Immunobiology of Transfer Factor

SECTION I

THE NATURE OF TRANSFER FACTOR

Outline

Chapter 1: THE IMMUNOLOGICAL ENIGMA OF TRANSFER FACTOR

Chapter 2: HUMAN TRANSFER FACTOR: SPECIFICITY AND STRUCTURAL MODELS

Chapter 3: HUMAN TRANSFER FACTOR: EXOGENOUS LABELLING, PURIFICATION, AND ROLE OF RIBONUCLEIC ACID SEGMENT

Chapter 4: SELECTIVE REMOVAL OF TRANSFER FACTOR ACTIVITY WITH ANTIGEN

Chapter 5: ANTIGEN-SPECIFIC INDUCER FACTOR IN HUMAN LEUKOCYTE DIALYSATES: A PRODUCT OF TH CELLS WHICH BINDS TO ANTI-V REGION AND ANTI-Ia REGION ANTIBODIES

Chapter 6: ANTIGEN-SPECIFIC SUPPRESSOR FACTOR IN HUMAN LEUKOCYTE DIALYSATES: A PRODUCT OF TS CELLS WHICH BINDS TO ANTI-V REGION AND ANTI-Ia REGION ANTIBODIES

Chapter 7: ISOLATION AND PURIFICATION OF ANTIGEN-SPECIFIC INDUCER AND SUPPRESSOR FACTORS FROM POOLED LEUKOCYTE DIALYSATES OF UNSELECTED DONORS BY AFFINITY ADSORPTION

THE IMMUNOLOGICAL ENIGMA OF TRANSFER FACTOR¹

Merrill W. Chase,     The Rockefeller University, New York, N. Y

Publisher Summary

This chapter discusses the development of the knowledge of biology of transfer factor (TF). The extracts of mononuclear cells, predominantly lymphocytes, release many intracellular products, of which some have special biological activities in in vitro systems. But the concern of the immunologist has to do with the biological basis for specificity of transfer, that is, the ability to confer, on a new individual, the DH+ reactivity of the cell donor. This problem has remained a conundrum, which seems closer to be solved now. The chapter discusses the specific and the nonspecific components of leukocyte lysates. TF from highly reactive donors can produce lasting reactivity. Positive skin transfer is the sole criterion of TF. TF is not provided by nonsensitive individuals. TF dialyzes through Visking tubing. Biologically active materials besides TF are diffusible. TFD passes 12,000D membranes and not 3500D membranes. The removal of 3500D diffusate (>90% of weight) raises peptide:RNA ratio.

I INTRODUCTION; EARLY HISTORY

As an outsider to your ranks, I speak with some feeling of reluctance, but not as a stranger to experimental design. Were we to meet in small groups around my desk, you would provide instant feedback and I would profit from the exchange.

Dr. Lawrence’s early evidence for a soluble transfer factor in man, and the challenging aspect of the basic biology underlying the effect, have brought much study, and finding of new facts which otherwise would scarcely have come to light. Dr. Lawrence well deserves our acclaim for his persistence in pursuing the phenomenon and in attracting such capable workers to his laboratory.

Extracts of mononuclear cells, predominantly lymphocytes, release many intracellular products, of which some have special biological activities in in vitro systems. But the concern of the immunologist has to do with the biological basis for specificity of transfer, that is, the ability to confer, on a new individual, the DH+ reactivity of the cell donor.* That problem has remained a conundrum, which we seem now to be closer to solving. To sharpen the issue of specificity, I use the term TFD+DLE, indicating both the specific and the nonspecific components of leukocyte lysates. Published data have not been helped, in my opinion, by accepting as positive reactions only 5mm in diameter, and by acceptance of the banal description indolent red spot. No developing skin reaction occurs idly.

Table I lists the attributes of TF. Only few of the statements deserve comment. The sole criterion of TF remains, as always, a positive skin transfer. Then, we recall that diffusate through a 3500 D (dalton) membrane contains 90% of the weight, including salts, so that only then – if one accepts TF, as first secured, as being >3500D – does dry weight on the retentate become useful. Next, TF is surprisingly hardy, resisting acetone and strong alcohol and 6M urea. At the bottom are two new items: immobilized antigen binds TF, and TF is accompanied by a suppressor in the same weight class.

TABLE I

Attributes of Human Transfer Factor

The earlier years of the TF story appear in Table II. At the start appears cellular transfer in guinea pigs. After the death of Landsteiner, I worked alone, and then transferred to the newly established laboratory of René Dubos in 1945. Dubos was surprised by the effectiveness of cellular transfer and he told William Tillett, his ex-Rockefeller Hospital colleague and friend, that here was something worth investigating. Bill, although he never came to see me, stimulated workers at the New York University School of Medicine to enter the field, in particular Drs. Lawrence and Herman Eisen. In Table II, note that the first report of leukocyte extracts in transfer appeared in 1955. The ability to use dialysates was not published, along with a Sephadex run, until 1965, but this work had been three years in the making. Dialysis through Visking membranes (tubing) yielded materials of about 16–20K dalton cut-off, whatever was being manufactured at the time for the sausage industry. This procedure was an advance of the highest importance, for previously lysates could contain HLA and other antigens, and other nondialyzable intracellular materials such as Interleukins (then unknown) as well as small molecules (the potential biological activity of which was not then appreciated).

TABLE II

a Developments in Delayed-Type Hypersensitivity

bThe first cmtisermi to TFD-MBSA was obtained by 0. J. Plescia, coupling being the method described by Plescia in Methods in Immlogy and Imnochemistry (C. A. Williams and M.W. Chase, eds.), Vol. I, p. 175, Academic Press, N. Y. (1967). Mention is made in Reference 69, pp. 269, 294.

cThe suggestia of using 3500D dialysis tubing was made by L.G. Foster, then in the laboratory of Dr. A. Arthur Gottlieb (pers. conwiunication).

aThe chosen entries are salient developments, without claim of priority.

Meanwhile, in vitro tests had uncovered PHA and PWM as cellular mitogens which caused DNA synthesis and blast transformation over a 3-day culture period, and blastogenesis was observed under similar test conditions when the cells of tubercular patients were incubated with PPD tuberculin. Macrophage inhibition, restraint of spreading growth, by specific antigen was discovered by George and Vaughan; and two years later MIF was shown to be a soluble material. An increasing number of soluble factors came to light, leading to the important Symposium of the American Association of Immunologists in 1968: In Vitro Correlates of Delayed Hypersensitivity. A year later, Dudley Dumonde coined the term which shortly was widely adopted: lymphokines. New workers entered this attractive field, for it appeared to be a way to study transfer factor without actual in vivo assessments of leukocyte dialysates.

The vertical line within the left-hand column points to initiation of therapy with TFD in patients defective in T-cell competencies. This idea had been in the works conceptually from 1960, but attempts at treatment were undertaken on increasing scale from 1969 onwards. The field appeared to be crystallizing; Academic Press started publication of Cellular Immunology, with Dr. and Mrs. Lawrence as the scientific editors. Two years later, Clinical and Experimental Pathology was launched under Dr. Fudenberg.

Transfer Factor Conferences date from 1973, bringing together persons of very different views as to specificity of TFD+DLE, and others with concern only for their patients and for potential benefits of TF therapy.

Controversy arose as to whether TFD was actually exacerbating existent but subclinical sensitivities of the recipients. To me, it seemed very unlikely that such a vast number of latent sensitivities existed in the populace at large, and that TF, selected especially because of a donor’s reactivities would exacerbate only those latent sensitivities which were shared with the donor. More recent reports, which may modify but not negate my conclusion, are discussed below (21, 22).

But in view of life’s experiences in an environment of microbes, non-microbial antigens were sought. The first neoantigen, ethylene oxide treated human serum albumin, remains the basis of a well-quoted paper, yet apparently its use has not been followed up. The newer candidate as neo-antigen, the super-molecule KLH (keyhole limpet hemocyanin) has been used for deliberate sensitization, and the making of TFD (KLH) for transfer. In vivo transfer is reported. One study suggested that this preparation can sensitize monkeys to reactivity to KLH, a conclusion which rests upon biopsy of skin sites, not palpable skin reactions. Still more recently, ferritin has been employed as another antigen.

In vitro studies were, overall, disappointing, since many suggested non-specificity. Quite evidently the cells engaged in in vitro correlates largely represented cell populations other than the subset engaged in DH+ capacity (cf. 105).

The prominent vertical line in Table II indicates the continuing use of leukocyte lysates for therapy. It became apparent that induced skin reactivity and clinical benefit in patients with T-cell incompetencies were usually transient, so that multiple doses would be needed. Leukopheresis could indeed secure the cells for processing. Of this era of wide panacea treatment I make no value judgement. It was a broad Phase I exploration. However, the ear of NIAID deafened, and several planned double blind trials were not funded. Indeed, DLE without any evident TF content could induce lymphocyte proliferation. Although the vertical line ends, such treatments have continued under individual physicians and groups. Lawrence summarized Phase I trials in 1974 (69).

Table III lists terms which have been used with TF, also abbreviations which appear in this chapter.

TABLE III

Abbreviations and Definitions

Terms used: Ab, antibody; Ag, antigen; BSA, bovine serum albumin; DH, delayed (-type) hypersensitivity; Diph. txoid, diphtheria toxoid; DNFB, dinitrofluorobenzene; DNP-, dinitrophenyl; DNTB, dinitrothiocyanobenzene; KLH, keyhole limpet hemocyanin; LIF, leukocyte inhibitory factor; LMI, direct: leukocyte migration inhibition; MAF, macrophage aggregating factor; MBSA, methylated BSA; MEM-S, [Eagle’s] minimal essential medium, spinner or suspension formulation; MIF, macrophage inhibiting factor; PBL, peripheral blood leukocytes, usually counted for lymphocyte types; PEC, peritoneal exudate cells; PPD, tuberculin of the purified protein derivative variety; SK-SD, streptokinase-streptodornase, enzymes (not separated) of the streptococcus; SRBC, sheep red blood cells; TBC, tubercle bacilli, mycobacterial cells, killed unless specified; Toxoid, tetanus toxoid unless specified; UdR, uridinedeoxyriboside.

It is unfortunate that cellular transfer of contact dermatitis in man has not received adequate study, for a way could be opened into an aspect of delayed-onset hypersensitivities where evidence for specificity of transfer could be sought under convincing conditions. There is one negative report (70).

Workers in the field usually take as 1 Unit the lysate of 5 × 10⁸ lymphocytes, perhaps rather constant as a measure of solids. Baram (8,9) introduced and continues to use as standard orcinol-reactivity (an estimate of ribose content) to compare different lysates. Empirically useful, neither method measures the TF content, which must vary with the donor.

II COMMENTARY AND QUESTIONS

First question: How many lymphocytes possess transfer capacity in a sensitive individual?

A few cogent studies have been designed to estimate the actual number of effector cells (DH+) among total lymphocytes. These cells are surprisingly few although the efficiency-per-cell is reportedly high. An instructive example is given by Gell and Godfrey (71–74), with guinea pigs rendered highly sensitive to thiocyanodinitrobenzene via mycobacterial cells. (Antibodies were essentially lacking.) These animals reacted in contact tests, their cells effected cellular transfer, and they responded to i.d. injections of DNP-BSA. In vitro, their LNC, exposed to DNP-BSA, produced MIF, macrophage agglutinating factor (MAF), blastogenesis; and DNP-sheep erythrocytes gave rosette-formation. But different clones of cells were involved. Immunoabsorbant cell-sorting was employed to isolate the DH+ cells. When the LNC were passed through a column of DNP-polyacrylamide beads (to which DNP groupings were attached in a variety of ways), cells capable of blastogenesis came through the column unhindered, as did also cells reactive with PPD. Of the retained cells, DNP-glycine discharged those active in cellular transfer and with capacity to produce DNFB-stimulated lymphokines. Still held by the column were cells forming rosettes with DNP-SRBC, and cells which could release a special variety of MAF upon contact with DNP-BSA. (Another affinity column could distinguish between these two sorts of cells.)

The DH+ cells represented, on average, one LNC in 700 with a spread from 1 in 200 to 1 in 5000, in the range reported by others (75,76). Cellular transfer was largely inhibited by brief contact with DNFB. This was striking information that DH cells bind to the prosthetic grouping of the antigen.

Rather remarkably, a cell-free extract, prepared by grinding LNC with KCl to 3M concentration, appeared to transmit contact-type DH+ to DNTB and intradermal reactivity to PPD, these constituents apparently being non-dialyzable (73, cf. 83).

These obcervations are worth pursuit but, I suggest, not with LNC. Oil-induced peritoneal exudate cells, with 23% of lymphocytes, have 6 to 12 times the capacity of LNC to effect cellular transfer, and these would offer a far richer source of DH+ cells.

Another approach to estimating the number of DH+ cells was made in Bloom’s laboratory, on the principle that resting cells do not replicate RNA viruses (vesicular stomatitis, newcastle disease), while cells activated by antigen do so. Counting then becomes possible by spreading the virus-treated cells, plus Ag, on monolayers of normal cells and counting the plaques which develop. Initially studied in tuberculin-sensitive guinea pigs, the work was repeated on PBL from sensitive human subjects (78). Both species yielded essentially equivalent numbers – about 1 cell in 300. This result may count more than just the DH+ cells since other clones can respond to antigen.

An earlier attack, crude by comparison, was to determine by radioautography the number of labeled cells arriving in skin sites in guinea pigs following transfer of thymidine-labeled lymphocytes (79). Although many cells become labeled in the donors, less than 1 in 100 of all marker cells participated in the local DH