a reaction that is due to cell-mediated immune mechanisms. As the authors acknowledge, there is an important omission in the experimental design. Ideally, they should have included a group that received transfer factor from beryllium-insensitive donors. If these recipients did not become reactive, the authors would have even stronger evidence both for specificity and for the requirement for priming exposures for transfer of contact allergic reactions with transfer factor. If, on the other hand, recipients of “beryllium-negative” transfer factor also became reactive, they would have good evidence for nonspecific activities. The importance of this point is illustrated by the experience with transfer factor-mediated delayed hypersensitivity in guinea pigs.” In this model, delayed allergic reactions occurred only in animals that had been “primed” with an intradermal dose of antigen, given a mixture of transfer factor and antigen, and then skin-tested. Subsequent experience showed that the system lacked antigenic specificity. I9 In spite of these shortcomings, Epstein and Byers have presented an important and useful model for future experiments. Their report also suggests some important questions about contact allergic reactions and why they appear to be more difficult to transfer than delayed responses to intradermal antigens. Transfer of delayed hypersensitivity with transfer factor does not appear to be under genetic restriction, but the possibility that transfer of contact allergy may be genetically modified has not been investigated. The transitory nature of the reactive state raises a question about the functional life span of the cells involved in mediation of contact allergic reactions. In addition, relatively little is known about the contributions of the various lymphokines to production of the inflammatory responses and epidermal proliferation that ac-
VOLUME NUMBER
Transfer
63 2
company cell-mediated immune reactions in the skin. If different lymphokines are essential for contact reactions than for delayed-type inflammations, then additional studies of the effect of transfer factor on production of individual mediators are warranted. Finally, there has been considerable progress toward isolation and characterization of the active component(s) in transfer factor. As the recent workshop,” several groups presented evidence that the active material was sensitive to proteases and to snake venom phosphodiesterase. The tentative model of the molecule includes a purine (probably inosine or inosine monophosphate), ribose, a phosphodiester bond, and a peptide. A theoretical model has been presented in which the peptide contains antigen-specific sites that are essential for initiation of the sensitive state.“’
Transfer
2. Lawrence HS, Al-Askari S, David J, Franklin EC, Zweiman B: Transfer of immunologic information in humans with dialysates of leucocyte extracts. Tram Assoc Am Physicians 76:84. 1963. 3. Jeter WS, Tremaine layed hypersensitivity pigs with leuctrytic 1954.
MM. Seebohm PM: Passive lransfer deto 2. 4-dinitrochlorobenzene in guinea extracts, Proc Sot Exp Biol Med 86:251,
4. Bloom ER, Chase MW: Transfer of delayed-type hypersensiti,vity: A critical review and experimental study in the &uinea pig, Prog Allergy 10: 151, 1967. 5. Basten A, Croft S, Edwards J: Experimental studies of transfer factor, in Ascher MS, Gottlieb AA, Kirkpatrick CH, editors: Transfer factor. Basic properties and clinical applications, New Yori, 1976, Academic Press, Inc., p 7.5. 6. Petersen EA. Frey JA. Dinowitz M, Rifkind D: Transfer of delayed hypersensitivity to mice with human immune cell extracts, in Ascher MS, Gottlieb AA, Kirkpatrick CH. editors:
Basic
properties
and clinical
8.
9. IO. I I.
12.
13.
14. IS.
I6
17. 18.
IO.
20.
73
applications,
New York. 1975, Academic Press, Inc.. p 387. 7. Rilkind D. Frey JA, Petersen EA, Dinowitz M: Transfer
REFERENCES I. Lawrence HS: The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with disrupted leukocytes, J Clin Invest 34:219, 1955.
factor.
factor
of
delayed hypersensitivity in mice to microbial antigens with dialyzable transfer factor, Infect Immun 16:258. 1977. Klesius PH. Fudenberg HH: Bovine transfer factor: In vivo transfer of cell-mediated immunity to cattle with alcohol precipitates, Clin Immunol Immunopathol 8:238, 1977. Khan A. Kirkpatrick CH, Hill NO, editors: Immune regulators in transfer factor, New York, Academic Press, Inc. (In press.) Maurer PH: Immunologic studies vvith ethylene oxide-treated human serum, J Exp Med 113: 1029. 1961. Zuckerman KS. Neidhart JA, Balcerzak SP, LoBuglio AF: Immunologic speciticity of transfer factor. J Clin Invest 54:947, 1974. Burger DR. Vandenbark AA, Finke P, Vetto RM: De nova appearance of KLH transfer factor following immunization. Cell Immunol 29:410, 1977. Eichberg JW, Steele RW, Kalter SS, Kniker WT. Heberling RL, Eller JJ, Rodriguez AR: Cellular immunity in gnotobiotic primates induced by transfer factor. Cell Immunol 26: 114, 1976. Kirkpatrick CH: Transfer of delayed cutaneous hypersensitivity with transfer factor, Cell Immunol 41:62, 1978. Landsteiner K, Chase MW: Experiments on transfer of cutaneous sensitivity to simple chemicals, Proc Sot Exp Biol Med 49~688, 1942. contact hypersensitivity in Brandriss, MW: Attempt tmnSfer man with dialysate of peripheral leukocytes, J Clin Invest 47:21X!. 1968. Arala-Chaves MP. Pinto AS: Transfer factor with dinitrochlorobenzene, Int Arch Allergy Appl Immunol 43:410, 1972. Welch TM, Triglia R, Spitler LE, Fudenberg HH: Preliminary studies on human “transfer factor” activity in guinea pigs. Clin Immunol lmmunopathol 5407. 1976. Vandenbark AA, Burger DR, Vetto RM: Human transfer factor activity in the guinea pig: Absence of specificity, Clin Immunol Immunopathol 8:7, 1977. Kirkpatrick CH, Smith TK: Specific and non-specific activities m dialyzable transfer factor, in Lucas DO. editor: Regulatory mechanisms in lymphocyte activation. New York, 1977, Hcademic Press, Inc.. p 171.
OF
ALLERGY AND
CLINICAL
IMMUNOLOGY NUMBER
VOLUME. 63
2
Editorials
‘Transfer of cellular transfer factor Charles H. Kirkpatrick,
immunity
M.D. &dzesdu, Md.
Since the original reports by Lawrence and associates’, 2 that it was possible to transfer delayed cutaneous hypersensitivity from sensitive human donors to insensitive recipients with lysates and dialysates of blood leukocytes, there has been controversy over the specificity, the mechanism, and even the existence of the phenomenon. The arguments against transfer factor derived from several sources. First, there was difficulty in confirming the reports that similar transfers could be achieved in guinea pigs.3* 4 Second, it was difficult to envision mechanisms by which molecules with molecular weights of 10,000 daltons or less could transfer the information required for reactivity against a vast repertory of antigenic determinants. Third, most of the experiments in man studied transfer of reactivity to ubiquitous microbial antigens such as tuberculin, streptococcal proteins, diphtheria toxoid, Can&&, etc., and it could be argued that the apparent transfers were due to enhancing or adjuvant-like activities of the dialysates in previously sensitized or “primed” recipients. A fourth argument reasoned that transfer factor contained minute amounts of “super-antigen” and that the apparently specific transfers were really the results of active immunization All of the arguments against transfer factor cannot be resolved at this time. but with the aid of properly
Reprint requests to: Charles H. Kirkpatrick, I IB- 13, National Institutes of Health,
with
M.D., Bldg. 10, Room Bethesda, MD 20014.
controlled studies in humans and with the recently developed animal models,“-g additional evidence for the phenomenon has accrued. For example, the crucial issue of specificity is unresolved, but recent findings are more compatible with specific than with nonspecific mechanisms. Maurer’O reported that lysates of peripheral blood leukocytes from donors with delayed hypersensitivity to ethylene oxide-treated human serum could transfer hypersensitivity to nonimmune recipients. Zuckerman and co-workers” were able to transfer delayed skin reactivity to keyhole limpet hemocyanin (KLH) from a KLH-sensitive donor to ten of ten nonimmune recipients with dialyzable transfer factor. Their donor was also reactive to purified protein derivative (PPD), and 8 of the 10 recipients also became responsive to this antigen. Two other transfer factor preparations, one from a KLH-negative, PPD-positive donor and the other from a pool of donors with unknown sensitivities, failed to transfer reactivity to KLH, but each of 5 recipients of the PPD-sensitive transfer factor developed reactivity to tuberculin. Recently, Burger and associates’* studied transfer of reactivity to KLH and added an important control. Transfer factor was prepared from the donors both before and after they were sensitized with KLH. The preimmunization transfer factor failed to sensitize any of the six recipients, while the postimmunization transfer factor sensitized 42 of 46 recipients. Other reports provide evidence that a previous “priming” exposure to an antigen is not essential for Vol.
63, No.
2, pp.
71-73
72
Kirkpatrick
passive transfer of delayed cutaneous reactivity to that antigen with transfer factor. Eichberg and co-workersL3 transferred delayed-type reactivity to primates that were housed in germ-free isolators. Kirkpatricki reported that 21 of 23 patients with chronic mucocutaneous candidiasis developed delayed hypersensitivity to Candidu after receiving transfer factor from Candidu-sensitive donors. In contrast, none of 5 candidiasis patients who were treated with transfer factor from Candidu-insensitive donors became reactive to Cundidu. This observation also argues against nonspecific, adjuvant-like activities being the only immunologic effect of transfer factor. In addition, there are no recognized genetic restrictions on this type of passive transfer in humans. Indeed, human transfer factor has been used to sensitize subhuman primates,13 mice,6 and cattle,x, g and murine and bovine transfer factorhas sensitized humans.” Since the original report by Landsteiner and Chase”’ that reactivity to tuberculin or chlorodinitrobenzene (CDNB) could be transferred from sensitive to insensitive guinea pigs with lymphoid cells, contact allergic reactions have been regarded as an expression of the cell-mediated immunity. In contrast to the relative ease of transferrring delayed cutaneous reactivity to intradermal tests with transfer factor, efforts to transfer reactivity to contact allergens have generally been unrewarding. One of the earliest studies was conducted by Brandriss,” who used dialyzable transfer factor from CDNB-sensitive, PPD-sensitive donors. Four of 7 recipients became reactive to PPD, but only 2 recipients developed “weak but definite reactions” to CDNB. Arala-Chaves and PintoI also attempted to transfer sensitivity to CDNB with leukocyte lysates. The experimental design was unique in that the challenge dose of antigen was applied to the skin either 72 hr before or at the same time that the transfer factor was injected. None of the 16 recipients who were challenged with 10 pg of CDNB before injections of transfer factor became reactive. Three of 19 (15.8%) recipients of both 10 and 20 pg challenge tests developed areas of erythema with papules or vesicles; similar reactions did not occur in any of the 21 control subjects challenged with the skin tests alone. Epstein Elsewhere in this issue of the JOURNAL, and Byers report another attempt to transfer contact allergy with dialyzable transfer factor. A central question in their study was the possibility that passive sensitization with transfer factor would occur only in persons who had been “primed” with subsensitizing doses of antigen (beryllium fluoride). The results were compatible with this model. None of the 20 subjects who were repeatedly patch-tested with BeFz
J. ALLERGY
CLIN. IMMUNOL. FEBRUARY 1979
and injected with Be0 became reactive. None of the 14 subjects who received transfer factor but did not have “priming” exposures of antigen became reactive. But 14 of 38 recipients (37%) who were both repeatedly exposed to beryllium salts and given transfer factor from beryllium-sensitive donors developed patch test reactivity to BeF,. Even then the sensitive state appeared to be transitory; 12 recipients were reactive one week after receiving transfer factor but only 4 persons were still reactive one week later. The finding that testing with BeFz at the same time that the transfer factor was injected did not produce positive responses confirms previously reported work in mice.7 In these experiments positive responses could not be obtained for at least 24 hr after injection of the transfer factor. It is also curious that only 3 recipients developed granulamatous responses to intradermal BeO, a reaction that is due to cell-mediated immune mechanisms. As the authors acknowledge, there is an important omission in the experimental design. Ideally, they should have included a group that received transfer factor from beryllium-insensitive donors. If these recipients did not become reactive, the authors would have even stronger evidence both for specificity and for the requirement for priming exposures for transfer of contact allergic reactions with transfer factor. If, on the other hand, recipients of “beryllium-negative” transfer factor also became reactive, they would have good evidence for nonspecific activities. The importance of this point is illustrated by the experience with transfer factor-mediated delayed hypersensitivity in guinea pigs.” In this model, delayed allergic reactions occurred only in animals that had been “primed” with an intradermal dose of antigen, given a mixture of transfer factor and antigen, and then skin-tested. Subsequent experience showed that the system lacked antigenic specificity. I9 In spite of these shortcomings, Epstein and Byers have presented an important and useful model for future experiments. Their report also suggests some important questions about contact allergic reactions and why they appear to be more difficult to transfer than delayed responses to intradermal antigens. Transfer of delayed hypersensitivity with transfer factor does not appear to be under genetic restriction, but the possibility that transfer of contact allergy may be genetically modified has not been investigated. The transitory nature of the reactive state raises a question about the functional life span of the cells involved in mediation of contact allergic reactions. In addition, relatively little is known about the contributions of the various lymphokines to production of the inflammatory responses and epidermal proliferation that ac-
VOLUME NUMBER
Transfer
63 2
company cell-mediated immune reactions in the skin. If different lymphokines are essential for contact reactions than for delayed-type inflammations, then additional studies of the effect of transfer factor on production of individual mediators are warranted. Finally, there has been considerable progress toward isolation and characterization of the active component(s) in transfer factor. As the recent workshop,” several groups presented evidence that the active material was sensitive to proteases and to snake venom phosphodiesterase. The tentative model of the molecule includes a purine (probably inosine or inosine monophosphate), ribose, a phosphodiester bond, and a peptide. A theoretical model has been presented in which the peptide contains antigen-specific sites that are essential for initiation of the sensitive state.“’
Transfer
2. Lawrence HS, Al-Askari S, David J, Franklin EC, Zweiman B: Transfer of immunologic information in humans with dialysates of leucocyte extracts. Tram Assoc Am Physicians 76:84. 1963. 3. Jeter WS, Tremaine layed hypersensitivity pigs with leuctrytic 1954.
MM. Seebohm PM: Passive lransfer deto 2. 4-dinitrochlorobenzene in guinea extracts, Proc Sot Exp Biol Med 86:251,
4. Bloom ER, Chase MW: Transfer of delayed-type hypersensiti,vity: A critical review and experimental study in the &uinea pig, Prog Allergy 10: 151, 1967. 5. Basten A, Croft S, Edwards J: Experimental studies of transfer factor, in Ascher MS, Gottlieb AA, Kirkpatrick CH, editors: Transfer factor. Basic properties and clinical applications, New Yori, 1976, Academic Press, Inc., p 7.5. 6. Petersen EA. Frey JA. Dinowitz M, Rifkind D: Transfer of delayed hypersensitivity to mice with human immune cell extracts, in Ascher MS, Gottlieb AA, Kirkpatrick CH. editors:
Basic
properties
and clinical
8.
9. IO. I I.
12.
13.
14. IS.
I6
17. 18.
IO.
20.
73
applications,
New York. 1975, Academic Press, Inc.. p 387. 7. Rilkind D. Frey JA, Petersen EA, Dinowitz M: Transfer
REFERENCES I. Lawrence HS: The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with disrupted leukocytes, J Clin Invest 34:219, 1955.
factor.
factor
of
delayed hypersensitivity in mice to microbial antigens with dialyzable transfer factor, Infect Immun 16:258. 1977. Klesius PH. Fudenberg HH: Bovine transfer factor: In vivo transfer of cell-mediated immunity to cattle with alcohol precipitates, Clin Immunol Immunopathol 8:238, 1977. Khan A. Kirkpatrick CH, Hill NO, editors: Immune regulators in transfer factor, New York, Academic Press, Inc. (In press.) Maurer PH: Immunologic studies vvith ethylene oxide-treated human serum, J Exp Med 113: 1029. 1961. Zuckerman KS. Neidhart JA, Balcerzak SP, LoBuglio AF: Immunologic speciticity of transfer factor. J Clin Invest 54:947, 1974. Burger DR. Vandenbark AA, Finke P, Vetto RM: De nova appearance of KLH transfer factor following immunization. Cell Immunol 29:410, 1977. Eichberg JW, Steele RW, Kalter SS, Kniker WT. Heberling RL, Eller JJ, Rodriguez AR: Cellular immunity in gnotobiotic primates induced by transfer factor. Cell Immunol 26: 114, 1976. Kirkpatrick CH: Transfer of delayed cutaneous hypersensitivity with transfer factor, Cell Immunol 41:62, 1978. Landsteiner K, Chase MW: Experiments on transfer of cutaneous sensitivity to simple chemicals, Proc Sot Exp Biol Med 49~688, 1942. contact hypersensitivity in Brandriss, MW: Attempt tmnSfer man with dialysate of peripheral leukocytes, J Clin Invest 47:21X!. 1968. Arala-Chaves MP. Pinto AS: Transfer factor with dinitrochlorobenzene, Int Arch Allergy Appl Immunol 43:410, 1972. Welch TM, Triglia R, Spitler LE, Fudenberg HH: Preliminary studies on human “transfer factor” activity in guinea pigs. Clin Immunol lmmunopathol 5407. 1976. Vandenbark AA, Burger DR, Vetto RM: Human transfer factor activity in the guinea pig: Absence of specificity, Clin Immunol Immunopathol 8:7, 1977. Kirkpatrick CH, Smith TK: Specific and non-specific activities m dialyzable transfer factor, in Lucas DO. editor: Regulatory mechanisms in lymphocyte activation. New York, 1977, Hcademic Press, Inc.. p 171.
