In Vitro Methods in Cell-Mediated Immunity

METHODOLOGY

SESSION I

METHODS FOR STUDY OF MEDIATORS

CHAIRMAN Barry R. Bloom

DR. BLOOM: The focus of this session will be the various systems which have been developed to study and assay cell-mediated immune reactions in vitro. The scope of these endeavors is summarized in Table 1 and indicates the variety of factors and activities found in the literature which may be related to the mediation of these reactions. Each factor is presently defined operationally by an assay. The factors in the first category can be elicited from specifically sensitized lymphocytes by contact with specific antigen in vitro - and therefore must be considered in the category of possible mediators. In addition, there are pre-existent materials which may be extracted from specifically sensitized cells, and these will be considered by Dr. Lawrence in the discussion of transfer factor. There is also a wealth of information on direct lymphocyte-target cell cytotoxicity, which is obviously involved in the effector process of cell mediated immune reactions. Different approaches have been chosen to measure these essential activities, and, as assays, they merit our consideration in terms of sensitivity, correlation with events in vivo, reproducibility, ease of performance, and application to different species, especially man. This then is the subject of the first session.

TABLE 1

PUTATIVE MEDIATORS OF CELLULAR IMMUNITY

FACTORS RELEASED UPON INTERACTION WITH ANTIGEN

Migration Inhibitory Factor (guinea pig, mouse, rat, human)

Macrophage Spread Inhibitory Factor (guinea pig, mouse)

Macrophage aggregating Factor (guinea pig)

Skin Reactive Factor (guinea pig)

Products of Antigen Recognition (PAR) (mouse, rat)

Lymphotoxin (mouse, human, guinea pig)

Cloning Inhibitory Factor (human)

Proliferation Inhibitory Factor (human)

Inhibitor of DNA Synthesis (human, mouse)

Chemotactic Factors (guinea pig)

Blastogenic Factors (guinea pig, mouse, human)

Interferon (human)

FACTORS PRE–EXISTENT IN CELLS

Transfer Factors (human)

Lymph Node Permeability Factor (rat, guinea pig)

Cytophilic Antibody (guinea pig, rat, mouse, rabbit)

DIRECT LYMPHOCYTE-TARGET CELL CYTOTOXICITY (mouse, human, guinea pig, rat)

In later sessions progress on the purification and chemistry of the factors responsible for the various in vitro activities as well as their relationship to the mechanisms of cell-mediated reactions will be considered.

Dr. John David will now outline the state of the first of the in vitro systems to be studied, namely, the inhibition of macrophage migration.

DR. DAVID: I will limit this discussion to the capillary tube migration system, although it is to be noted that the tissue explant system is being used successfully by Johanovsky and his colleagues in Prague, as well as in other laboratories¹.

The capillary tube system described by George and Vaughan (Proc. Soc. Exp. Biol. 111: 514, 1962) has several advantages as an in vitro system for the study of cellular reactions. These include the ease of quantification and the ability to watch the cells in the populations to be tested. Because of this flexibility, the assay has proved useful in dissecting the mechanism of cellular reactions.

The correlation of this method with cellular hypersensitivity in vivo has been established as follows:

1. Animals must be sensitized either by protein antigens in complete Freund’s adjuvant or by living BCG. Human subjects are sensitized by natural infection. Peritoneal cells from sensitized animals are inhibited by antigen from migrating in vitro; and lymphocytes from such animals or from sensitive human subjects produce migration inhibitory factor (MIF) when stimulated by antigen in vitro.

2. Guinea pigs sensitized with antigen-antibody complexes in complete Freund’s adjuvant by the method of Uhr, Salvin and Pappenheimer (J. Exp. Med. 105: 11, 1957) demonstrate delayed hypersensitivity without detectable antibody. Peritoneal exudate cells from such animals are also inhibited from migrating by specific antigen.

3. Guinea pigs immunized by the i. v. or i. m. route without complete Freund’s adjuvant, which produce certain antibodies but do not exhibit delayed hypersensitivity, yield peritoneal exudate cells which are not inhibited by antigen. In addition, lymphocytes from such animals produce no MIF.

4. The specificity of inhibition of migration has been studied with hapten-protein conjugates and chemically defined hapten-oligopeptides (David and Schlossmann, J. Exp. Med., 128: 1451, 1968). The carrier specificity found correlates with that of delayed hypersensitivity reactions in vivo, and does not exhibit the specificity of the reaction between antigen and humoral antibody.

5. Peritoneal exudate cells are not inhibited from migrating by specific antigen when the donor animals have been made partially tolerant, i.e., that they have diminished or absent delayed hypersensitivity but are still producing antibody.

The reaction involves two cell types. First, sensitive lymphocytes interact with antigen to produce a soluble mediator, MIF. The mediator in turn inhibits the migration in vitro of macrophages (Bloom, B.R., and Bennett, B., Science 153: 80, 1966; David, J., Proc. Nat. Acad. Sci. 56: 72, 1966). It should be pointed out that it is likely that the in vitro system is measuring only some of the initial steps that occur in in vivo reactions of cellular hypersensitivity. It does not take into account the possible release of lysosomal enzymes from macrophages and the subsequent reactions following such release, nor does it assess the effect of other mediators, blood clotting factors or local tissue conditions such as the condition of blood vessels. This point is emphasized by two circumstances in which one may observe inhibition of migration of peritoneal exudate cells from animals which have been sensitized but exhibit negative skin tests. For example, newborn guinea pigs, following sensitization with complete Freund’s adjuvant, exhibit poor skin reactions. Their cells, however, are capable of transferring delayed hypersensitivity into adult guinea pigs. Further, Janicki and his group have shown that peritoneal exudate cells from such sensitized young animals are inhibited from migrating in vitro, suggesting that sensitized cells and reactive macrophages are present, but some other necessary component for the skin reaction is absent. Similarly, scorbutic guinea pigs, when sensitized with complete Freund’s adjuvant, display poor to no skin reactions to PPD. Zweiman and his co-workers (Zweiman et al., J. Immunol. 96: 296, 1966) have shown that cells from such animals are capable of transferring delayed hypersensitivity into normal adults, and William Reed in our laboratory found that peritoneal exudate cells from sensitized scorbutic guinea pigs with markedly diminished skin reactions were nonetheless inhibited from migrating in vitro. However, it was especially interesting that Reed found only about one-tenth as many cells in the peritoneal exudates from scorbutic animals as compared to control guinea pigs. Thus, the lack of skin reactivity might be secondary to a poor exudative response stemming from the known vascular pathology in scurvy. These, then, are but two examples of how the assay might be used to dissect out the etiology of negative skin reactions in anergic states and other situations.

Inhibition of migration is now used extensively as an assay for presence of sensitive lymphocytes or for MIF. Several workers have used alveolar macrophages in such studies, but the peritoneal exudate is still the most common source of target cells.

The sources of sensitive lymphocytes include lymph nodes, peritoneal exudates, spleen and blood. It is of interest that guinea pig lymph node lymphocytes, when packed into capillary tubes, are not themselves inhibited from migrating by antigen; on the contrary, they migrate out of the tube in a monolayer. However, if the same sensitive lymphocytes are added to normal peritoneal exudate cells, containing predominantly macrophages, the resulting population is inhibited by antigen. Thus, the macrophages are the cells affected in this system.

The migration inhibition method can be used to get semiquantitative results in the following ways:

1. When peritoneal exudate cells from sensitized guinea pigs are used (this has been called the direct assay), the medium used to fill the chambers can be. made to contain various dilutions of antigen to determine the least amount of antigen necessary to produce significant inhibition.

2. If one of the various populations of lymphocytes are used, they can be added to normal peritoneal exudate cells in stepwise dilutions to determine the smallest number needed to obtain inhibition of migration with a standard dose of antigen. Further, the amount of escape from inhibition when measured at 48 hours may also reflect the degree of sensitivity of the original cell population.

3. When MIF is to be assayed, it can be diluted in various proportions with tissue culture media to determine the greatest dilution which will inhibit peritoneal exudate cells.

The capillary tube migration assay has been used with a variety of antigens including bacteria, virus and fungal antigens, soluble proteins and synthetic oligopeptides, and carbohydrates. It has been used successfully to detect transplantation antigens by Al-Askari et al. (Nature, 205: 916, 1965) and others, and also, it has proved useful for studying problems with tissue antigens, including brain and thyroid. With appropriately sensitized animals, it may prove useful for assaying the purification of various antigens. Recently, it has been used to detect chemically-induced tumor antigens on cells by Kronman et al. (Science, 165: 296, 1969) and solublized tumor antigens by Bloom et al. (Proc. Nat. Acad. Sci., 64: 1176, 1969).

The assay is especially useful when one is dealing with antigens that give nonspecific skin reactions or when animals have a strong Arthus reaction which makes the reading of the delayed skin reaction difficult. In human subjects it provides a way to study cellular hypersensitivity to tissue or tumor antigens which one would not inject into the skin.

Until this point, I have been discussing the in vitro reactions in this system occurring in specifically sensitized cells stimulated by antigen. It is also of interest to know whether nonspecific stimulation by mitogens would also result in the production of MIF and inhibition of macrophage migration. We have tried to produce MIF both with PHA and pokeweed mitogen. Although it appeared that these mitogens did produce MIF it was difficult to be certain that there was no mitogen left in the materials being tested. These might agglutinate the macrophages directly producing artefactual inhibition. Recently, Pick et al. (J. Immunol. 104: 265, 1970) have independently been studying the effects of Concanavalin A on the production of a skin reactive factor and of MIF by lymphocytes. Concanavalin is especially useful as it is a plant mitogen with the property of binding to carbohydrates, which also enables it to be removed from the solutions being studied, e. g., by binding to Sephadex. We have found that supernatants obtained from lymphocytes stimulated by Concanavalin and then fractionated on Sephadex G 100 yield fractions which are inhibitory to macrophage migration. Similar fractions from control supernatants, to which Concanavalin is added after the cells are centrifuged, are not inhibitory. The peak of activity is found in the fraction eluting from acrylamide gels just after albumin. One might ask whether this is the same MIF as that produced by antigen, or if there is more active material made by this method. These questions are presently under study and the properties of Concanavalin-produced MIF are being compared to antigen-produced MIF. Such studies must include further determination of molecular weight, assessment of electrophoretic properties, sensitivity to chymotrypsin and neuraminidase, buoyancy on CsCl, and heat and pH stability, a project which in our lab is being carried out by Dr. Heinz Remold. If the factors turn out to be the same material but produced in larger quantity, Con A should be most useful in obtaining larger quantities of material to characterize.

At this juncture, I should consider some conditions under which cell migration can be inhibited other than by MIF. It should be apparent that any material that is toxic to macrophages and/or kills them will also inhibit migration. Further, it has been shown by several workers that antigen-antibody complexes, when made in antigen excess using large amounts of antibody, will prevent normal peritoneal exudate cells from sensitized animals, but it must be considered in any in vitro cell inhibition study. It is also possible that cytophilic antibody may affect this system. Although many workers have tried and have been unable to sensitize normal cells with cytophilic antibody, several investigators, including Amos et al. (Int. Arch. Allergy, 32: 496, 1967) have found that certain cytophilic antibodies will sensitize normal cells to PPD. It is of interest that Amos sensitized guinea pigs according to three different schedules, with BCG and PPD. Only one of these immunizing schedules gave cytophilic antibody which sensitized normal cells. However, peritoneal exudate cells from guinea pigs sensitized by any of the three schedules were inhibited from migrating by antigen, demonstrating a clear dissociation between the presence of detectable cytophilic antibody and inhibition of migration, which has been reported by others. In Amos’ experiment, it is possible that the cytophilic antibody was eluted from the cells when they were placed in tissue culture medium containing normal serum, and that antigen-antibody complexes were formed with the added antigen which in turn inhibited the migration of macrophages.

The cell migration system has been applied successfully in several animal species. The most extensively studied has been the guinea pig. There are some studies in rats, although several workers have found it difficult to get consistent migration from rat peritoneal exudate cells. The system has been used also with cells from mice, hamsters, and more recently, man. A notable new addition to this list from the work of Prendergast is perhaps the sea star (Forbesi et al., Fed. Proc. 29: 2963, 1970).

Thor and Dray placed human lymph node cells in capillary tubes and found they were inhibited from migrating by specific antigen, but only if the cells had been preincubated for three days prior to placing them in the capillary tubes; this requirement for lymph node cells limited the usefulness of the test. However, the same workers went on to find that supernatants obtained after the culture of blood lymphocytes with antigen and concentrated, inhibited the migration of normal guinea pig peritoneal exudate cells. (Thor et al., Nature, 219: 5155, 1968). The ability of MIF to cross species barriers was of interest in itself but also makes possible a useful assay in man. I would stress that human MIF can be assayed on guinea pig cells, but it must be concentrated to be detected; this may represent a species difference.

In our laboratory, Rocklin has confirmed Thor’s work and is now using the method as a routine assay for delayed hypersensitivity in the investigation of various clinical problems (Rocklin et al., J. Immunol. 104: 95, 1970). The assay correlates very well with delayed hypersensitivity as measured by skin test, is reproducible, and the area of migration of guinea pig cells in human supernatants is easy to read. However, there are some disadvantages to the technique, namely that it takes a considerable amount of time and work, as concentration of supernatants is necessary, and requires a large number of cells. Rocklin has found that lymphocytes from patients with agammaglobulinemia who have delayed sensitivity will produce MIF; in contrast, lymphocytes from patients with Di George syndrome, with no delayed sensitivity but able to make antibody, produce no MIF. Lymphocytes from one such patient was found to produce MIF after immunologic reconstitution with a thymus transplant.

In addition, Rocklin has been using these methods for the study of patients with anergy. One of his recent findings is especially interesting: an apparent dissociation is seen between the skin test and MIF production on the one hand, and thymidine uptake by lymphocytes on the other. In studying over 30 patients with various diseases associated with anergy, he has found 10 persons (1) who have negative skin test to SK-SD and PPD; 2) whose blood lymphocytes produce no detectable MIF in response to these antigens, but 3) whose lymphocytes respond well in vitro to these same antigens when the incorporation of ³H-thymidine is being measured. Thus, as far as the assays presently used can measure, cells may proliferate in response to antigen even when obtained from patients with negative skin test and negative MIF production. Although it is possible that the incorporation of thymidine is just a more sensitive assay, the normal thymidine incorporation in some of these patients in the presence of negative skin test and MIF makes this unlikely.

Another type of assay for human hypersensitivity is that described by Søborg and Bendixen (Acta Med. Scand. 181: 47, 1967, and Method 9). They packed blood buffy coat cells directly into capillary tubes and demonstrated inhibition of migration using Brucella antigen. The observed inhibition of migration correlated with delayed sensitivity to Brucella as measured by skin test. They also have reported success with this technique using several other tissue antigens, but most of the antigens have been particulate. This has become a controversial subject because a number of investigators have been unable to obtain consistent results when using soluble antigens in this assay system. When I was at New York University, Dr. Lawrence and I tried many times to assay buffy coat cells. Occasionally the results were spectacular and had us all dancing, but unfortunately they were never consistent.

After the reports of Søborg and Bendixen, these efforts were renewed, but Meyers and I were again unsuccessful. Recently, there have been two reports published, one by Kaltrieder et al. (J. Immunol. 103: 179, 1969) and another by Lockshin (Proc. Soc. Exp. Biol. Med., 132: 928, 1969) reporting negative results using PPD in the buffy coat system. It would indeed be curious if PPD, which works so well in other systems of delayed hypersensitivity, did not work in this one.

Recently Dr. Steven Rosenberg in our laboratory has been working on this assay, hoping to have a 24-hour assay for human delayed hypersensitivity. The first 62 patients he studied, using several protocols (including those of Kaltrieder and Lockshin) were either negative or inconsistent. More recently, after changing several parts of the technique, and increasing the doses of antigen, he has obtained quite consistent results in 25 consecutive patients.

DR. BLOOM: Dr. Chase would like to comment on his studies on migration inhibition in guinea pigs.

DR. CHASE: Dr. Marcus and I undertook to ascertain the time at which macrophage inhibition could be demonstrated in relation to the onset of allergic conversion. We chose the tuberculin system and the outcome was rather different from results reported to date in the literature.

Intradermal sensitivity to tuberculin is established in a few guinea pigs on day 5, perhaps in 40% by day 6, and in up to 85% by day 7. Dr. Marcus has found that macrophage inhibition is demonstrable on day 6 in the 40% of the animals which convert by this time.

For this investigation, paraffin oil was introduced intraperitoneally on day 4, a tuberculin test (0.5 ug) was made on day 5, tuberculin-reactors were chosen on day 6 and cells harvested. Findings have been parallel in both strains of guinea pigs used to date – the Rockefeller University albino strain and Wright’s strain XIII. Sensitization was established by injecting, at one time, five intradermal sites, each with 5 ug of mycobacteria H37Ra in 0.05 ml of medium-weight paraffin oil. Some animals were sensitized with half this weight of mycobacteria. This method of sensitization is highly efficient, and it allows rather satisfactory determination of the time of allergic conversion by daily inspection of the local sites.

Apart from the method of sensitization, several features of the techniques used are worth recounting. The cells are not cooled at any time during the 3 to 4 hours allowed for setting up a test and they are not handled in refrigerated centrifuges. To save time, cells are not counted by hemocytometer; since macrophages essentially determine the turbidity of washed peritoneal exudates, cell harvests from individual donors are determined from spectrophotometer readings at 660 mu on side portions of suspensions diluted in 3% acetic acid. Cells harvested from individual donors are suspended in 5 ml of gelatin-Hanks solution and are diluted 1:5 in 3% acetic acid in 10 ml Perfect-Round Coleman Jr. curvettes. The final suspensions (4 ml) are swirled just before reading so that the cells are in motion. A standard curve is used as reference, prepared from hemocytometer-counted cells.

Weybridge PPD, human type, is added to the extent of 20 µg/ml in the medium, not only in the final chamber but also in the suspension being centrifuged within the capillary tubes, following advice from Dr. Bloom.

Serum for inclusion in culture medium (15%, in MEM containing L-glutamine) is inactivated and is taken from guinea pigs screened according to the criteria of Dr. J. R. Battisto, in order to exclude serum of animals possessing serum factor. (Such samples of serum cause reactions when injected intradermally into guinea pigs of the so-called isohypersensitive type.

It may be useful to state the criteria which Dr. Marcus and I accept for significance in evaluating differences in migration observed when cells are compared in outgrowth in the presence and absence of PPD. These conclusions were reached after studying migration from 5 through 52 days following sensitizing injections on day 0. As customary, projections are compared of the areas of migration out of capillary tubes, calculations being made both after 24 and 48 hours of incubation.

Variation among control samples is in the range of 5-10%, so the 10% level is excluded as significant. At the 20% level, an interpretation of inhibition must be regarded as suggestive only. We accept inhibition as indicating hypersensitivity whenever either of two criteria are met by cells of sensitized animals: a) normal cells migrate within the normal range in the presence of PPD and b) migration at 18 hours in the presence of allergen is found to be between 50% and 70% of the control, providing that migration at 48 hours is still not greater than 70% of the control. We have noted no disparities between quadruplicate tests excepting when alkaline pH shift occurred rarely in single chambers.

The finding that allergic conversion and the property of macrophage inhibition arise at the same time reinforces the concept that macrophage inhibition is a correlate of delayed hypersensitivity. At the very early period of allergic conversion, inhibition of migration is expressed clearly and well after 24 hours of incubation. In the next 24 hour period, outgrowths of cells from the capillary stubs occur to a greater extent, on average, than are found one or two weeks later.

Macrophage inhibition is a more sensitive tool than has been appreciated generally; it should serve for primary investigation in many systems.

DR. THOR: In first trying to develop a technique which would allow us to apply migration inhibition to man, the biggest problem we faced was in obtaining a migrating cell population which gave reproducible results. Initially we used a peritoneal exudate system in humans, using exudates from patients that were on peritoneal dialysis. These initial experiments failed to give reproducible results, probably because of the type of patients which provided the exudate cells, i. e., either uremics or others with primary diseases which caused difficulties in studying migration inhibitory factors.

While there have been some recent reports now that peripheral buffy coat cells will work in the migration inhibition system, we tried this on numerous occasions and had difficulty in obtaining reproducible results. We went from this system to the use of lymph node biopsies, and found initially that lymph nodes also did not migrate or inhibit well. That is why we chose to use the 72 hour tissue culture system-carrying lymph node cells in culture for 72 hours until they would migrate. Using such cells it was possible to get migration inhibition in reproducible fashion. I think it is most interesting now that Dr. Glade and others are finding that continuous lymphocyte cell lines will migrate and possibly work in this system. In our hands also these 72 hour lymphocyte cultures also produced considerable amounts of migration inhibitory