Studies on in vitro models of cellular immunity: The role of T and B cells in the secretion of lymphotoxin

Abstract When normal murine spleen cells were treated with anti-theta serum and complement, they failed to produce LT or synthesize cellular DNA when stimulated in vitro with PHA. Theta-positive cells were responsible for LT production in spleens removed from X-irradiated and bone marrow- or thymus-reconstituted animals. Finally, spleens from congenitally athymic Nu/Nu mice failed to produce LT when stimulated with pokeweed mitogen or phytohemagglutinin.

Understanding the contrihtioii of a specific Iyinpl~oitl cell or cell type in the generation of an iinn~uine response is of central importance.Attempts to provide a functional characterization of lpphoitl cells have been in large measure restricted to immediate-type responses in mice ( 1,2).Recoustitutin g iiiimunologically deficient animals with in~munoc0~77j~etei~t transplants has revealed that an interaction is required between tllymus-depentlent (T cell) , and thymus-intlel)ell~lent (13 cell) lymphocyte suhpopulations before stinmlation of antibody synthesis to certain antigens caii occur (3).Adtlitional studies are required to determine the role of B aid T cells in delayed-type hypersensitivity reactions.
The tlevelopent of itz zfitro methods for the study of cell-mediated iinniune (CMI) reactions has increased our understantling of the niechanisnis involvetl iii direct lymphocyte cytotosicity (4)) tumor inimunity (5)) and delayed hypersensitivity states (6).The work of numerous investigators, using these same methods, ha\-e led to the coiiceld of the l~m~hol;ine s):stem (7  To obtain a single cell suspension the mixture was removed from the petri by pipet, and placed in conical centrifuge tubes at 4°C.The suspension I\-as then permitted to stand for 10 min, after which the single cell-rich supernatant fluid was decanted into a second conical centrifuge tube and spun at 300g for 10 min.The cell pellets obtained by this procedure were resuspended in 1640 + SC-/o FBS.Aliquots of the preparation were taken and diluted in 0.1% Eosin Y, placed in a hemocytometer, and both the differential ant1 total cell counts determined.The majority of these cells were small lymphocytes, and \vere 85-95 "/c viable. Mitogens.Phytohemagglutinin-P (PHA) was obtained from Difco Labs, Detroit, MI (Lot 533815)) and pokeweed mitogen (PWM) (control No. 29094 H) was obtained from Grand Island Biological Company, Oakland, CA 94609.
Prefiaration of anti-theta serum.Anti-theta sera were induced by repetitive injections of C3H/St mouse (L.Strono Res.Found., San Diego, CA) thymocytes into AKR mice according to the method originally described by Reif and Allen (11).Inasmuch as immunized animals varied considerably in their individual antiserum titers against the theta antigen, they were bled individually and each serum sample assayed for anti-theta activity, using release of 51Cr from labeled thymus cells as a measure of cytotoxicity (12).Only sera with a reciprocal titer greater than 300 were pooled and used in the present studies.
Anfiserzm treatment of lpphoid cells.Eight million spleen cellls were suspended in 1 ml RPM1 1640, containing 13% immune or control antiserum.After 45 min at 4"C, the cells were washed once in 1640 by sedimentation and resuspension.The cell pellet was then resuspended in 1 ml of an Sojo solution of normal guinea pig serum in saline and further incubated at 37°C for 30 min.The cells were washed once in 5 ml 1640 and total viable cell counts performed in O.l(h Eosin Y. Tlzy~nectowked, X-irradiated, bone wzarrom-reconstituted mice.Male A/J mice were thymectomized as adults (6-7 weeks of age) according to the method of Miller (13).Two weeks after surgery, the animals were given 1000 R whole-body X-irradiation using opposing 220 kV Picker machines set 160 cm apart.Operations took place at 210 kV and 15 mA with an inherent filtration equivalent to 0125 cm Cu and 1 mm Al.Within 3-4 hr after irradiation, the A/Jax mice were injected intravenously with syngeneic 15 x 10" bone marrow transfer.At the time of sacrifice (12 weeks postgrafting), each animal was checked for the presence of thymus remnants and mice with remnants were not employed in these studies.Lynzphotoxin assail.The amomlt of LT in test and control media was determined by ascertaining its cytolytic activity on target L cell monolayers.Duplicate tube cultures were exposed to cell-free supernatant fluids from the different representative cultures.Test and control media were in continuous contact with assay monolayers for 48-72 hr, and thereafter replaced with 0.5 pCi/ml l"C-amino acid (Schwartz Bioresearch, Van Nuys, CA) labeling medium.This assay is based on the ability of the remaining viable cells to incorporate %-labeled materials into TCA-precipitable protein over an interval of 30 min as previously described (9).Toxicity is expressed in direct counts or as the percentage value of cpm test/cpm control X 100.Both diluted and undiluted supernatant fluids were tested for activity.In kinetic experiments, it was required that a number of the first supernatant fluids collected be stored at 4°C until samples of all the various points were collected.
Assay for DNA synthesis.
The incorporation of tritiated thymidine ("H-TdR) into DNA was assayed as previously described ( 15).Two tenths-milliliter volume of a 20-PCi "H-TdR/ml solution (Schwartz Bioresearch, Van Nuys, CA) \vas added to the different cell cultures 4 hr before their termination.Elsewhere, it has been demonstrated that this level of the radioisotope does not adversely affect normal cell functions (16).Nucleic acids were extracted by the hot phenol method of Sherrer and Daniel1 (17), and the amount of radioactive material present was tletermined in a Beckman Scintillation Counter, as has been previously described (9).

RESULTS
The first experiments were designed to determine the levels of PHA which induce maximum in z&o LT production and lymphocyte DNA synthesis.The spleen cells from untreated A-strain mice were cultured at a density of 5 x 10" cells in l-ml cultures in the presence of various doses of PHA.Nucleic acid synthesis was measured by the addition of 2 &i/ml tritiated thymidine (3H-TdR) directly to the tube cultures 68 hr after their initiation.The amount of label present in the phenolextracted cell pellet was determined after 3 hr of incorporation.The supernatant media was assayed for LT activity.The data shown in Fig. 1, A and B, are the results of one of five separate tests.In each experiment replicate tubes were included at each dosage.From these data, it is clear that DNA synthesis and LT production reached maximal levels in cultures stimulated with IO-20 pg PHA/ml.
The next experiments were designed to attempt to determine the type of lymphoid cell responsible for the initiation and/or production of LT in mitogen-stimulated cultures.The spleen cells taken from A-strain mice were treated with antitheta serum and guinea pig complement in order to prepare a B cell-rich cell suspension.These cells were placed in culture with 20 pg/ml PHA, using the above conditions, and were later assayed for both DNA synthesis and LT production.The results of three separate experiments, including replicate tubes at each point have been   The previous data indicated that PHA-induced DNA synthesis and LT secretion required the presence of theta-positive cells.However, it seemed appropriate to employ another test system to further substantiate this observation.Cell suspensions rich in T or B cells were obtained from normal or immunologically reconstituted irradiated A/Jax animals.As controls, bone marrow, spleen, and thymus cells acquired from normal mice were cultured with various levels of PHA.These experimental findings were then compared with the results obtained with spleen cells from the reconstituted animals.The data shown in Fig. 3, reveal that mitogeli-stimulated spleen cells obtained from animals which were reconstituted with B cell-rich bone marrow transplants, synthesized less cellular DNA than did their normal controls.However, the levels of LT production appeared to be similar in PHAstimulated cultures obtained from either hone marrow reconstituted or normal mice.In contrast, the spleen cells from mice restored with transplants of syngeneic thymocytes did not respond to PHA stimulation.When bone marrow cells and thymo-cytes taken from untreated donors were placed directly in culture with different amounts of PI-IA, there was no evidence of the activation of either DNA synthesis or LT secretion.
The observation that spleen cells from bone marrow-restored animals were able to produce LT as normal spleen cells was further substantiated by a series of kinetic experiments.The experiments were designed as follows: lo-ml cultures were initiated with mitogen in Falcon plastic flasks, then Z-ml aliquots were removed at 12-hr intervals, cleared of both cells and debris by centrifugation, and stored at 4°C until all samples were collected.The cytotoxicity assays were performed as usual with the exception that both test and control media were serially diluted by Z-fold steps with fresh media to determine the level of LT production.The end point was the dilution which resulted in reduction of cell numbers below 507% of the untreated control.While these data are not shown, spleen cell cultures from either normal or bone marrow-reconstituted animals, when cultured with PHA secreted identical levels of I,T.To eliminate any possible contaminating T cells from the spleens of B cell-reconstituted animals in the next experiment, the spleen cell suspensions were first exposed to antitheta serum and complement, then cultured with PHA.The Eecause of the antiserum treatment used in the al)o\-e esl)erinients to eliminate theta-positive cells, the possibility remained that surviving cells failed to secrete TAT, hecause of nonsl)ecific alterations iii their intlucil~ility.LAlternatively, the unrespon- siveness could simply reflect the inefficacy of PHA to stimulate B cells (19).To attempt to clarify the above situation, we decided to examine the reactivity of splecll cells from naturally occurrin g tlipi71"s-deficieiit niicc.Rlice congenitally athynlic (nude mice), and their normal (Ku/+ or +/t-) siblings were employed in the following studies.The culture conditions and methods of culture were essentially identical to those when normal spleen cells were examined ; however, in addition to PHA, poken.eedmitogen \vas used, because it has been reported to stimulate B cells (19), and it also stimulates LT secretion.The results shown in Table 1 represent the averages obtained in two separate tests.Each employed two nude mice and two of their normal siblings.In each experiment, as many as six replicate cultures were included to represent each test condition.These data reveal that spleen cells taken from nude mice failed to produce LT or undergo DNA synthesis h\-hen cultured in the presence of either mitogen.\Vhereas their normal siblings gave a vigorous response.

DISCUSSION
The optimal activating dose of PHA for normal murine spleen cells in vitr-o ranged between 10 and 20 pg/ml.However, thymus cells cultured with the same levels of mitogen did not respond with either I>I\'A synthesis or LT secretion.The observation that thymus cells are refractory to mitogen or antigen activation is in agreement with the results of numerous investigators measuring the capacity of these cells to participate directly in other immunologic reactions (4,(20)(21)(22).
Removal of theta-positive cells by antiserum treatment completely ablated the capacity of PHA to induce murine spleen cells to LT secretion or DNA synthesis ilz vitro.
However, spleen cells from thymus-reconstituted X-irradiated syngeneic C57BL/6 mice \?-ere refractory to PHA stimulation.This was surprising and can only be explained on the basis that the transplanted thymus cells were not at the level of differentiation to allow a response.However, these experiments were difficult to interpret, because of nonspecific toxicity in supernatant fluids from both the stimulated and unstimulated cultures.In contrast, spleen cells from irradiated animals reconstituted with syngeneic bone marrow were inducible with PHA to DNA synthesis and LT secretion.This response also required T cells, for when they were removed with antitheta serum, the response was completely ablated.This finding was interesting, for only 5% of the cells taken from spleens of the marrow reconstituted animals were destroyed by the antitheta serum treatment.In comparison, 50% of the cells obtained from the spleens of a normal animal are sensitive.Yet, even though there were only 10% of the reactive T cells present in the marrowreconstituted animal, the actual amount of LT released was the same as that found in stimulated cultures from the normal animal.If release is due solely to activated T cells, they must only be a small percentage of the total T cell population present in the normal spleen.The other alternative is that a very small number of T cells are required as helper cells to initiate the B cell, which then secretes LT.
In order to rule out any nonspecific effects of antiserum and complement treatment, we tested the reactivity of congenitally athymic (Nu/Nu) mice.The spleen cells obtained from these animals were completely unresponsive to treatment with PHA or PWM.In contrast.both DNA synthesis and LT secretion occurred at normal levels in P\NM-and PHA-stimulated cultures from the normal sibling heterozygous (Nu/+ ) mice.
The present results obtained using both in Z~&XJ and in z&o techniques unequivocally demonstrate that the release of LT by mitogen-stimulated spleen cells requires T cells.Moreover, it seems that only a small percentage of the T cell population are required for the response.These cells may be the LT-secreting cell.However, we cannot as yet rule out the possibility that they act as helper cells to initiate B cells, which then secrete LT.T cells have been shown to be central as helper cells in the initiation of antibody formation in z&o (3).It appears that this helper function involves synthesis and secretion of soluble effector molecules, \vliich in some way permit induction of the B cell.It has been suggested that killer lymphocytes in mice are T-type cells (22).The finding that LT secretion requires T cells suggests that the killer T cell and tile LT-secreting cell may be one and the same, and that the mechanism ( s) of destruction may, indeed, involve local secretion of a soluble cell toxin (s) .
FIG. 1.The effect of different doses of PHA on cellular DNA synthesis and in vitro secretion of LT by nonimmune A/Jax lymphoid cells: (A) DNA synthesis was assayed at 72 hr as described in the text, (B) LT secretion, the amount of LT in the medium as determined by measuring the capacity of I* cell monolayers to incorporate 1°C.AA, as described in the text.
averaged and are shown in Fig.2, A and B. The results indicate that antitheta serum and complement treatment removed or inhibited the cells required for PHAinduced in vitro DNA synthesis and LT secretion.
FIG. 3. Comparison of the capacity of normal thymus cells or spleen cells from bone marrowreconstituted mice to synthesize DNA and secrete LT after stimulation in vitro with PHA. A. DNA synthesis.PHA and control cells were assayed for capacity to incorporate 'H-TdR into cellular DNA after 72 hr.B. LT secretion.The cell-free media from stimulated and nonstimulated controls Lvere tested for activity on target L cell monolayer cultures as described in Methods.

then the tube was gassed with a mixture of 556 COr in air, capped, placed at a 5-degree angle, and incubated at 37°C. After lS-24 hr, each tube culture was examined and only those having uniform monc)- layers were used.
( 15 )roup of cell-free sub-ronc;tllavalill A (COll-.\), (, 1( 15 ).\\'hile cell-free medium from a sing-le activated lymphocyte culture, Iiiriltil)le activities C~II