Transfer factor: Recent developments in the pursuit of an idea

CELLULAR

Transfer

62, 301-308 (1981)

IMMUNOLOGY

Factor:

Recent Developments

H. S. LAWRENCE’

in the Pursuit of an Idea’,’

AND WILLIAM

BORKOWSKY

Department of Medicine. Infectious Diseases and Immunology Division, and the Department Pediatrics, New York University School of Medicine. New York, New York 10016 Received April

of

13, I981

When I came to Sir Peter’s laboratory in 1959 the work on transfer factor had firmly secured an unenviable position of provoking either heated controversy or polite disdain among the members of the immunological establishment of my own country. In the face of this chill climate of opinion my resolve was beginning to falter and I considered abandoning the pursuit of the whole idea and getting on to some other more socially acceptable field of endeavor. How heartening, then, to be welcomed by Peter and to find such a warm and loyal friend whose generous mind and whose constant support and encouragement revived my flagging spirit. Peter’s acceptance of the idea and his analytical approach to its solution bolstered my confidence in myself and in the problem, cheering me on with renewed vigor and purpose to see it through. It is no wonder that I spent the happiest days of my career in Peter’s laboratory. It was a rich, rewarding, and enjoyable experience from which I learned not to quarrel with nature over the cards dealt but to get on with the game we call scientific enquiry with zest and purpose. And so I owe a tremendous debt to Sir Peter whom we all honor today-it is a debt that I am proud to acknowledge and try to repay daily in the quality of the science achieved. Hence, it is a privilege to be able to report on recent work from our laboratory which promises to lead to the denouement of the perplexing engima posed by the discovery of transfer factor. TRANSFER

FACTOR-INDUCER

HELPER ACTIVITY

We have reported recently (1) on the adaptation of the leukocyte migration inhibition test (LMI)3 as a reliable and reproducible in vitro assay for the anti* This paper was presented at the Symposium, “Advances in Immunology: A Meeting in Honor of Sir Peter Medawar,” held at The Royal Society, 6 Carlton House Terrace, London, on July 18, 1980. ’ The work discussed here was supported in part by United States Public Health Service research Grants 2 ROI Al/CA 02154-24A1, SPOI CA 16247-07, and in part by the Cancer Immunotherapy Research Foundation. ’ To whom reprint requests and all correspondence should be sent. ’ Abbreviations used : LMI, leukocyte migration inhibition; DLE, dialyzable leukocyte extract; DTH, delayed-type hypersensitivity; TOX, diphtheria toxoid; MI, migration index; TF, transfer factor; SKSD, streptokinase-streptodornase; PPD. purified protein derivative of tuberculin. 301 0008-8749/8l/l203Ol-08$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

302

LAWRENCE 1.2 Y ii " 1.0 1

0 00:

AND BORKOWSKY

y

3-4+ PPD

i

:r .tf

++t 4

0

0.2L

.

+ + tt: ttt +t +t$ 4

0

.

0

3-4+

3-4+ SKSD

D.TOX

0

3-4+

CANDIDA

FIG. 1. Skin test reactivity of DLE donor. Migration indices of nonimmune leukocytes in the presence of antigen after treatment with DLE obtained from individuals unreactive (0) or highly reactive (3 to 4+) to the respective antigen (PPD. diphtheria toxoid, SK-SD, or Candida). P c 0.001 for the degree of migration induced by unreactive and highly reactive donors of DLE. Reprinted, with permission, from Ref. (1).

genspecific activity in human leukocyte dialysates (DLE) containing transfer factor. The main manipulations of the assay consist of pulsing nonimmune leukocyte populations with leukocyte dialysates obtained from immune donors, centrifuging, and resuspending the cells in media containing the specific or control antigen before placing the cell suspension in agarose wells to measure migration indices 16 hr later. The critical target is the nonimmune mononuclear cell which is converted to a state of immune reactivity by the antigen-specific activity in DLE which, in the presence of the related antigen but not unrelated antigens, produces leukocyte

0 +o .+

7 .

0.8 -

.

0 : + .

0

l

:.

00

08 .

0

0 . A + II

AJ NL 16 HF LF

.

TA

L

0 CA

0.6 -

u o-1+ PPD

3-4+ o-I+4* D.TOX

I o-1+

I

3-4*

SKSD

0-b

3-4+

CANDIDA

FIG. 2. Skin test reactivity of DLE donor. Migration indices of nonimmune leukocytes in the presence of different antigens noted in the abscissa (PPD. diphtheria toxoid, SK-SD, and candida) after treatment with selected DLE preparations from donors with a known profile of reacitivty to these antigens (e.g., AJ is minimally reacitve to diphtheria toxoid and SK-SD (0 to l+) and highly reactive to PPD (3 to 4+) whereas NL is minimally reactive to PPD and SK-SD but highly reactive to diphtheria toxoid). Migratibn inhibition by a particular DLE preparation is evident only when the test antigen correlates with the antigen to which the DLE donor is highly reactive. Reprinted, with permission, from Ref. (1).

TRANSFERFACTOR:RECENTDEVELOPMENTS

303

TABLE 1 Comparison of Antigen-Specific Effectsof CrudeDLE and Fractions Redialyzed Using 3500MW PoreSizeMembrane

DLE

MW

3500 MW retentate of dialysis tubing

0.95 0.99 0.86 0.98 0.94 0.96 0.87 0.85

0.87 0.83 0.79 0.85 0.82 0.85 0.74 0.53

0.81 0.87 1.03 0.85 0.82 0.88 0.77 0.46

0.89 1.20 1.18 1.13 0.93 1.09 0.98 1.24

0.93 + 0.06

0.79 + 0.11

0.81 I!Z0.16

1.08 f 0.13

Control-no Expt

Mean+ SD

Crude DLE < 12,000

3500 MW dialysate of dialysis tubing

Source. Reprinted, with permission, from Ref. (1).

inhibitory factor and results in inhibition of leukocyte migration from the agar well. Essentially similar results using the LMI test for the assay of antigen-specific activity in DLE have been reported by Wilson and Fudenberg (2). In the LMI assay system we find that only those DLE preparations which are obtained from donors with an intense degree of delayed cutaneous reactivity to antigen are capable of causing antigen-specific inhibition of migration consistently (see Fig. 1). DLE preparations obtained from donors with moderate or absent reactivity are generally inactive. The capacity of the antigen-specific activity to convert nonimmune cells to immune reactivity is also dose dependent. These two variables (degree of donor reactivity and dose dependency) have been shown repeatedly to be critical requirements for successful in vivo transfer of cutaneous DTH reactions with transfer factor preparations (3). The pulsing of nonimmune migrating cell populations with immune DLE for 30 to 60 min is an additional requirement for maximum effects to be achieved. With careful attention to these experimental details antigen-specific inhibition of migration is conferred by immune DLE on nonimmune cell populations which is concordant with the cutaneous DTH reactivities of the donor of DLE (see Fig. 2). We have also found that the antigen-specific activity in DLE resides in the >3500 and ~12,000 MW dialysis fraction and not in the ~3500 MW fraction which contains the bulk of the pharmacologically active molecules capable of exerting nonspecific effects (e.g., serotonin, histamine, ascorbate, nicotinamide, cyclic nucleotides, and prostaglandin E). These results are summarized in Table 1. The antigen-specific activity does not behave like a superantigen nor a chemotactant in the LMI test. Moreover, the antigen-specific activity is inactivated by treatment with the enzymes Pronase and phosphodiesterase I and it is unaffected by RNase I (Borkowsky and Lawrence, unpublished)-sharing additional properties with antigen-specific TF activity in vivo (4). We also reported on the detection of suppressor activity in the same crude leukocyte dialysates which contained the antigen-specific activity. In experiments de-

304

LAWRENCE

AND BORKOWSKY TABLE 2

Antigen-Specific Effects of Murine DLE” Migration index with specific antigen DLE treatment

Candida

Expt I No DLE Candida-immune DLE TOX-immune DLE

0.97 0.80

1.01

TOX

1.oo 0.88 0.75

Expt II No DLE Candida-immune DLE TOX-immune DLE

0.85 0.96

0.93 0.96 0.83

Expt III No DLE Candida-immune DLE TOX-immune DLE

0.91 0.77 0.98

0.93 0.93 0.76

Expt IV No DLE Candida-immune DLE TOX-immune DLE SK-SD-immune DLE

0.93 0.79 0.94 0.95

0.93 0.99 0.78 0.95

1.oo

SK-SD

0.96 0.93 1.05 0.77

Source. Reprinted, with permission, from Ref. (5). Note. TOX = diphtheria toxoid. o MI, migration with DLE/migration without DLE; MI < 0.85 is statistically significant (P -z 0.05).

signed to augment the inhibition of migration of immune cell populations in the presence of antigen, the related immune DLE was added to such immune cells and abrogation of their response to antigen was observed instead (1). We have since separated the suppressor activity from the antigen-specific inducer activity and are studying its properties. ANTIGEN-SPECIFIC ACTIVITY DETECTED IN MURINE &ENRICHED LYMPH NODE CELL DIALYSATES In an interesting adaptation we went on to evaluate antigen-specific activity in DLE prepared from inbred mice and assayed on human migrating cell populations in the LMI test (5). When nonimmune human leukocyte populations were pulsed with lymph node cell DLE preparations obtained from BALB/C or SJL mice immunized with diphtheria toxoid, candida, or SK-SD respectively, their migration was inhibited in the presence of the related antigen but not unrelated antigens as summarized in Table 2. The antigen-specific activity in murine DLE was found to be present in lymph node cell preparations but was not detected in spleen cell preparations obtained from the same donor. Additionally, the antigen-specific activity in DLE preparations obtained from immune lymph node cells was found to be present in the e-cell-enriched, nonadherent cell population following passage through nylon wool columns. Moreover, the antigen-specific activity of murine

TRANSFER

305

FACTOR: RECENT DEVELOPMENTS TABLE 3

Deletion of Antigen-Specific Activity from DLE after Absorption by Polystyrene-Bound Specific Antigen Antigenic specificities of DLE

Polystyreneantigen Immunoadsorbant

No DLE TOX+ CAN+ TOX+ CAN+ TOX+ CAN+

None None TOX CAN

Migration indices” For CAN 0.98 0.75 0.76 0.95

r+ .04' f .09 k .09' f .08g

For TOX 0.95 0.77 0.97 0.79

+ k f +

.05d .09' .03* .os

Source. Reprinted, with permission, from Ref. (7). Note. CAN = candida; TOX = diphtheria toxoid. a Mean MI + 1 SD for 6 separate experiments by paired t-test analysis. kit vs b (P c 0.01); e vs d (P < O.Ol);fvs c, g vs b, h vs d, and i vs d (not significant).

DLE, as we reported for human DLE, was found to reside in the >3500 and
306

LAWRENCE

AND BORKOWSKY TABLE 4

Recovery of Antigen-Specific Activity Deleted from DLE from Polystyrene-Antigen Complex by Treatment with 8 M Urea Preparation added to respective nonimmune cell populations plus related antigen No DLE

MI for CAN (N = 6)

0.97 + 0.03 No DLE

MI for TOX (N = 4)b

MI for specific antigen pooled data (N = 10)

CAN(+)DLE

0.81 + 0.05 Toxoid(+)DLE

0.99 f 0.02

0.76 f 0.05

No DLE

Immune DLE

0.98’ + 0.03

0.79d + 0.06

CAN(+)DLE after absorption with CAN

CAN specific activity recovered with 8 M Urea

0.99 + 0.04

0.84 k 0.12

Toxoid(+)DLE after absorption with toxoid

Toxoid specific activity recovered with 8 M urea

1.00 + 0.08

0.84 k 0.12

DLE after antigen absorption

l.OV + 0.05

Antigen-specific activity recovered with 8 M urea 0.84’ + 0.12

Source. Reprinted, with permission, from Ref. (7). Note. CAN = candida; Toxoid = diphtheria toxoid. ’ Mean f 1 SD for 6 separate experiments. b Mean + 1 SD for 4 separate experiments. ‘-/Using the paired t test: c vs d (t = 9.04; P -z 0.001); c vs e (t = 0.83; not significant); d vs f (t = 1.31, not significant); c vsf(t = 3.37; P c 0.01).

candida and diphtheria toxoid. This preparation of candida-immune, toxoid-immune DLE was found to be selectively depleted for either candida-specific activity or toxoid-specific activity after absorption with candida-coated or toxoid-coated polystyrene, respectively; and upon recovery the absorbed DLE retained its candidaspecific activity following absorption with toxoid and its toxoid-specific activity following absorption with candida. Thus we found the antigen-specific activity in DLE binds to the related antigen but not to unrelated antigens as summarized in Table 3. Additionally it was determined that such antigen-specific activity for candida or for toxoid following absorption could be recovered from the respective antigenpolystyrene complex following treatment with 8 M urea as summarized in Table 4. Our data suggest that the antigen-specific activity in DLE preparations functions to convert nonimmune cells to a state of immune reactivity by virtue of its capacity to bind antigen. We postulate that the antigen-specific activity engages and arms a subpopulation of T cells either directly or indirectly via macrophages with a

TRANSFER

FACTOR:

RECENT

DEVELOPMENTS

307

specific antigen-binding moiety. This assumption is based on the finding that the antigen-specific activity: (i) converts nonimmune cells to an antigen responsive state promptly and in a dose-dependent fashion; (ii) activity can be absorbed by passage through Ficol-Hypaque-purified mononuclear cells. The facts that the antigenspecific activity does not behave as superantigen in this system and that it does not bind to a high-titer antibody of related specificity tend to exclude the presence of dialyzable antigen fragments as a possible explanation of the observed specificity. We interpreted these findings to suggest that the antigen-specific activity may be either a dialyzable fragment of an immune T-cell antigen receptor site, or a portion of its V-region gene product or perhaps a unique T-cell Ir gene product that assists in antigen presentation to other T cells (7). At the practical level the finding that the antigen-specific activity can be separated from all contaminating molecules in crude leukocyte dialysates by binding to a specific antigen-immunoadsorbent and the bound activity can be subsequently recovered following treatment with 8 M urea has provided an important advance in the isolation, purification, and characterization of such activity for testing in in vitro and in vivo assay systems. In the course of our in vitro studies detailed above (8) two groups of investigators have reported independent observations on the binding of the antigen-specific activity in dialyzable TF preparations to the related antigen as measured by the neutralization of the transfer of cutaneous DTH responses in vivo. Peterson et al. (9) have reported that prior incubation of murine DLE with the related but not unrelated antigens blocked the transfer of footpad swelling between C57BL6 or BALB/c mice. Burger et al. (10) also have reported that prior absorption of KLHspecific human TF preparations with KLH immunoabsorbent colums resulted in a marked diminution of the capacity to transfer cutaneous reactivity to KLH in vivo between human volunteers. We are currently pursuing the possibility that the antigen-specific activity in DLE may represent a dialyzable fragment of a T-cell receptor or its V region. Hence the studies in progress are evaluating the capacity of column-purified antiVu chain and anti-V, chain antibodies prepared and generously donated by Dr. Givol to bind the antigen-specific activity. Similar studies are underway using antiVH chain and anti-V, chain antibodies prepared and generously donated by Dr. Eisen. The results to date are promising and warrant detailed careful evaluation. The relationship between the antigen-specific activity detected in vitro and the antigen-specific effects of transfer factor in vivo remain to be established. In any event to recall Sir Peter’s felicitous phrase, the pursuit of this idea has moved to “The Art of the Soluble” and the facts emerging may yet gain for transfer factor its rightful place in the philosophy of immunology. And so, Sir Peter, I salute you and acknowledge with deep gratitude your understanding, support, and encouragement. We wish for you all the happiness and joy and high spirit which you have given so freely over the years to all who are privileged to honor you here today. REFERENCES 1. Borkowsky, W., and Lawrence, H. S., J. Immunol. 123, 1741, 1979. 2. Wilson, G. B., and Fudenberg, H. H., J. Lnb. Clin. Med. 98, 819, 1979.

308

LAWRENCE

AND BORKOWSKY

3. Lawrence, H. S., In “The Harvey Lectures,” Ser. 68, pp. 239-350, Academic Press, New York, 1974. 4. Burger, D. R., Vandenbark, A. A., Dunnick, W., Kraybill, W., Daves, G. D., and Vetto, R. M., J. Immunol. 122, 1091, 1979. 5. Borkowsky, W., and Lawrence, H. S., J. Immunol. 126, 80, 1981. 6. Lawrence, H. S., and Pappenheimer, A. M., Jr., J. Clin. Invest. 36, 908, 1957. 7. Borkowsky, W., and Lawrence, H. S., J. Immunol. 126, 486, 1981. 8. Borkowsky, W., and Lawrence, H. S., Clin. Rex 28, 550(A), 1980. 9. Petersen, E. A., Greenberg, L. E., and Kirkpatrick, C. H., Fed. Proc. 39, 1160, 1980. 10. Burger, D. R., Vandenbark, A. A., and Vetto, R. M., In “Proceedings, Serono Symposia” (F. A. Aiuti and H. Wigzell, Eds.), Vol. 38, pp. 431-439, Academic Press, New York, 1980.