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' . g . - --- n - ' . - - n - . ‘ q o _ . - . . c.‘ . . . u . . .. ‘ - , '. a - ~ ‘ _ . ‘ ' ‘ .. . ~ . . ‘ .\ .‘. ' ' ' - . 4' . I - i ' I - . I ~ - . . t- . .. . _ . . . u . . - . . . ' . ‘ , I I . I , _ - . . - O . * u , ' - . - p - . . ‘ . ‘ ‘ . . . Q. ~ I . . _ o ' . . ' o 0 V l . . ‘ ' . I c n ' - ' . . ,, c . . . . o « o . " Q o . . . . . . . . . . - . \ . . o . ‘ o u . ' o o " I - - . u . ..- I . ‘ ’ r - ‘ o ‘ u ' n . . - , ~ . I - . A . ' ’ . . a x . | ' , . I ‘ t . t ' . ’ , ,. - I ' ‘ ~ _ I . v - - - _ . . w - ' n ‘ 0 .. ’ . 0v ‘ . ‘ ¢ . D ' ~ , . . o . o I o n ' . ‘ - - o u ' 0 c I ' o b . ‘ . . u . I a . ‘ u ‘ . . . . . . O . . . l I . O u - o . \ I ; LIBRARY Michigan State g University 3‘ nERY INC. B'NF7R5 J ' may I ; :2. Bnnwu ABSTRACT QUANTITATION 0F CONTACT SENSITIVITY RESPONSES IN THE MOUSE BY 3H-THYMIDINE LABELING OF LYMPHOCYTES By Erich Eipert Quantitation of delayed hypersensitivity responses has usually re- quired the measurement of crude skin reactions. In a new ear assay for delayed contact sensitivity, mice were sensitized to sheep red blood cells (SRBC) and injected with 3H thyminedeoxyribose (3H TdR) to label antigen sensitive cells. The accumulation of labeled cells in ear challenge sites at twenty-four hours was then assessed by scintillation counting. Posi- tive responses were obtained when one ear was challenged with SRBC and the other with phosphate buffered saline as a control. However, a consistent response could not be demonstrated when non-crossreacting erythrocytes were used in the control challenge. Modification of the assay permitted the detection of contact sensitivity induced by dinitrochlorobenzene (DNCB) Skin painting. The method was shown to be capable of detecting this cellular response by producing consistent responses. Specificity was tested in two ways. Following challenge, DNCB sensitized mice showed an increased response to DNCB when the control was provided by phenylene- diamine (PD), a second contact sensitizing chemical. If the animals were sensitized to PD however, response to DNCB was minimal. When the animals were made tolerant to DNCB by a 15 mg intravenous injection of dinitro- benzene sulfonic acid (DNBS), the response was again largely erased. fl .4.) \ } f. C;- Unexpectedly, subtolerogenic (1 mg) amounts of DNBS were found to enhance {2 o l l L") response to DNCB if injected intravenously during or prior to sensitization. It was concluded that the assay is antigen specific and is detecting labeled cells, probably of the recirculating lymphocyte type. QUANTITATION OF CONTACT SENSITIVITY RESPONSES IN THE MOUSE BY 3H-THYMIDINE LABELING OF LY‘MPHOCY'I‘ES By Erich Eipert A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1974 TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF THE LITERATURE . . . . . . . . . . . . . . . . . . . . . 3 Historical Perspective of Delayed Hypersensitivity . . . . . 3 Recoqnition of the Phenomenon . . . . . . . . . . 3 Recognition of the Cell Type Involved in Delayed Responses . . . . . 5 Characterization of Delayed Hypersensitivity . 6 In Vitro Measurement of Delayed Hypersensitivity . 7 Blast Transfbrmation . . . . . . . 8 Inhibition of Macrophage Migration . 9 Macrophage Activation . . 9 Interferon . . . . . . . . . . . . . . 9 cytotoxicity and Inhibition . . . . . . . . . . . . . 10 In Vivo Measurement of Delayed Hypersensitivity . . . . . . 10 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 14 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Irradiation . . . . . . . . . . . . . . . . . . . . . . . . l4 Antigens . . . . . . . . . . . . . . . 14 Cell Suspensions and Transplantation . . . . . . . . . . . . 15 Immunization . . . . . . . . . . . . . . . . . . . . . . . 15 Radioactive Labeling . . . . . . . . . . . . . . . . . . . . 15 Ear Assay . . . . . . . . . . . . 15 Measurement of Cell 3H TdR Incorporation . . . . . . . . . . 16 Assay for Plaque- forming Cells . . . . . . . . . . . . . . . 16 Evaluation of Ear Responses . . . . . . . . . . . . . . . . 17 Coupling of DNBS to SRBC . . . . . . . . . . . . . 17 Preparation and Use of Antithymocyte Serum . . . . . . . . . 17 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Evaluation of SRBC Ear Assay . . . . . . . . . . 19 Measurement of Delayed Contact Sensitivity by Ear Assay . . 21 Intensity and Specificity of Response to DNCB . . . . . . . 22 Determination of the Mediator Cell Source ,. . . . . Detection of Antibody Producing Cells . . . . . . . . . . . 32 ii DISCUSSION . SUMMARY APPENDIX 3 BIBLIOGRAPHY . H TdR Incorporation by:Normal and Sensitized Mice . iii Page 33 39 4O 43 Table LIST OF TABLES Comparison of ear assay responses for routine and low dose immunized mice challenged with PBS and SRBC . Ear assay responses of mice immunized sq with a routine dose of SRBC and challenged with CBRC and SRBC . . . . . . . . . . . . . . . . . . . . . . . Comparison of responses in SRBC and DNCB immunized mice challenged by a control and a test antigen Ear responses obtained from DNCB sensitized mice: effect of varying DNBS injections . . . . . . . DNCB re5ponses obtained in A) PD sensitized mice, B) nonsensitized mice and, C) DNCB sensitized mice challenged with DNCB and olive oil . Effect of azathioprine on ear response in DNCB sensitized mice Effect of in vivo administered ATS on response of DNCB sensitized CBA mice Day 6 plaque-forming cells/spleen of DNCB skin painted BCFl mice . . . . . . . . . . . 3H TdR incorporated by normal and sensitized mice iv Page 20 21 23 24 27 30 31 32 42 INTRODUCTION Cell mediated immunity and delayed hypersensitivity (DH) have been studied in many systems, but after decades the most widely used in vivo detection methods are still those which measure visible or gross manifes- tations. Because of the complexity of cell mediated reactions and the lack of production of any one definitive soluble mediator in these reac- tions, macroscopic tests such as measurement of induration, footpad swelling, and ear thickening, in all likelihood will be in use for years to come. Nevertheless, there exist at present a number of alternate techniques. The subject of this study involves one such method in which uptake of the radioactively labeled nucleotide 3H thyminedeoxyribose (3H TdR) by proliferating lymphocytes is used as an index of deoxyribonucleic acid synthesis and has been shown to correlate to DH (62). The validity of the labeling procedure rests on two premises: 1) the labeled cells or at least some of them are specifically sensitized to antigen and 2) the labeled cells largely retain their label during the course of the experi- ment. Indeed, these assumptions have been borne out in several studies (7,9,68) and are the subject of an extensive review (27). Evidence exists that animals with competent, circulating, labeled lymphocytes, up- on appropriate challenge, exhibit a characteristic localized accumulation of labeled cells (29,42,63). In a test designed to measure delayed re- Sponses to purified protein derivative (PPD), the observations concerning cell labeling were applied to sensitized cells in guinea pigs (62) and 2 recently extended to measuring the delayed response to Sheep red blood cells (SRBC) in mice (73). Unfortunately, specificity was not satisfac- torily tested in this latter report. Experiments reported herein, using both SRBC and a control antigen challenge in each mouse, could not sub— stantiate claims of sensitization to SRBC. Recently, contact sensitivity to simple chemicals has emerged as one of the most widely used means of studying DH as well as T cell Specificity and function in the mouse. Advantages of this system are 1) sensitivity is easily induced, 2) a nearly pure T cell response is evoked, 3) the antigenic determinant is simple and defined, and 4) a gross response measureable by conventional means is produced. Several recent reports have utilized this system to study various aspects of contact sensitivity (3,4,65,66) by measurement of ear thickness of challenged non—sensitized animals vs challenged sensitized animals as a gauge of response. The work described in this paper represents the adaptation of the 3H thymidine assay to the measurement of contact sensitivity in a proce- dure employing more rigorous controls than those used previously (5,65, 73) by providing both a test and control challenge in each mouse. Spec- ificity has been tested by a reverse procedure in which mice are sensi- tized to the control antigen and has also been tested in animals made tolerant to the sensitizing agent. The finding that subtolerogenic levels of DNBS, used to induce tolerance, actually enhances in vivo re- sponses is discussed. REVIEW OF THE LITERATURE Historical PerSpective of Delayed Hypersensitivity Recognition of the Phenomenon Cellular hypersensitivity, a form of cellular immunity, may be de- fined as an inflammatory reaction which is immunologically specific, takes hours to reach maximum intensity and occurs in the absence of demonstrable antibody. Because they meet this definition, reactions such as homograft rejection, tumor suppression, contact hypersensitivity, experimental auto- allergy, and bacterial hypersensitivity are now generally included in this category. Extensive study of the latter in the classic tuberculin reac- tion has led to the term delayed hypersensitivity which is often applied to this class as a whole. Various aspects of DH have been touched upon or described throughout the history of medical science. Jenner was proba- bly the first to describe the reaction under controlled conditions. He noticed a 'reaction of immunity' in revaccinated persons which reached a peak intensity within twenty-four to seventy-two hours. In 1890, Koch provided one of the earliest descriptions of the hypersensitivity reaction produced by subcutaneous injection of tuberculin in tubercular patients. He was also the first to recognize the diagnostic value of the reaction. Although he used fever to detect reactions, the skin response became the standard in the study of tuberculosis for the next 30 years. Zinsser (80) recognized that the tuberculin reaction represented a 3 4 principle applicable to bacterial infections in general. This work was supported by Dienes (21) who studied the histological aspects of the tuber- culin reaction which he characterized as a slow exudative phenomenon with an early infiltration by mononuclear cells. He contrasted this to the anaphylactic reactions in which there was a rapidly developing edema fol- lowed by intense polymorphonuclear infiltration. He found that ordinary proteins can produce much the same effect and the best method of producing this DH was to inject the antigen directly into the tuberculous focus (20). Another form of DH is the skin reaction resulting from contact by simple chemicals. Although cases of contact sensitivity to such diverse materials as East Indian Satin wood and mercury were reported in the early 1900's, the mechanism of this sensitivity was not systematically investi— gated until Landsteiner's work in the 1930's (46). Working with a number of chloro- and nitro- substituted benzene products, he found that sub- stances containing loosely bound Cl' and N05 which would interact with amino groups of the organic base aniline were the substances that produced contact sensitivity in guinea pigs. The analogy drawn was that the sim- ple chemicals were built up into antigens by attachment to proteins in vivo. Landsteiner was also able to produce contact sensitivity in guinea pigs by infecting picryl chloride conjugated erythrocyte stroma along with tubercle bacilli as adjuvant. This suggested to him that the reac- tion must be related to bacterial type allergies. The fOrmal link was provided when Landsteiner and Chase (47) passively transferred contact sensitivity to picryl chloride in guinea pigs by peritoneal exudate (PE) cells as was done for tuberculin hypersensitivity (18). Brent (15) demonstrated that homograft reactions were also related to these phenomena. Skin grafts normally tolerated by the host were S rejected if the host were first injected with lymphoid cells sensitized to donor skin. Furthermore, an inflammatory response resembling the tuber- culin reaction was produced when lymphoid cells from a homograft recipient were injected into the skin of the donor. Recognition of the cell Type Involved in Delayed Responses The cell eventually implicated as the antigen sensitive cell in cellular responses was the circulating small lymphocyte. Some of the earliest evidence of this was provided by experiments in which it was shown that passively transferred small lymphocytes obtained from the thoracic duct or peripheral blood of mice and rats were capable of pro- ducing a graft vs host (GVH) response or enhancing homograft rejection (10,34). Abundant evidence now exists equating these recirculating lymphocytes with thymus derived (T) cells. This point is most strikingly emphasized by the reduction or ablation of cellular reactions such as homograft rejection and GVH reaction in T cell suppressed, neonatally thymectomized, or congenitally athymic animals (34,44). The class of cells designated as T cells is today under intense scrutiny. Various types of heterogeneity has been ascribed to these cells as a result of these studies. Raff and Cantor (67) have proposed an arbitrary designation of T1 and T2 cells on the basis of migration in irradiated recipients, ability to react to histoincompatible cells in a GVH reaction, and amount and type of surface antigens by reaction against specific antisera. Blomgren and Andersson (12) have found in mice a cor- tisone resistant fraction of thymus cells which accounts for only 3-5% of the total thymus, yet displays in GVH and antibody helper function as- says a response equal to that of an intact thymus. Discontinuous density gradient separation of thymus cells by Levey and Burleson (49) has revealed 6 further heterogeneity. When several parameters, including 3HTdR uptake in response to antigen stimulation, cell proliferation in response to mito- gen stimulation, and ability to mount a GVH response were applied, only three of nine fractions were active in one or more of these properties. On another level Cantor and Asofsky (16) have presented evidence for the synergism of two types of T lymphocytes in GVH reactions in mice. In a regulatory role, suppressor T cells have been reported (31) which are able to reduce specifically both antibody response and T cell prolifera— tion. These cells may represent another subpopulation of T cells. Characterization of Delayed Hypersensitivity A typical delayed reaction can be described as follows: the reaction begins at four to six hours and peaks at twenty-four to forty-eight hours. Gross manifestations are induration, erythema, and even necrosis in severe reactions (76,77). One of the earliest effects is an increased permeability of blood vessels resulting in the accumulation of fluid and cells in the tissue containing antigen. The area of infiltration is de- termined largely by the extent of antigen diffusion. Infiltration by mononuclear cells increases rapidly over twenty-four hours. At the peak of infection 90% of these cells are mononuclear. In a study by Najarian and Feldman (62), guinea pigs sensitized to PPD or other proteins, in- jected with 3H TdR and subsequently skin tested, have been shown by auto- radiographic techniques to contain fewer than 10% specifically sensitized lymphocytes at the site of inflammation. Kosunen et al. (43) in a similar type of experiment have reported higher values. Remaining cells were mainly macrOphages which exhibit activation characterized by increased size, a higher level of pinocytosis, enzyme changes, and stickiness (77). The aSpect of DH most vigorously pursued in recent years has been 7 the mechanism responsible for this type of reaction. At least partial success in this regard can be claimed in the discovery of a number of sol- uble mediators of inflammation and tissue damage expressed in delayed re- actions. In early studies, little importance was placed on making a dis- tinction between lymphocytes and macrophages involved in DH reactions. The work by Rich and Lewis (69) 40 years ago showed that when spleen or lymph node cells from sensitized guinea pigs were put in vitro with anti- gen, cell migration was inhibited. More recently David (19), George and Vaughan (30), and Bloom and Bennet (13) showed that this inhibition is the result of macrophage inhibition factor (MIF) elaborated by lymphocytes. Since the discovery of MIF, it has been demonstrated that antigen contact with sensitized lymphocytes is the initiating event for the production of a number of other solUble factors as well. Among these are chemotactic factors (78), lymphotoxic factors (71), growth inhibitory factors (48), and mitogenic factors (48). Discovery of these mediators has strengthened the link between the various forms of cellular hypersensitivity and has served to explain various manifestations of DH, but consequently has prov- en to be both a blessing and a curse. Their varied effects have opened a number of investigative avenues of approach to DH but at the same time have added another dimension to its complexity. In Vitro Measurement of Delayed Hypersensitivity Elucidation of events associated with antibody production came about in a very short time. This was due largely to the development of the hemolytic plaque assay (40) which allows precise quantitation of anti- body producing cells. Unfortunately, no comparable technique exists for measurement of DH, largely because of the lack of a mediator produced in 8 stoichiometric amounts and the sheer complexity of the reaction. Con- sequently a great deal of effort has been devoted to the study of various aspects of this reaction in vitro. A comprehensible review cannot attempt to cover 4%? research conducted in this area but may point out key experi- ments, p tgications and reviews. The essential criterion in all of the Iarget cells. Particular systems vary from laboratory to laboratory ‘:d ideally attempt to measure one element of DH. These can be catego- ; ized by five general parameters. Blast Transformation Upon confrontation with antigen or mitogens, a certain number of exposed lymphocytes undergo a blast transformation. These cells differen- tiate to a lymphoblastic form characterized by the appearance of nucleoli, increased basoPhilia, diffuse chromatin, and eventually increased numbers of mitotic figures (50). The assessment of blast transformation is tech- nically very simple, and is usually run by placing lymphoid cells in cul- ture with antigen or mitogen. After seventy-two hours, transformation is evaluated by staining and observing morphological features (vide supra) or monitoring increased RNA, DNA, or protein synthesis by measuring up- take of radioactive precursors. Supernatants of such cultures may also be tested fOr 'blastogenic factor' by addition to cultures of normal lymphocytes and observing the effect (22,56). Inhibition of.Macrophage Migration Early studies of tuberculin sensitivity indicated that addition of antigen to cultures of leukocytes from tuberculin sensitized animals was more cytotoxic than addition of antigen to normal cultures (38). Rich and Lewis (69) found that tuberculoprotein also caused an inhibition of migration of cells from spleen and buffy coat. George and Vaughan (3) developed the capillary tube method to make the procedure more quantita- tive. David (19) was able to show that this effect was due to the soluble mediator MIF, and was produced by sensitized lymphocytes eight or more hours after antigen contact. Macrophage Activation Activation has been described by various parameters such as in- creased adherence to glass, cell size, particle uptake, lysosomal enzymes, and ameboid activity. These changes have been linked to release of sol- uble factors by lymphocyte-antigen interaction (1,60), but the number of factors, the specificity of each, and the isolation of each are problems yet to be resolved. Increased activity in clearance of bacteria and other particulate antigens has generally been considered nonspecific (52), although an increasing number of reports have appeared attesting to spec- ificity in antigen clearance (8) as well as cytotoxicity (25). Interferon Certain antigens or mitogens upon contact with sensitized lympho- cytes cause the release of a factor known as interferon. This factor bestows on lymphocytes a protective effect against a number of viruses and is measured by plaque reduction or end point dilution (36). 10 cytotoxicity and Inhibition Cytotoxicity and inhibition have been the most frequently-used in vitro methods because of technical ease and the obvious parallel of tis- sue damage and cell death in the in vivo situation. A number of individ- ual methods exist for determining cytotoxicity including gross observa- tion (33), enumeration of plaques in a monolayer (51), cell counts (79), and isotope release (64). ‘These methods have been employed by many re- searchers in a variety of systems. Confusion exists because of the large variety of techniques in use. This introduces many variables, most of which concern the purity of the effector cell pOpulation and the type of target cells. For example, the use of spleen, lymph node, thoracic duct, or PE as a source of effector cells by different investigators affects re- sults because these sources differ in prOportion of contaminating bone marrow derived (B) cells and may well differ in T cell activity. Various systems also differ as to whether macrophages, sensitized lymphocytes, or a combination of the two effect cytotoxicity. The combination of these unanswered questions and the inherent difficulty in correlating numbers of cells killed in these different systems to an in vivo delayed reaction makes interpretation of results difficult. Obviously the above methods are useful for quantitating responses within a particular system. However, by way of generalization it may be said that while in vitro methods exhibit a broad correlation to the in vivo phenomena, meaningful quantitative extrapolation to the in vivo situation is difficult. In Vivo Measurement of Delayed Hypersensitivity Cell mediated immunity is demonstrated in vivo by a number of dif- ferent measureable reactions. These include GVH reactions, transplantation ll rejection, tumor suppression, and skin reaction. All have been success- fully induced in humans, guinea pigs, and rats. While the mouse is the classic animal for transplantation and tumor studies (11,57), delayed skin reactions, which have proven to be one of the most useful DH.manifesta- tions in guinea pigs, are not consistently produced (5,35). This problem has been circumvented to some extent by measuring footpad swelling or ear thickening instead of measuring an area of induration on the skin, al- though success has been claimed with skin reactions (32). It is quite evident that assays of this type in any experimental situation are quali- tative because the measureable effects of DH are produced largely by non- specific cells. These methods are influenced by the activity of the lymphokine-producing cells and the number of macrophages participating in the reaction. In attempts to obtain more quantitative information about delayed reactions, several researchers have made use of 3H TdR uptake by proliferating cells as a measure of DNA synthesis. Tritiated thymidine uptake by lymphocytes responding to a variety of antigens has been demon- strated both in vivo and in vitro (7,23,63). Studies indicate that in a syngeneic system, labeled lymphocytes are stable and reutilization pre- sents little problem for several days (9,27,68). Several experiments have been described in which guinea pigs were sensitized to PPD, an anti- gen evoking a good delayed response, and then injected with 3H TdR. Lymphocytes passively transferred to normal recipients, upon ear challenge were found to produce visible skin reactions. Correlation of 3H TdR up- take to DH was provided by autoradiographic techniques which demonstrated that 2-12% of the responding mononuclear cells were labeled while a maxi- mum of only 0.8% were labeled in normal animals when challenged with the same agent (61,62). Two other groups were unable to confirm these 12 observations (37,41). McCluskey et al. (54) found labeled cells at specif- ic challenge sites to average about 4% of the total mononuclear infil- trate. This group concluded that the small number of specifically labeled cells necessary to elicit a delayed response made a mechanism of specific attraction for these cells unnecessary. Najarian and Feldman (61) further explored the problem by injecting mixtures of labeled tuberculin sensitive cells and unlabeled dinitrochlorobenzene (DNCB) sensitive cells into nor- mal guinea pigs and studying the appearance of labeled cells at reaction sites. When the test antigen corresponded to the competency of the la- beled cells, a greater percent of the infiltrating cells was labeled. These data indicated that cells may accumulate at the site randomly but sensitized cells are retained more efficiently. Sprent, Miller and Mitchell (75) in an adoptive transfer system were also able to show lo- calization of sensitized circulating lymphocytes in lymph nodes shortly after intravenous (i.v.) injection of sheep red blood cells (SRBC). Ford and Marchesi (29) have similarly demonstrated localization and the selec- tive disappearance of antigen sensitive thoracic duct cells in mice when fOotpads were injected with SRBC. Early in vivo studies of DH were carried out largely with tubercu- lin, bacteria, or contact sensitizing agents. A number of other antigens have since been employed in several systems. Asherson and Ptak (5) re- ported ear swelling in mice with protein antigens in addition to a num— ber of contact sensitizing chemicals. It has been reported that low doses of some antigens stimulate T cells while B cells are left unaffect- ed (59, 74) and that this same dose optimally produces DH in mice (42). This was demonstrated in the latter report by fOOtpad swelling in mice sensitized with a low dose of SRBC. Sabolovic et a1. (73), in mice, have 13 used SRBC and 3H TdR labeling in an ear assay similar to that used earlier with PPD in guinea pigs (62). SRBC were injected subcutaneously (sq) and one day later injected with 3H TdR intraperitoneally (ip). Ears were then challenged by intradermal (id) injection of the test antigen and the saline control. TWenty-four hours later the ear tissue was removed and prepared for scintillation counting. An increase of activity in the anti- gen challenged ear was shown; however, specificity of the response was not demonstrated because only saline was used as a control. In a variation of the ear assay, Asherson and Allwood (3) prepared picryl chloride sen- sitized lymph node cells, labeled them nonspecifically in vitro with 51Cr and injected these cells into recipients. Assay showed that cells from immune animals accumulated at a site of challenge to a much greater de- gree, but this method did not distinguish antigen-sensitive cells and hence the question of specificity was not answered. In summary, it is clear that cellular responses are varied and complex. While several forms of the classical skin reaction have been and still are widely used to measure this type of response in vivo, it must be recognized that results obtained are at best only qualitative. Quantitation is difficult since no one factor is produced in amounts easily measureable. Only relatively recently have investigators utilized radioactive labeling in this problem. This was done either by specifical- ly or nonspecifically labeling lymphocytes which are capable of passively transferring DH, and then measuring accumulation of label in locally challenged tissue. Unfortunately, controls have often been neglected in this type of experiment, leaving the procedure short of proven and speci- ficity of the results in doubt. MATERIALS AND METHODS Mice (C3H/He x C57BL/102)F1, abbreviated BCFl, and CBA/J female mice, two to four months old, were used for all experiments. BCFl mice were obtained from Health Research Inc., Seneca, New York or Cumberland View Farms, Clinton, Tenn. and CBA mice from Jackson Laboratories, Bar Harbor, Maine. Irradiation 60 . . Mice were exposed to 1000 rads of Co y-radiation from an em1tter contained in the Department of Food Science facility at Michigan State University. Mice were rested at least 4 hours before transplantation. Antigens Sheep red blood cells (SRBC), chicken red blood cells (CRBC), and goat red blood cells (GRBC) were preserved in Alsever's solution and washed three times with phosphate buffered saline (PBS) before use. l-chloro-2,4 dinitrobenzene (DNCB) and 2,4-dinitrobenzene sulfonic acid (DNBS) were purchased from Eastman Kodak Co., Rochester, New York and the latter was recrystallized as follows. Fifteen grams of the salt was dissolved in a liter of 95% ethyl alcohol by heating to 70° C. Powdered charcoal was added and the mixture stirred and filtered through three thicknesses of Whatman No. 3 paper on a Buchner funnel. DNBS crystals appeared on slow cooling. This procedure was repeated a total of three 14 15 times. Phenylenediamine (PD) was purchased from Fisher Scientific Co., New Jersey. Cell Suspensions and Transplantation Nucleated spleen, bone marrow and thymus cells were suspended in Eagles medium (MEM) and counted with the aid of an eosinophil counting chamber. 6.7 x 107 cells were injected into a lateral tail vein of ir- radiated mice. The DNCB sensitization procedure was begun two days later. Immunization SRBC immunization was carried out by iv or sq injection of the antigen as indicated. 5 x 108 SRBC were considered a routine dose and 2 x 105 SRBC a low dose. Animals were injected once at the beginning of the experiment. Hapten sensitization was carried out by painting the clipped abdomen of mice with four drops of a 1.5% solution (3% in experi- ment reported in Table 3) of DNCB or PD in olive oil on days 1 and 3. The solution, in olive oil, was dropped onto the skin by syringe and 21 gauge needle. Radioactive Labeling 3H TdR, specific activity 20 Ci/mmole, was purchased from New England Nuclear, Boston Mass., and diluted to 10 u Ci/ml shortly before use. Mice were given three 1.5 u Ci injections ip at eight to ten hour intervals beginning 1 1/2 days before challenge unless otherwise indi- cated. Ear Assay Challenge with the erythrocyte antigens was done by id injection of 5 u l of a 50% suspension in the pinna of the ear by means of a microliter 16 syringe and 30 gauge needle. Challenge by DNCB or PD in olive oil was accomplished by applying one dr0p of a 1.5% solution of the agent to the ear and spreading over both surfaces with the flat of the needle. TWenty- four hours after challenge, pinnae were excised with a scissors along the base of the ear and solubilized in 1 ml of Soluene (Packard Co., Downers Grove, 111.). Following solubilization at room temperature, 15 ml scintillation fluid consisting of 4 g 2,5-diphenyloxazole (ppo) and 50 mg 1,4—bis[2-(4-methyl-5-phenylorazolyl)] benzene (dimethylpOpOp; Research Pro- ducts International Corp., Elk Grove, Ill.) in one liter toluene was added. The vials were stored in the dark for one week to eliminate chemolumi- nescence (27) and counted in a Packard model 3320 scintillation counter. Measurement of Cell 3H TdR Incorporation Cells were harvested from mice at the time the ear assay would normally be performed. Pooled spleen suspensions for each group were prepared in MEM and counted with the aid of an eosinophil counting cham- ber. PE cells were harvested by injecting donor mice ip with three ml of PBS shortly before sacrifice. The cell suspension, which was first aspi- rated by syringe from the abdominal cavity upon sacrifice, was passed through a glass wool column formed from a 10 ml syringe to remove adher— ent cells. Aliquots of 0.1 ml were solubilized in 1 ml of Soluene and prepared for scintillation counting in the same manner as ear tissue (vide supra). AsSay for Plaque-forming Cells The number of direct (198) and indirect (7S) plaque-forming cells in spleens of DNCB sensitized mice was determined on day 6 by the Jerne hemolytic plaque method (40) as modified for use with agarose gel on 17 glass microscope slides. Details of the procedure are described by Miller and Cudkowicz (55). Evaluation of Ear Responses The following formula was used in the calculation of specific challenge responses: 0 6 INCREASE = CPM (TEST EAR) - CPM (CONTROL EAR) x 100 CPM (CONTROL EAR) Negative responses were obtained when counts per minute (CPM) in the con- trol ear exceeded CPM in the test ear. An increase of 10% or greater was designated a positive response and is based largely upon the residual response obtained from tolerant animals and animals sensitized to PD, the control antigen. Couplingof DNBS to SRBC The procedure followed is described by Rittenberg and Pratt (70). Briefly, PBS washed SRBC are further washed three times in modified bar- bital buffer (M88) and 3 ml of the erythrocytes were added to a solution of 60 mg DNBS in 21 ml cacodylate buffer. The mixture was magnetically stirred for 10 minutes and washed three times in cold MBB containing glycyl-glycine. Although cells were stable for 3-4 days, they were washed again if not used immediately. Preparation and Use of Antithymocyte Serum Rabbit antithymocyte serum (ATS) was prepared by injecting two rabbits twice with 109 BCFl thymocytes iv at an interval of two weeks. Rabbits were bled following the second injection. ATS was heat inacti- vated by incubating for one hour at 56° C and then adsorbed by incubating 18 for one hour at 40 C at a volume ratio of 1:3 with BCF1 liver previously washed three times in PBS. Mice were injected iv with 0.1 ml of undiluted serum on days 1 and 3 of sensitization. RESULTS Evaluation of SRBC Ear Assay The 3 H TdR ear assay in CBA mice has been reported to be antigen specific (73) when animals are sensitized to SRBC. This antigen has also been used in another system whereby doses as low as 105 SRBC have been re- ported to produce an optimal sensitization for DH when measured by footpad swelling (42). To test this ear assay as well as low antigen sensitiza— tion, SRBC in both routine and low doses were used to sensitize two groups of BCF1 mice. A third control group received no antigen. All animals were injected with 2 u Ci 3H TdR on day 2, challenged with PBS in the left ear and SRBC in the right ear on day 4, and assayed on day 5. The results, expressed in Table 1, Show a greater accumulation of counts in antigen challenged ears than in PBS injected control ears for both sensitized_ groups while virtually no increase was seen in the nonsensitized group. In a second experiment, a protocol similar to that used by Sabolovic (73) was used in which a group of CBA mice was immunized by a sq injection of SRBC. Following 3H TdR labeling, the animals were challenged with CRBC as the control in place of PBS. Responses, displayed in Table 2, indicate no pattern of increased label accumulation in the SRBC challenged ears oc- curred when a non-crossreacting RBC served as a control. Similar prelimi- nary experiments employing either GRBC or CRBC as the control also failed to provide evidence of selective label accumulation in SRBC challenged ears and hence claims of specificity within this system remain unsub- stantiated. 19 Table 1. 20 Comparison of ear assay responses for routine and low dose immunized mice challenged with PBS and SRBC @ Treatment+ CPM/PBS Group CPM/SRBC % Increase Fraction positive ear ear (>10%) 6 .Mean 3 SE A SRBC* 4706 5072 7.7 3/5 (routine dose) 5073 5431 6.2 4409 4882 10.6 3570 5601 57.0 4389 5278 19.8 20.2 i 9.5 B SRBC* 4111 4327 5.2 3/4 (low dose) 3686 6487 76.5 3316 3986 20.5 3760 5051 34.3 33.9 i 15.4 C No antigen 2566 2333 -8.2 0/4 3430 3418 0.0 3995 4373 9.5 4234 4352 2.8 1.0 1 3.7 + CBA mice; 2 uCi @ Counts per minute * Injected iv; assayed day 5 3H TdR injected ip day 5 21 Table 2. Ear assay responses of mice* immunized sq with a routine dose of SRBC and challenged with CRBC and SRBC Mouse CPM/CRBC CPM/SRBC % Increase Fraction positive number ear ear (>10%) Means: SE 1 1175 981 —16.5 1/10 2 1805 1528 ~15.3 3 1324 1120 -15.4 4 1729 1495 -13.5 5 1410 1798 27.4 6 1069 1119 4.6 7 1673 1580 -5.5 8 1569 1456 —7.2 9 1476 1579 7.0 10 1068 895 -16.1 -6.5 i 4.5 * CBA mice injected with S x injected ip day 2; assayed day 5 IO8 SRBC; 1.5 uCi 3H TdR Measurement of Delayed Contact Sensitivity by Ear Assay Upon analysis of the previous experiments, failure to demonstrate specificity was attributed to one or more of a number of possibilities: I) an untenable assay, 2) inability of the SRBC antigen to induce ade- quate DH, 3) interference by the trauma of ear challenge whereby tissue damage results in nonspecific accumulation of labeled cells, and 4) leak- age from the challenge site resulting in unequal antigen distribution from mouse to mouse. It was reasoned that another type of delayed response, contact sensitivity to DNCB, would provide a more objective evaluation of 22 the assay by eliminating the last three possibilities since DNCB is known to induce a delayed response and can be applied topically. With reference to Table 3, groups A and B were sensitized with low or routine doses of SRBC as before while a third group was skin painted with DNCB. Animals received two injections of 3H TdR in an eight hour period. Groups A and B were challenged with CRBC and SRBC by id injection and group C was chal- lenged by painting the ears with either PD or DNCB. PD was chosen as the control in this system because of its simple chemical nature and its abili- ty to produce an inflammation of the same magnitude as DNCB (5). One and four negative re5ponses were obtained for the low and routine dose SRBC sensitized groups respectively. DNCB sensitization however, resulted in all positive responses. Furthermore the mean response was 149%, indicating that the system was capable of providing more dramatic and consistent re- sponses than the SRBC system and warranted further study. This consist- ency was indeed demonstrated in the next experiment depicted by group A of Table 4. CBA mice were sensitized with DNCB, injected three times with 3H TdR, challenged with DNCB and PD and assayed on day ten. Eight of nine mice responded positively within a narrow range. Intensity and Specificity of Response to DNCB Many studies have been carried out using skin reactive haptens to produce contact hypersensitivity. Two recent reports have emerged indi- cating tolerance to DNCB and dinitrofluorobenzene has been induced by iv injection of the sulfonic acid form of these chemicals, DNBS (5,65). In the system being described, specificity would be demonstrated if normal response upon challenge could be abrogated by tolerance induction. Mice in group A of Table 4 were tested by the standard procedure while mice in groups B-D, in addition to DNCB sensitization, were injected with DNBS in 23 Table 3. Comparison of Responses in SRBC and DNCB immunized mice challenged by a control and a test antigen Group Treatment Mouse % Increase Fraction positive number (>10%) 6 Mean i SE A* SRBC l 94 4/5 (low dose) 2 —54 3 146 4 l6 5 59 52 t 34 B* SRBC l -49 1/5 (routine dose) 2 -37 3 -32 4 -52 5 76 -18 i 24 c+ DNCB l 250 5/5 2 214 3 155 4 24 5 103 149 i 40 * BCFI mice injected iv; challenged with CRBC and SRBC; assayed on day S + BCF mice Skin painted by 3% DNCB in olive oil on days 1 and 3; challenged with PD and DNCB; assayed on day 7 24 Table 4. Ear responses obtained from DNCB sensitized mice: effect of varying iv DNBS injections Group Treatment — Day Mouse % Increase Fraction positive number (>10%) Mean : SE A* DNCB 1,3 1 35.2 8/9 6 2 -25.2 Assay 10 3 34.8 4 30.5 5 42.9 6 41.9 7 31.3 8 10.2 9 24.0 25.0 t 7.0 B* DNCB 1,3 1 15.9 8/9 G 2 177.7 DNBS 6,8 3 32.4 (1 mg) 4 15.3 G 5 3.9 Assay 10 6 63.4 7 110.0 3 130.6 9 245.3 88.3 t 27.7 25 Table 4. (cont'd) Group Treatment - Day Mouse % Increase Fraction positive number (>10%) 8 Mean 1 SE C* DNCB 1 78.2 10/10 8 2 72.8 DNBS -6,-5, 3 23.7 (1 mg) -3,1,3 4 83.0 G 5 74.7 Assay 9 6 52.1 7 75.8 8 113.7 9 137.0 10 80.5 79.1 i 9.7 0* DNCB 1,3 1 6.8 3/8 6 2 -l 9 DNBS 7 3 0.1 (15 mg) 4 —2.1 5 10.5 6 —0.4 7 31.6 8 11.4 7.0 i 4.0 * CBA mice + BCF mice; assayed on day 7 in protocol similar to that in earIier experiment employing BCF1 mice (Group C, Table 3) 26 a progressively more rigorous regimen for the purpose of inducing tolor~ ance. Group B was injected with 1 mg iv twice during the DNCB sensitiza- tion period and group C three times before and twice during sensitization. The data demonstrate no suppression occurred and in fact, enhancement was observed. Mean responses were increased at least two-fold for each group and with a remarkable consistency as attested to by the fact that there were no negative responses in nineteen animals tested. Eighteen of these exhibited responses greater than 10%. Group D mice were injected with 15 mg of DNBS 7 days before DNCB sensitization. Due to the unavailability of CBA mice, BCF mice were used in this group and assayed on day 7 in— l stead of day 10 because this had been the protocol in an earlier experi- ment involving BCFI mice (Table 3, Group C). Only one response was well above the 10% level and the mean was 6.9% for the group, indicating that a state of specific unresponsiveness had been induced and had resulted in the abrogation of ear response to DNCB. In a subsequent experiment shown in Table 5, group A was sensitized with PD instead of DNCB. Mice were challenged on day 8 with PD and DNCB as before. Although reSponse to DNCB was not completely eliminated, in— dividual response and the mean were considerably reduced and are at the same level as the residual response found in tolerant animals. This small positive response may reflect cross reactivity at the T cell level where- by PD sensitive cells are able to recognize DNCB as well as PD, but not vice versa. An alternative explanation may be that this response was merely the result of lower immunogenicity for a comparable amount of the chemical, hence a poor stimulation. Group B consisted of nonsensitized normal mice challenged with PD and DNCB. The responses are more varied than those of sensitized animals and the mean of —l3.6% favors PD 27 Table 5. DNCB response obtained in A) PD sensitized mice, B) non- sensitized mice and, C) DNCB sensitized mice challenged with DNCB and olive oil Group Treatment Mouse CPM/left CPM/right % Increase Fraction positive number ear ear (>10%) E Mean 1 SE A* PD 1 1550 1583 2.1 6/12 (day 1,3) 2 2021 2659 24.0 G 3 1803 1862 3.2 Assay (day 9) 4 2068 2412 14.4 5 1912 2427 21.1 6 2467 2450 -0.7 7 2087 2007 -4.0 8 1933 2285 -15.4 9 2001 2290 12.6 10 2092 2112 0.9 11 1997 2287 12.7 12 1875 2156 8 8 9.2 1 2.5 3* No sensi- 1 2348 2441 4.0 1/5 tization 2 2695 1298 -51.8 G 3 2314 1475 -36.3 Assay (day 9) 4 2070 2665 28.7 S 2319 2027 -12.6 —13.6 t 13.4 28 Table 5. (cont'd) Group Treatment Mouse CPM/left CPM/right % Increase Fraction positive number ear ear (>10%) 6 Mean 1 SE 0* DNCB 1 2856 4017 40.7 4/4 (day 1.3) 2 2010 2988 48.7 6 3 2069 4160 101.1 Assay (day 9) 4 2380 4017 58.5 62.2 1 13.4 * CBA mice; challenged with PD (left ear) and DNCB (right ear) + CBA mice; challenged with olive oil (left ear) and DNCB (right ear) challenge. Group C mice were sensitized with DNCB and challenged with olive oil, the suspending medium used for sensitization and challenge, in addition to DNCB. This mean compared to the mean response obtained in the routine procedure (Table 4, Group A) is within the same range, indi- cating that PD cross reactivity does not significantly affect DNCB response. Determination of the Mediator Cell Source To determine which type of cell could account for the accumulation of 3H labeled cells in ear responses, the following experiment was per- formed. Spleen, bone marrow, and thymus cells were collected from normal BCF1 mice and 6.7 x 107 of each type were injected into irradiated recipi- ents. These mice were then sensitized to DNCB by skin painting and on day 7, spleens of each group were collected and cell su5pensions prepared and pooled. Results showed that 3.5 x 10‘3, 2.4 x 10-3, and 4.5 x 10'3 CPM/cell were incorporated by spleen, bone marrow, and thymus cells 29 respectively. Although these results indicated slightly more label was taken up by thymus cells, analysis showed that a moderate CPM increase of test ear over the control ear required an unreasonably large number of cells at this level of incorporation. In a subsequent experiment, normal mice were sensitized to DNCB and on day 6 both spleen and PE cells were collected and pooled separately. Peritoneal cells were further treated by filtering through a glass wool column to remove macrophages. Aliquots of both spleen and peritoneal cells were then optically counted and prepared for scintillation counting. 2 CPM/cell while the value Peritoneal cells were found to yield 2.2 x 10- for spleen cells was 4.2 x 10.4 CPM/cell. This suggested that peritoneal lymphocytes, which consist of the circulating type T cell as opposed to the non-circulating type found in the spleen, are a likely source of the reactive cells in the ear response. Furthermore, a reasonable number of these cells contain enough label to account for the increases of 3H ob- served in ear responses. Inhibition provides another means of determining the source of cells mediating this sensitivity. An agent at least partially selective in suppressing T cells should inhibit the response to DNCB if this is a T cell phenomenon. Azathioprine (AZT), a 6-mercapt0purine analog, is one such drug which has been shown to inhibit certain types of DH responses (6). Table 6 indicates the responses obtained when mice received none, one, or two sq injections of AZT at the time of DNCB sensitization. Re- sponses were considerably reduced in the mice receiving the drug treat- ment. However, the dosage used, 0.15 mg/g body weight, did cause several deaths and weight loss in the survivors. In another experiment mice were treated with ATS in an effort to lllllllll).\II A 30 Table 6. Effect of azathioprine on ear response in DNCB sensitized mice Treatment Mouse number % Increase Mean No AZT 1 114.2 128.7 2 143.5 lAZT + injection 1 45.5 42.3 2 33.7 3 47.9 2 AZT 1 61.4 69.2 injections+ 2 77.0 * CBA mice skin painted with DNCB on days 1 and 3 + 2.2 mg of a 2 mg/ml solution ip/injection show that reduction of T cells results in a coincident reduction of re- sponse since recirculating cells have been shown to be sensitive to ATS in vivo (44). Eight mice were sensitized to DNCB by skin painting on days 1 and 3 and were subsequently injected iv with 0.1 ml of ATS on days 6 and 8. As seen in Table 7, with the exception of animals 1 and 2, which may actually have been stimulated, responses were not significantly affected when compared to the control group with which this experiment was concurrently run (Table 4, Group A). Lack of effect by the treatment may have been due to an insufficient amount of ATS and/or injection of the antiserum at an ineffective point in the sensitization procedure. The Optimal time of injection may be prior to antigen contact (16). 31 Table 7. Effect of in vivo administered ATS on response of DNCB sensitized CBA.mice Mouse ATS+ CPM/PD CPM/DNCB % Increase Fraction positive number ear ear (>10%) 1 - 3142 4249 35.2 8/9 2 - 2143 1660 -25.2 3 - 2743 3697 34.8 4 - 3354 4377 30.5 S - 2486 3552 42.9 6 - 2342 3314 41.9 7 - 2205 2896 31.3 8 - 2893 3183 10.2 9 - 3160 3919 24.0 25.0 i 7.0 l + .745 2817 278.1 8/8 2 + 1603 3159 97.1 3 + 3170 4110 29.7 4 + 2111 2553 20.9 5 + 2410 3430 42.3 6 + 2465 3489 41.5 7 + 2660 3277 23.2 8 + 1971 2390 21.3 69.2 i 31.1 * This experiment run concurrently with experiment reported in Table 4, Group A and assay protocol is identical; mice 1-9 served as controls in both experiments + 0.1 m1 ATS administered iv on days 1 and 3 32 Table 8. Day 6 plaque-forming cells/spleen of DNCB skin painted@ BCFl mice Indicator cell DNP—SRBC SRBC * Direct PFC 87 90 Indirect PFC 15 22 *Plaque-forming cell @DNCB sensitization on days 1 and 3 Although not demonstrating suppression, this experiment does reinforce the response consistency obtained by this procedure. Detection of Antibody Producing_Cells To explore the possibility that skin sensitization resulted in sub- sequent induction of antibody-producing cells and perhaps influenced the experimental results, a group of four mice was sensitized by DNCB skin painting. Spleens were collected on day 6 and assayed for hemolytic plaques using DNP-SRBC as a specific indicator and SRBC alone as a back- ground control. Table 9 depicts the results. substraction of SRBC back- ground plaques from DNP-SRBC plaques indicates DNP-specific antibody pro- duction is not stimulated. DISCUSSION The objective of this study was to assess DH in mice by means of a quantitative assay. The T cells responsible for DH have two known major functions; that of collaborating with B cells through the induction of antibody responses following antigen stimulation as well as mediation of cellular immune responses. The first of these functions can be quanti- tatively and qualitatively assessed by ability to induce B cells into antibody production as detectable by the hemolytic plaque assay. There is yet no adequate method of quantitating the second function despite the numerous manifestations of cellular responses. A good test fOr DH should provide quantitative, non-subjective results. Current in vivo methods used in the mouse include measuring either footpad thickening, ear swell- ing or the degree of skin reaction--none of which yield information on cell numbers or mechanisms involved in a particular reaction. The first assay studied was a novel technique described by Bloom et a1. (14) for enumerating antigen sensitive lymphocytes obtained from tuberculin sensitized guinea pigs. The approach was designed to detect intrinsic changes in these antigen activated lymphocytes rather than to measure products or gross macroscopic changes and was based on the follow- ing observations. While it was well known that resting lymphocytes were refractory to a number of RNA viruses, phytohemagglutinin activated cells were not and permitted replication of the virus. It was reported that the same principle applied to tuberculin sensitized guinea pig lymphocytes which were placed in culture both with or without antigen for a period of 33 34 up to four days. Virus was allowed to adsorb, excess virus neutralized by antiserum, and cells plated in agar over a monolayer of virus- susceptible target cells. Adaptation of this procedure to murine cells was the first consid- eration in preliminary experiments. Using bovine gamma globulin (BGG) as antigen, spleen, lymph node, and PE cells were prepared by culturing normal and sensitized cells of each with or without antigen prior to assay. Although a high background existed, generally higher responses were obtained with sensitized cells. Spleen cells produced the highest responses and were found to be the only practical source of cells due to the large number of cells required. Later it was found that background could be reduced by use of virus specific rabbit antiserum for excess virus neutralization during the procedure. During the course of exten- sive subsequent testing in many experiments, a number of variations were attempted with this assay because of a high incidence of technical fail- ures and inconsistent results. These modifications included: 1) testing a number of antigens including 866, heat aggregated 866, human gamma glob- ulin, EGG-SRBC, BGG-trinitrophenol, and SRBC in both routine and low doses, 2) elimination of the in vitro culture step to improve cell via- bility, and 3) preparation and comparison of various controlled cell types in lethally irradiated mice i.e., thymus, spleen, bone marrow. For the following reasons, consideration of an alternate procedure was necessary. An assay suited to the objective of this study with regard to the investigation of T cell functions would necessarily be technically simple enough to allow testing a number of individual animals in addition to yielding results which are consistent and reproducible. This proce~ dure clearly did not satisfy these requirements. Response and background 35 varied considerably from experiment to experiment and readable results were obtained in only approximately one—third of the experiments. Viable cells remaining after in vitro culturing may-not reflect the true in vivo population of cells. In addition the time consuming nature of the assay and the requirement for an initially large number of cells made the test- ing of other than pooled sources of cells impractical. The assay described by Sabolovic (73) and the subject of this pa- per is the second method evaluated. Use of SRBC as antigen posed several immediate problems. Ear challenge requires id injection of SRBC, a very delicate Operation. It was found that some tissue damage, puncture of tiny blood vessels, and leakage of antigen from the injection site is unavoidable, thereby introducing a number of sources of variation. A response could be demonstrated for SRBC in primed animals when PBS was used as a control challenge but not when non-crossreacting erythro- cytes were used in place of PBS as the control challenge. This suggested that response measured in this procedure may not be antigen specific. The use of low doses of SRBC described as producing optimal DH without antibody production (26,42) was also inconclusive with regard to demon- stration of specificity. To avoid pitfalls tied to the use of SRBC, the study was pursued with DNCB, a contact sensitizing agent known to elicit a high level of a persistent sensitivity as a result of skin painting (5,24,47). Quanti- tation of contact sensitivity response to challenge is conventionally measured by gauging ear thickness with a micrometer. This assay has an intrinsic shortcoming as do other assays: namely, it measures a largely non-specific effect presumable initiated by Specific antigen sensitive cells. Asherson and Ptak (5) by-this method have shown that mice 36 sensitized either to picryl chloride or oxazolone respond favorably to challenge by these haptens with non—immunized animals as controls. Phanuphak et al., using the same procedure attempted to demonstrate speci~ ficity in another manner. Mice injected iv with a large amount of DNBS became tolerant to dinitrofluorobenzene (DNFB) but not oxazolone. How- ever, mice sensitized to either DNFB or oxazolone were not tested with an agent other than the sensitizing agent. An important modification in the current study was the addition of a specificity control for each individual animal. One ear was challenged with DNCB and the other with PD. Specificity was apparent for l) DNCB sensitized, 2) PD sensitized, and 3) DNCB tolerant animals. The fact that a small residual response remains in tolerant or cross sensitized animals may reflect a less than complete tolerance to DNCB or less than optimum sensitization by PD in the respective experiments i.e., PD may be less inflammatory than an equal concentration of DNCB. The alternative possibility is that some degree of non-specific inflammatory potential is associated with DNCB. Results of the experiment comparing label incorporation indicated that peritoneal cells contained fifty times as much activity as spleen cells from the same animal. Since peritoneal lymphocytes consist primari- ly of recirculating T cells (34), the implication is that these are the reactive cells in this resPonse. In further studies, by the use of auto- radiographic techniques, the percent of labeled cells from this source can be determined and correlated to CPM for a known number of cells. When used in conjunction with a passive transfer system to reduce back- ground, numbers of participating cells could be determined. The procedure described in this report, due to its dependence on 37 the uptake of radioactive precursor, measures a cellular response. A num— ber of studies have implicated the recirculating T cell as the likely me- diator of this response (34,76). If T cells indeed are responsible fer initiating this reaction, the results of this study may lend support to the case for hapten specificity at the T cell level. The existence of hapten specific T cells at the present is one of the nebulous areas of cellular immunology. Janeway (39) pointed out that much of the evidence against hapten specific T cells is an inability to demonstrate function or a coincident involvement of antibody in a particular reaction. This can be traced back to early studies in which contact sensitivity could be induced successfully by id injection or skin painting but not by iv injection of DNP-conjugates. Mitchison (57) has described a system in which anti-lymphocyte serum treatment of protein primed donor mice was fOund to depress adoptive secondary antibody reSponse to that particular protein. Response could be restored by adding hapten primed cells to the adoptively transferred cells and then boosting the recipients with the hapten-protein conjugate. It was later shown that this effect can be accounted fer by specific antibody (39). Nevertheless, evidence fer hap- ten specificity of T cells is now mounting. A study using azobenzene—p- arsonate has demonstrated hapten specific helper function and cellular immunity (2). It has also been shown that DNP-KLH primed spleen cells from mice were capable of binding DNP-BSA coated nylon fibers and is sub- ject to inhibition by DNP-lysine or anti—theta serum (72). Phanuphak et al. (65,66) reported that 3H TdR uptake in vitro by DNP—BSA primed lymph node cells was DNP specific and subject to specific tolerance. Further- more, DNBS could induce lymphocyte proliferation in vitro in the absence of serum protein binding. While this does not eliminate the possibility 38 that DNBS is binding non-specific cells or cell products, it does point out the possibility that T cells may be capable of recOgnizing independent haptenic determinants. In the current in vivo study, small doses of DNBS stimulated cellu- lar responses while large amounts produced tolerance. The mechanism by which these chemicals affect the lymphocyte response is unknown but proba- bly related to the fact that as a class they are very reactive and form protein conjugates readily (24). However, skin and serum proteins differ and this study Shows iv injection of DNBS strengthens measured response to DNCB skin challenge. While the presence of cross-reactive DNP-protein conjugates in skin and serum cannot be ignored, it is possible that the carrier protein may play no part in specificity. The inability of a num— ber of investigators to produce contact sensitivity by DNP-protein conju- gates may then be explained by failure to provide a strong enough stimulus or the right kind of stimulus. In conclusion, the data provided by this and other studies cited, in addition to studies concerning T cell recep- tors, serve to point out that at present our knowledge of T cells at best is incomplete and warrants further research. SUMMARY A delayed hypersensitivity assay relying on the detection of accumu- lated radioactively labeled lymphocytes in a tissue challenge site was tested. Mice were sensitized to low or routine doses of SRBC, injected with 3H TdR to label antigen sensitive cells, and then challenged in the pinna of the ear. Each mouse was challenged on one ear with SRBC and on the other with PBS. Upon ear tissue solubilization and scintillation counting, increased accumulation of the 3H label was observed. However, this accumulation could not be demonstrated if the control ear was Chal- lenged with a non-crossreacting erythrocyte. The assay was subsequently modified to test contact sensitivity to DNCB, induced by skin painting. This system eliminated several possible sources of variation to provide a more objective test of this assay. Positive responses were consistently obtained by challenge consisting of DNCB, and PD as the control antigen. Specificity was demonstrated by showing that induction of tolerance to DNCB by DNBS injection largely abrogated re5ponse to DNCB challenge. Sensitization to PD and subsequent challenge by DNCB and PD, while not producing a response to PD, did largely eliminate response to DNCB. It was also shown that sensitization results in an increased uptake of 3H TdR and that the label is found in cellular DNA. 39 APPENDIX APPENDIX 3H TdR IncorporatiOn bnyorm l and Sensitized Mice To properly assess the activity measured in the ear assay it was necessary to compare 3H TdR uptake by normal and sensitized mice to deter- mine whether more label was actually incorporated by lymphocytes of antigen stimulated mice than by lymphocytes of non-stimulated mice. One group of three BCF1 mice was painted with DNCB. Beginning on day 5, this group and a second normal group was injected three times with l u Ci 3H TdR at an 8- 10 hr interval. PE or spleen cells were collected in phosphate buffered saline on day 7. Half of the PE cell suspension from both groups was fil- tered through glass wool to remove macrOphages. All cells were subse- quently washed with PBS and counted with the aid of an eosinophil counting chamber. Duplicate 0.1 ml aliquots of each sample were centrifuged, the supernatants discarded, and the cells lysed by the addition of 1 drop 1 M NaOH. Volume was brought to 1 ml with saline and 0.2 ml of 50% trichloro- acetic acid (TCA) added. This mixture was incubated 10 min at 4° C, the insoluble material collected on glass fiber filters, the filters dried and scintillation counting carried out in 10 ml toluene and omnifluor. Two additional aliquots of the DNCB stimulated PE sample were lysed with distilled water, incubated with 10 pg DNase for 30 min at 370 C and insolu- ble material collected on glass fiber filters for scintillation counting. Results showed DNCB treatment increased 3H TdR incorporation in both spleen and PE by approximately 50%. PE cells however, contained 20 40 41 times as much label as Spleen. Glass wool filtering of both PE samples resulted in an 85% loss of cells, which indicated a large number of lympho- cytes were lost in addition to macrophages. Consequently no conclusion can be drawn from this result. DNase treatment of a DNCB stimulated PE aliquot produced a 75% reduction in TCA precipitated counts indicating that at least the major portion of the label had indeed been incorporated into cellular DNA. Remaining counts could be attributed to incomplete lysis by the procedure used and a resultant inaccessibility of DNA to DNase. As a whole these data imply that l) the peripheral recirculating type lymphocyte is stimulated to a greater degree than the splenic type cell by hapten skin painting, 2) normal cells also incorporate label, but to a lesser degree, and 3) 3H TdR is incorporated into DNA by these cells as expected. Table 9. 42 Comparison of spleen and PE 3H TdR label incorporation in normal and DNCB sensitized mice Cells Cell CPM CPM/Cell Number PEDNCB* 7.2 x 105 839 11.60 x 10-4 -4 PENORMo 5.3 x 105 390 7.37 x 10 Gw*-pEDNCB 9 1 x 104 41 4.55 x 10"4 GW—PENORM 7.4 x 104 22 2.45 x 10'4 DNCB 6 -5 SPL 1.1 x 10 52 4.86 x 10 SPLNORM 1 0 x 10 31 3.12 x 10’5 DNase@-pEDNCB 7 2 x 105 207 2.8 x 10"4 BCF donors; sensitized days 1,3; l u Ci TdR 3 timAs days 5,6; cells collected day 7 BCF donors; 1 0 Ci TdR 3 times days 5,6; cells colIected day 7 adsorbed on glass wool @ PEDNCB aliquot treated with DNase BI BLIOGRAPHY 10. BIBLIOGRAPHY Adams, D. 0., J. L. Biesecker, L. G. Koss. 1970. 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