CEILI-L.ZR
I\IXUKOLOGT
27, 323-3-77(1976)
Serial Transfer of Delayed Hypersensitivity Dialyzable Transfer Factor CITARLES
Lnboratory
H.
KIRIS~\TRICIC,
of Clinical Imvstigatiorz, ;\:atiosal Institutrs
AND
Xatio+lal
Institute
TERRILL
K.
of Allergy
of Health, Betlacsda, Maryland Rccci-,vd April
with
SMITH
afld Infectious 20014
Discascs,
16, 1976
A patient with defective cell-mediated immunity was reconstituted with dialyzable transfer factor. Subsequently, this patient served as a transfer-factor donor and this material was used to passively sensitize two additional patients with cellular immunodeficiency. These experiments confirm an earlier report of serial transfer of cellular immune responses with transfer factor.
IiYTRODUCTIOX Tn 1935, Lawrence (1) reported that delayed cutaneous reactivity to streptococcal ilI substance and tuberculin could be passively transferred to skin-test-negative, normal human recipients with the lysates of leukocytes from skin-test-positive human donors. In his study, two subjects who became skin-test-positive after receiving the lysates subsequently served as donors of leukocytes, the lysates of which passively sensitized other skin-test-negative recipients. These observations suggested that passive transfer of delayed hypersensitivity with nonliving cellular material was not due to contaminating antigenic components. The purpose of this report is to describe another instance of serial transfer of delayed hypersensitivity xvith leukocyte extracts. In this case, however, the material used was dialyzable transfer factor and the recipients were immunodeficient patients with chronic mucocutaneous candidiasis (2). METHODS
AND
MATERIALS
Szrbjccts a& cxperimnfd design. The initial transfer-factor donor was a normal adult male who fulfilled all of the medical criteria for donors in the NC1 Plasmapheresis Unit. The recipient of this transfer factor (primary recipient) was a 17year-old male with mucocutaneous candidiasis. The immunologic investigation of the primary recipient disclosed that he had cutaneous anergy and impaired antigeninduced MIF production (Table 1). However, when his lymphocytes were stimulated in vitro with the same antigens, brisk increases in DNA synthesis were observed (Table 2). After he received the transfer factor from 6 X lo8 lymphocytes, both delayed skin reactivity and MIF production were demonstrable. In contrast, there were no significant changes in the magnitude of the lymphocyte transformation responses. 323 Copyright @ 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
N.T.
N.T.
4.5
0
Histoplasmin Trichophytin 1:30
Primary
0
0
0 0 0
D.H. 0
N.T.
N.T.
3 0 N.T.
MIF 0
TABLE
1
Hypersensitivity
N.T.
N.T.
23 80 N.T.
MIF 0
N.T.
N.T.
0 0 N.T.
MIF 0
of induration
0
0
0 0 0
D.H. 0
Pre TF
with Dialyzable
responses in centimeters
0
0
0.7 1.3 0.9
D.H. 0
Post TF
recipient
Cutaneous
Pre TF
of Delayed
a D.H.-Delayed cutaneous hypersensitivity responses. Maximum * MIF-Percentage inhibition, mean of quadruplicate assays. c N.T.-Test not done.
32 40 N.T.”
2.5 2.0 3.0
Candida 1: 100 SK-SD 40/10 Mumps
MIF* 0
donor
D.H.a 0
Primary
PPD (intermed)
Antigen
Serial Transfer
0
0
0.8 1.0 0.8
D.H. 0
N.T.
N.T.
27 30 N.T.
MIF 0
Post TF
Secondary
Factor
at 24 or 48 hr.
1
Transfer
0
0
0 0.6 0.5
D.H. 0
N.T.
N.T.
0 41 N.T.
MIF 0
Pre TF
recipients 2
0
0
1.; 1.-l 0.5
D.H. 0
N.T.
N.T.
2.i 16 N.T.
51IF 0
Posr TF
$ m
; R c z ?+ ;j
WI tl 0 2
Six \!T!ckS ;Ifter Wccivillg tllc tr;ulsTc~l-f;tctr)r ;llltl wliilc llis illj~jl~~tl~]qic resputlsrs \vWe intact, he served as a Irr~kncytr donor. ‘I’hr rcripients (secondary recipients _ I antI 2) of tllc lraiisfrr factor lq)nrr(l frcjm llis cells \vcrc a 23-scar-old male and a 14year-old felllale, htll of \vlloln llad clironic n~ucocutaneous candidiasis and evidence of impaired cellular immunity (Table 1). Both also received the dialyzable material from G X lo* lymphocytes. The pre- and post-transfer-factor immunologic data summarized in Table 1 were obtained within 2 weeks of administration of the transfer-factor preparations. The studies of lymphocyte transformation were performed within 1 month of administration of transfer factor. These studies were conducted in accord with a protocol that had been reviewed and approved by the appropriate NIH Human Experimentation Committees. I?n$?zzinologic stzidics. The initial leukocyte donor and each of the recipients were evaluated with both in V~V,Oand in vifro tests of immunologic function. The skin-test antigens and assessment of cutaneous responses and the technique for performing the studies of lymphocyte transformation were the same as described previously (3). Antigen-induced production of macrophage migration inhibitory factor (MIF) was determined by the indirect assay of Rocklin ct al. (4). Pr+~ution of diaZy&& transfer- frrcfor. The technique for preparation of dialyzable transfer factor has been described in a previous publication (3). In brief, the leukocytes were collected with a continuous-flow blood cell separator (American Instrument CO., Silver Spring, Md.), washed with Hanks’ balanced salt solution, and lysed with multiple cycles of freezing and thawing. The lysate was dialyzed against sterile, pyrogen-free distilled water, the dialysate was lyophilized and the residue was dissolved in sterile saline so that the material from 3 X lo8 lymphocytes was contained in each milliliter. The final product was sterilized by Millipore filtration, bottled, and stored at -30°C until used. Note that DNase digestion was not done. RESULTS Prior to receiving transfer factor, the primary recipient and secondary recipient 1 did not show delayed hypersensitivity to any of the skin-test antigens (Table 1). Secondary recipient 2 responded to both SK-SD and mumps test antigen. None of the three subjects developed significant amounts of macrophage migration inhibitory factor (MIF) 1% 71 len their cells were stimulated with PPD or candida. Only secondary recipient 2, who had a positive delayed response to SK-SD, produced MIF when her cells were stimulated ipl vitro with this antigen. As shown in the representative experiments in Table 2, before receiving transfer factor both the primary recipient and secondary recipient 2 responded to in vitro stimulation with candida and SK-SD with significant increases in DNA synthesis. Secondary recipient 1 was virtually unresponsive to these antigens. The effects of administration of dialyzable transfer factor are shown in Table 1. Following injection of transfer from the normal donor, the primary recipient could develop positive delayed cutaneous responses to candida, SK-SD, and mumps; reactivity to histoplasmin was not transferred. The negative skin tests of the donor remained negative in the recipient. In addition, after the transfer-factor injection, blood lymphocytes from the recipient produced MIF in response to candida and SK-SD. The transfer factor from the primary recipient was active in the secondary recipients. All three delayed cutaneous responses of the primary recipient were
326
SHOR’k
COMikilJNICATION.$
TAI3LE Antigen-induced
Lymphocyte Transformation in the Recipients Prior to Receiving Transfer Factor
Subject
Primary
Secondary
Secondary
2
recipient
recipient-l
recipient-2
Antigen
Candida 10 pg/ml SK-SD b 100/25 units/ml Candida 10 a/ml SK-SD 100/25 units/ml Candida 10 wdml SK-SD 100/25 units/ml
Control dpma
Stimulated dpma
1,390
48,706
1,390
75,074
2,540
6,363
2,540
2,068
929
18,414
929
7,122
a Mean of triplicate cultures. b SK-SD = streptokinase-streptodornase.
transferred to secondary recipient 1. Moreover, his lymphocytes could now produce MIF when stimulated with candida and SK-SD. Prior to receiving transfer factor, secondary recipient 2 had positive skin tests to SK-SD and mumps and her SKSD-stimulated lymphocytes produced MIF. After receiving the transfer factor she could develop positive responses to candida in viva and in vitro. In contrast, the lymphocyte transformation response by the primary and secondary recipients did not show significant changes after administration of transfer factor. DISCUSSION Since publication of the original observation in the 1950’s (1), several laboratories have confirmed that, in man, cell-mediated immune responses may be passively transferred to unresponsive recipients with cell lysates and dialysates. The chemical composition and mechanism of action of the active substance in this transfer factor have not been defined. One possible mechanism was that the lysates were contaminated with antigens in highly immunogenic forms, perhaps as antigenpolynucleotide complexes. The finding that delayed cutaneous hypersensitivity to streptococcal M substance and tuberculin could be transferred sequentially from recipients of transfer factor to other skin-test-negative subjects argued against antigenic contamination. Instead, it seemed more likely that the active material was derived from donor leuko:ytes. The experiments described in this report confirm the previous report by Lawrence (1). In addition, they show that serial transfer may be achieved with a dialyzable material and thereby present additional evidence against a role for contaminating antigens. The results summarized in Table 1 illustrate the apparent specificity of passive transfer with transfer factor. Delayed reactivity to candida, SK-SD, and mumps, but not histoplasmin, was transferred from the primary donor to the primary recipient. Difficulty in passively transferring cutaneous reactivity to histoplasmin leukocytes (6)) and dialyzable transfer factor (7) with living cells (5)) refrigerated
SITORT
COhIXU~ICATIOiXS
327
has been reported previously e\-en when transfer of lymphokine production to this: antigen occurred (7). The primary recipient, in turn, transferred the same three reactivities to one secondary recipient and reactivity to candida to the other secondary recipient; responses that were negative in the primary recipient did not become positive in the secondary recipients. A crucial question concerning transfer factor is its mechanism of action. One suggestion is that it contains specific genetic or instructional information that endows previously unresponsive cells with the ability to respond to certain antigens. Another model proposes that transfer factor functions by amplifying or “unmasking” subthreshold or latent responses by previously sensitized cells (8). The observations in the subjects of this study are more compatible with the second proposal. Each patient had chronic mucocutaneous candidiasis and had had prolonged exposure to candida antigens. It is also probable that each had had prior exposure to streptococcal antigens. Indeed, the positive responses to candida and SK-SD by the primary recipient and secondary recipient 2 in the lymphocyte transformation test indicate the presence of antigen-responsive cells even though the patients had negative skin tests and did not produce MIF (prior to receiving transfer factor). Delayed reactivity to these ubiquitous antigens appeared in each recipient after administration of transfer factor. On the other hand, none of the recipients had is common and none became resided in areas where exposure to H. ca~psulnfwc responsive to this antigen after receiving transfer factor. These experiments also show that an immunodeficient subject can, after being reconstituted with transfer factor, serve as a source of transfer factor for others. Lf:hile this experiment suggests that the ability to produce transfer factor was a consequence of the immunologic reconstitution, this conclusion is tentative because it was not shown that the primary recipient was incapable of producing transfer factor prior to receiving transfer factor. REFERENCES 1. 2. 3. 4. 5. 6.
Lawrence, H. S., J. Clin. Imvst.
34, 219, 1955. Kirkpatrick, C. H., and Montes, L. F., J. Cutan. Pathol. 1, 211, 1974. Kirkpatrick, C. H., Rich, R. R., and Smith, T. K., J. Cli%. Invest. 51, 2948, 1972. Rocklin, R. E., Meyers, 0. L., and David, J. R., J. Iulntnrm~[. 104, 95, 1970. Kirkpatrick, C. H., Wilson, W. E. C., and Talmage, D. W., J. Exp. Med. 119, 727, 1964. Jensen, K., Patnode, R. A., TownsIcy, H. C., and Cummings, 91. M., Awcr. Rev. Rcsp. Dis. 85, 373, 1962. 7. Graybill, M. R., Ellenbogen, C., Drossman, D., Kaplan, P., and Thor, D. E., I+$ “Transfer (M. S. Ascher, A. A. Gottlieb, and Factor. Basic Properties and Clinical Applications” C. H. Kirkpatrick, Eds.), pp. 509-514. Academic Press, New York, 1976. 8. Kirkpatrick, C. H., J. Allergy Clis. Imnmnol. 55, 411, 1975.
I\IXUKOLOGT
27, 323-3-77(1976)
Serial Transfer of Delayed Hypersensitivity Dialyzable Transfer Factor CITARLES
Lnboratory
H.
KIRIS~\TRICIC,
of Clinical Imvstigatiorz, ;\:atiosal Institutrs
AND
Xatio+lal
Institute
TERRILL
K.
of Allergy
of Health, Betlacsda, Maryland Rccci-,vd April
with
SMITH
afld Infectious 20014
Discascs,
16, 1976
A patient with defective cell-mediated immunity was reconstituted with dialyzable transfer factor. Subsequently, this patient served as a transfer-factor donor and this material was used to passively sensitize two additional patients with cellular immunodeficiency. These experiments confirm an earlier report of serial transfer of cellular immune responses with transfer factor.
IiYTRODUCTIOX Tn 1935, Lawrence (1) reported that delayed cutaneous reactivity to streptococcal ilI substance and tuberculin could be passively transferred to skin-test-negative, normal human recipients with the lysates of leukocytes from skin-test-positive human donors. In his study, two subjects who became skin-test-positive after receiving the lysates subsequently served as donors of leukocytes, the lysates of which passively sensitized other skin-test-negative recipients. These observations suggested that passive transfer of delayed hypersensitivity with nonliving cellular material was not due to contaminating antigenic components. The purpose of this report is to describe another instance of serial transfer of delayed hypersensitivity xvith leukocyte extracts. In this case, however, the material used was dialyzable transfer factor and the recipients were immunodeficient patients with chronic mucocutaneous candidiasis (2). METHODS
AND
MATERIALS
Szrbjccts a& cxperimnfd design. The initial transfer-factor donor was a normal adult male who fulfilled all of the medical criteria for donors in the NC1 Plasmapheresis Unit. The recipient of this transfer factor (primary recipient) was a 17year-old male with mucocutaneous candidiasis. The immunologic investigation of the primary recipient disclosed that he had cutaneous anergy and impaired antigeninduced MIF production (Table 1). However, when his lymphocytes were stimulated in vitro with the same antigens, brisk increases in DNA synthesis were observed (Table 2). After he received the transfer factor from 6 X lo8 lymphocytes, both delayed skin reactivity and MIF production were demonstrable. In contrast, there were no significant changes in the magnitude of the lymphocyte transformation responses. 323 Copyright @ 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
N.T.
N.T.
4.5
0
Histoplasmin Trichophytin 1:30
Primary
0
0
0 0 0
D.H. 0
N.T.
N.T.
3 0 N.T.
MIF 0
TABLE
1
Hypersensitivity
N.T.
N.T.
23 80 N.T.
MIF 0
N.T.
N.T.
0 0 N.T.
MIF 0
of induration
0
0
0 0 0
D.H. 0
Pre TF
with Dialyzable
responses in centimeters
0
0
0.7 1.3 0.9
D.H. 0
Post TF
recipient
Cutaneous
Pre TF
of Delayed
a D.H.-Delayed cutaneous hypersensitivity responses. Maximum * MIF-Percentage inhibition, mean of quadruplicate assays. c N.T.-Test not done.
32 40 N.T.”
2.5 2.0 3.0
Candida 1: 100 SK-SD 40/10 Mumps
MIF* 0
donor
D.H.a 0
Primary
PPD (intermed)
Antigen
Serial Transfer
0
0
0.8 1.0 0.8
D.H. 0
N.T.
N.T.
27 30 N.T.
MIF 0
Post TF
Secondary
Factor
at 24 or 48 hr.
1
Transfer
0
0
0 0.6 0.5
D.H. 0
N.T.
N.T.
0 41 N.T.
MIF 0
Pre TF
recipients 2
0
0
1.; 1.-l 0.5
D.H. 0
N.T.
N.T.
2.i 16 N.T.
51IF 0
Posr TF
$ m
; R c z ?+ ;j
WI tl 0 2
Six \!T!ckS ;Ifter Wccivillg tllc tr;ulsTc~l-f;tctr)r ;llltl wliilc llis illj~jl~~tl~]qic resputlsrs \vWe intact, he served as a Irr~kncytr donor. ‘I’hr rcripients (secondary recipients _ I antI 2) of tllc lraiisfrr factor lq)nrr(l frcjm llis cells \vcrc a 23-scar-old male and a 14year-old felllale, htll of \vlloln llad clironic n~ucocutaneous candidiasis and evidence of impaired cellular immunity (Table 1). Both also received the dialyzable material from G X lo* lymphocytes. The pre- and post-transfer-factor immunologic data summarized in Table 1 were obtained within 2 weeks of administration of the transfer-factor preparations. The studies of lymphocyte transformation were performed within 1 month of administration of transfer factor. These studies were conducted in accord with a protocol that had been reviewed and approved by the appropriate NIH Human Experimentation Committees. I?n$?zzinologic stzidics. The initial leukocyte donor and each of the recipients were evaluated with both in V~V,Oand in vifro tests of immunologic function. The skin-test antigens and assessment of cutaneous responses and the technique for performing the studies of lymphocyte transformation were the same as described previously (3). Antigen-induced production of macrophage migration inhibitory factor (MIF) was determined by the indirect assay of Rocklin ct al. (4). Pr+~ution of diaZy&& transfer- frrcfor. The technique for preparation of dialyzable transfer factor has been described in a previous publication (3). In brief, the leukocytes were collected with a continuous-flow blood cell separator (American Instrument CO., Silver Spring, Md.), washed with Hanks’ balanced salt solution, and lysed with multiple cycles of freezing and thawing. The lysate was dialyzed against sterile, pyrogen-free distilled water, the dialysate was lyophilized and the residue was dissolved in sterile saline so that the material from 3 X lo8 lymphocytes was contained in each milliliter. The final product was sterilized by Millipore filtration, bottled, and stored at -30°C until used. Note that DNase digestion was not done. RESULTS Prior to receiving transfer factor, the primary recipient and secondary recipient 1 did not show delayed hypersensitivity to any of the skin-test antigens (Table 1). Secondary recipient 2 responded to both SK-SD and mumps test antigen. None of the three subjects developed significant amounts of macrophage migration inhibitory factor (MIF) 1% 71 len their cells were stimulated with PPD or candida. Only secondary recipient 2, who had a positive delayed response to SK-SD, produced MIF when her cells were stimulated ipl vitro with this antigen. As shown in the representative experiments in Table 2, before receiving transfer factor both the primary recipient and secondary recipient 2 responded to in vitro stimulation with candida and SK-SD with significant increases in DNA synthesis. Secondary recipient 1 was virtually unresponsive to these antigens. The effects of administration of dialyzable transfer factor are shown in Table 1. Following injection of transfer from the normal donor, the primary recipient could develop positive delayed cutaneous responses to candida, SK-SD, and mumps; reactivity to histoplasmin was not transferred. The negative skin tests of the donor remained negative in the recipient. In addition, after the transfer-factor injection, blood lymphocytes from the recipient produced MIF in response to candida and SK-SD. The transfer factor from the primary recipient was active in the secondary recipients. All three delayed cutaneous responses of the primary recipient were
326
SHOR’k
COMikilJNICATION.$
TAI3LE Antigen-induced
Lymphocyte Transformation in the Recipients Prior to Receiving Transfer Factor
Subject
Primary
Secondary
Secondary
2
recipient
recipient-l
recipient-2
Antigen
Candida 10 pg/ml SK-SD b 100/25 units/ml Candida 10 a/ml SK-SD 100/25 units/ml Candida 10 wdml SK-SD 100/25 units/ml
Control dpma
Stimulated dpma
1,390
48,706
1,390
75,074
2,540
6,363
2,540
2,068
929
18,414
929
7,122
a Mean of triplicate cultures. b SK-SD = streptokinase-streptodornase.
transferred to secondary recipient 1. Moreover, his lymphocytes could now produce MIF when stimulated with candida and SK-SD. Prior to receiving transfer factor, secondary recipient 2 had positive skin tests to SK-SD and mumps and her SKSD-stimulated lymphocytes produced MIF. After receiving the transfer factor she could develop positive responses to candida in viva and in vitro. In contrast, the lymphocyte transformation response by the primary and secondary recipients did not show significant changes after administration of transfer factor. DISCUSSION Since publication of the original observation in the 1950’s (1), several laboratories have confirmed that, in man, cell-mediated immune responses may be passively transferred to unresponsive recipients with cell lysates and dialysates. The chemical composition and mechanism of action of the active substance in this transfer factor have not been defined. One possible mechanism was that the lysates were contaminated with antigens in highly immunogenic forms, perhaps as antigenpolynucleotide complexes. The finding that delayed cutaneous hypersensitivity to streptococcal M substance and tuberculin could be transferred sequentially from recipients of transfer factor to other skin-test-negative subjects argued against antigenic contamination. Instead, it seemed more likely that the active material was derived from donor leuko:ytes. The experiments described in this report confirm the previous report by Lawrence (1). In addition, they show that serial transfer may be achieved with a dialyzable material and thereby present additional evidence against a role for contaminating antigens. The results summarized in Table 1 illustrate the apparent specificity of passive transfer with transfer factor. Delayed reactivity to candida, SK-SD, and mumps, but not histoplasmin, was transferred from the primary donor to the primary recipient. Difficulty in passively transferring cutaneous reactivity to histoplasmin leukocytes (6)) and dialyzable transfer factor (7) with living cells (5)) refrigerated
SITORT
COhIXU~ICATIOiXS
327
has been reported previously e\-en when transfer of lymphokine production to this: antigen occurred (7). The primary recipient, in turn, transferred the same three reactivities to one secondary recipient and reactivity to candida to the other secondary recipient; responses that were negative in the primary recipient did not become positive in the secondary recipients. A crucial question concerning transfer factor is its mechanism of action. One suggestion is that it contains specific genetic or instructional information that endows previously unresponsive cells with the ability to respond to certain antigens. Another model proposes that transfer factor functions by amplifying or “unmasking” subthreshold or latent responses by previously sensitized cells (8). The observations in the subjects of this study are more compatible with the second proposal. Each patient had chronic mucocutaneous candidiasis and had had prolonged exposure to candida antigens. It is also probable that each had had prior exposure to streptococcal antigens. Indeed, the positive responses to candida and SK-SD by the primary recipient and secondary recipient 2 in the lymphocyte transformation test indicate the presence of antigen-responsive cells even though the patients had negative skin tests and did not produce MIF (prior to receiving transfer factor). Delayed reactivity to these ubiquitous antigens appeared in each recipient after administration of transfer factor. On the other hand, none of the recipients had is common and none became resided in areas where exposure to H. ca~psulnfwc responsive to this antigen after receiving transfer factor. These experiments also show that an immunodeficient subject can, after being reconstituted with transfer factor, serve as a source of transfer factor for others. Lf:hile this experiment suggests that the ability to produce transfer factor was a consequence of the immunologic reconstitution, this conclusion is tentative because it was not shown that the primary recipient was incapable of producing transfer factor prior to receiving transfer factor. REFERENCES 1. 2. 3. 4. 5. 6.
Lawrence, H. S., J. Clin. Imvst.
34, 219, 1955. Kirkpatrick, C. H., and Montes, L. F., J. Cutan. Pathol. 1, 211, 1974. Kirkpatrick, C. H., Rich, R. R., and Smith, T. K., J. Cli%. Invest. 51, 2948, 1972. Rocklin, R. E., Meyers, 0. L., and David, J. R., J. Iulntnrm~[. 104, 95, 1970. Kirkpatrick, C. H., Wilson, W. E. C., and Talmage, D. W., J. Exp. Med. 119, 727, 1964. Jensen, K., Patnode, R. A., TownsIcy, H. C., and Cummings, 91. M., Awcr. Rev. Rcsp. Dis. 85, 373, 1962. 7. Graybill, M. R., Ellenbogen, C., Drossman, D., Kaplan, P., and Thor, D. E., I+$ “Transfer (M. S. Ascher, A. A. Gottlieb, and Factor. Basic Properties and Clinical Applications” C. H. Kirkpatrick, Eds.), pp. 509-514. Academic Press, New York, 1976. 8. Kirkpatrick, C. H., J. Allergy Clis. Imnmnol. 55, 411, 1975.
