CELLS MEDIATING SPECIFIC IN VITRO CYTOTOXICITY : I. DETECTION OF RECEPTOR-BEARING LYMPHOCYTES

Spleen cells from mice immunized with allogeneic tumor cells are incubated on different fibroblast monolayers. The nonadsorbed cells are tested for cytotoxicity against ⁵¹Cr-labeled target cells. The cytotoxicity of nonadsorbed cells is much lower after incubation on fibroblasts syngeneic to the immunizing tumor cells than after incubation on fibroblasts syngeneic to the immune cells. This specific decrease of cytotoxic activity depends on the duration and temperature of incubation on monolayers. After incubation the monolayers are trypsinized and pure populations of adsorbed lymphocytes isolated by density gradient fractionation. The cytotoxicity of such trypsin-eluted, gradient-purified lymphocytes is much higher when these lymphocytes are isolated from fibroblasts syngeneic to the immunizing tumor cells than when they are isolated from fibroblasts syngeneic to the immune cells. These experiments demonstrate specific adsorption of immune cells onto fibroblasts carrying the immunizing antigens, and thus prove the existence of specific receptors at the surface of these immune cells. Spleen cells from mice immunized with two types of allogeneic tumor cells bearing different H-2 antigen alleles are incubated on different fibroblast monolayers. The results of such experiments show a differential specific adsorption pattern, suggesting independent adsorption of two populations of immune cells bearing receptors directed against either one or the other immunizing H-2 antigen. The existence of at least a majority of cells, each of which is homogeneous as to the specificity of its receptors, makes it likely that specific receptors are synthetized by the cells that bear them. The role of specific receptor-bearing cells in the killing process is discussed.     

ANTIGENIC MODULATION : LOSS OF TL ANTIGEN FROM CELLS EXPOSED TO TL ANTIBODY. STUDY OF THE PHENOMENON IN VITRO

Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions.     

ANTAGONISTIC EFFECTS OF HUMORAL ISOANTIBODIES ON THE IN VITRO CYTOTOXICITY OF IMMUNE LYMPHOID CELLS

The ability of specifically immunized lymphoid cells to kill H-2 incompatible target tumor cells in tissue culture was shown to depend on the source of the lymphoid tissue (spleen versus lymph nodes). Marked cytotoxic effects were obtained with regional lymph node cells 7 to 10 days after primary immunization, whereas spleen cells from the same animals had little or no effect. Hyperimmunization did not decrease the cytotoxic efficiency of lymph node cells. Experiments were performed to test the possibility that the weak effect of spleen cells is a result of humoral antibody production, antagonizing the cell-bound immunity. Humoral antibodies were cytotoxic in vitro in the presence of complement only. Their effect was manifested after 2 hours, whereas immune lymph node cells did not require complement and cytotoxicity was not expressed until 24 to 48 hours' incubation. Tumor cell cultures treated with specific humoral antibodies in the absence of complement became resistant to the cytotoxic effect of subsequently added immune lymph node cells, while no such protection was seen when normal serum was added. Thus, humoral antibodies led to an "efferent" inhibition of cell-bound immunity in vitro, in analogy with previous results in vivo.     

SYNERGY BETWEEN SUBPOPULATIONS OF MOUSE SPLEEN CELLS IN THE IN VITRO GENERATION OF CELL-MEDIATED CYTOTOXICITY : Evidence for the Involvement of a Non-T Cell

Two subpopulations separated from normal spleen have been shown to synergize as responding cells in the in vitro induction of specific cell-mediated cytotoxicity during the mixed lymphocyte culture (MLC). The synergizing populations are a nylon wool column-adherent and a nylon wool column-nonadherent fraction, enriched for B lymphocytes and T lymphocytes, respectively. When a mixture of these fractions is used as the responding cell population in MLC, greater cytotoxicity is generated than would be expected from the sum of activities generated in the two subpopulations sensitized separately. The synergy appears to occur at the sensitization rather than the effector phase. The synergizing cell which is contained in the nylon-adherent subpopulation is distinct from the cytotoxic effector T lymphocyte, is resistant to lysis by rabbit antimouse brain serum, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by either plastic-adherent spleen cells or peritoneal exudate cells. These results suggest a role of a non-T-cell nonmacrophage population in the generation of cytotoxic activity.     

LYMPHOCYTE-MEDIATED CYTOTOXICITY IN VITRO : EFFECT OF ENHANCING ANTISERA

The ability of antisera to suppress immune responses either in vivo or in vitro is well known. A variety of lymphocyte-target cell systems have been employed to demonstrate inhibition of cell-mediated immunity by antisera in vitro, and skin, tumor, and kidney graft survival have been prolonged by passively administered antiserum in vivo. An in vitro lymphocyte-tumor cell assay system was developed for the purpose of studying the effects of enhancing antisera (in vivo) on lymphocyte-mediated cytotoxicity in vitro. The characteristics of this system with respect to route of immunization, time of harvest of immune cells, lymphocyte:tumor cell ratio, and effect of nonimmune or nonspecifically immune lymphoid cells are presented. Sera capable of enhancement in vivo were tested in this system and shown to inhibit cell-mediated immunity in vitro. Further, in both instances the immunosuppressive effect is mediated by antigen-antibody complexes and not by free antibody alone. Experiments were also carried out to determine the site of action of these suppressive antigen-antibody complexes. Presensitized lymphocytes were exposed to antigen-antibody complexes, washed, and then allowed to interact with fresh tumor cells (not antibody treated). Lymphocytes treated in this manner are incapable of exhibiting cell-mediated immunity in vitro. This evidence supports the concept that the antigen-antibody complexes have a direct immunosuppressive effect on the lymphocyte.     

CELLS MEDIATING SPECIFIC IN VITRO CYTOTOXICITY : II. PROBABLE AUTONOMY OF THYMUS-PROCESSED LYMPHOCYTES (T CELLS) FOR THE KILLING OF ALLOGENEIC TARGET CELLS

In order to investigate whether only T cells are involved in a cell-mediated cytotoxic system in vitro, we tested the cytotoxicity of immune killing cell populations as deprived as possible of B cells. Educated thymus cells, immune spleen cells purified by filtration through a column of beads coated with antimouse Ig antiserum, and finally educated thymus cells further purified by filtration through such a column fully retained their specific cytotoxic activity. This very strongly suggests that only T cells are involved in the killing of target cells by allogeneic immune cells in vitro, in this system. Receptor-bearing cells involved in killing in the present system are thus very probably T cells. This point was further strengthened by the demonstration of specific adsorption, on the relevant monolayers, of each of the three above mentioned killing cell populations.     

SPECIFIC ADHERENCE OF IN VITRO DIFFERENTIATED LYMPHOCYTES TO TARGET CELLS

Blast cells which were derived from rat lymphocytes by stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM) transformed within 2–3 days into a new type of lymphocytes when plated without mitogen on embryo fibroblast monolayers. These lymphocytes were termed secondary lyrophocytes. Upon addition of PWM to PWM-secondary lymphocytes a marked adherence to fibroblast monolayers was observed. The degree of adherence was estimated (a) by direct count of the lymphocytes in the medium and in the trypsinized fibroblast fraction, and (b) by using ⁵¹Cr-labeled lymphocytes. The adherence process required incubation at 37°C. The process started immediately after the addition of PWM and reached a plateau at 6 hr. At this time more than 80% of the lymphocytes adhered. In the absence of PWM only 12% of the lymphocytes were found in the fibroblast fraction. Unlike PWM-lymphocytes. Con A-lymphocytes, PHA-lymphocytes, and ordinary lymphocytes taken directly from the rat lymph nodes adhered only slightly more in the presence of PWM (10–20% adherence of ordinary lymphocytes) than in its absence (8% adherence). The adherence of the secondary lymphocytes and the ordinary lymphocytes was also studied in the presence of Con A and PHA. These mitogens induced high rate of adherence and they did not demonstrate specificity in their action. The adherence was accompanied by transformation of the lymphocytes to blast cells endowed with target-cell lytic ability. This transformation occurred mostly in the adhering fraction of the lymphocyte population. The results support the notion that target-cell recognition and destruction in cellular immunity involve contact between the cells.     

POLYMORPHONUCLEAR LEUKOCYTES IN IMMUNOLOGIC REACTIONS : THE DESTRUCTION OF VASCULAR BASEMENT MEMBRANE IN VIVO AND IN VITRO

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.     

IN VITRO STIMULATION OF ANTIBODY FORMATION BY PERITONEAL CELLS : I. PLAQUE TECHNIQUE OF HIGH SENSITIVITY ENABLING ACCESS TO THE CELLS

An improved method for the short-term culture of mouse peritoneal cells in a medium containing carboxymethylcellulose (CMC), sheep erythrocytes (SRBC), and guinea pig complement is described. It involves preparation of microcultures, of thickness 12–15 µ and volume 3.6 µl, under paraffin oil. With such cultures, peritoneal cells from normal, unimmunized young male CBA mice give about 3000 hemolytic plaques per million cells cultured, this figure being attained within 24 hr. The plaque detection method is about four times as sensitive as the Jerne technique. A method is described whereby such plaque-forming cells (PFC) can be transferred, by micromanipulation, to fresh monolayer cultures containing SRBC, CMC, and complement. In this fashion, the secretory capacity and susceptibility to inhibitors of peritoneal PFC can be tested in detail. Using this technique, evidence is presented that the hemolytic substance responsible for plaque formation is actually secreted by the cell at the center of the plaque, and is not a complement component but probably an antibody. Studies on the time of plaque appearance after cell transfer, and the subsequent growth rate of the zone of hemolysis, have been performed. They speak against the idea that the PFC is either a reservoir of cytophilic antibody or a "background" PFC. Rather they suggest that active antibody secretion is induced in the cell at some defined time point in culture. Detailed kinetics of the rate of appearance of plaques in peritoneal cell cultures revealed an exponential phase lasting from about 3 to about 13 hr with a doubling time of 2 hr. The reasons for this are not known. A greatly heightened reactivity was shown in peritoneal cells of mice that had been pregnant several times. Cultures of such cells showed more rapid plaque appearance and a peak activity about 20 times higher than with cells from young male mice. Cultures in which 1 cell in 10 formed a plaque were not infrequent. A series of experiments on germ-free mice showed reactivity similar to that of conventional mice from the same strain and source. The significance of the findings for cellular immunology are discussed.     

CONTRIBUTIONS TO THE BIOLOGY OF TISSUE CELLS : I. THE RELATION OF CELL CROWDING TO TISSUE GROWTH IN VITRO.

1. A method has been described by which it is possible to scatter isolated tissue cells in a suitable medium. 2. Experiments were undertaken to determine whether minute fragments of fibroblastic culture are able to persist and multiply when transferred into a new culture medium. 3. Cell divisions were not observed in isolated cells under the conditions of the experiment. Growth took place only when the tissue cells were numerous and close to one another.     

PRIMARY STIMULATION AND MEASUREMENT OF ANTIBODY PRODUCTION TO SHEEP RED BLOOD CELLS IN VITRO

A method for the primary stimulation and measurement of antibody production to sheep red blood cells in vitro has been described. In this system when mouse spleen cells are incubated with sheep erythrocytes large complement-dependent plaques are formed. The number and size of plaques increases from day 1 to day 3 of incubation with an average of 4.4 plaque areas per 1 x 10⁶ cells plated at day 3. There is a linear relationship between the number of spleen cells plated and the number of plaques formed. Plaque formation is inhibited by colchicine, actinomycin D, and rabbit anti-mouse globulin. This system offers a possible means for the direct in vitro measurement of the number of cells in a population susceptible to antigenic stimulation by sheep erythrocytes.     

RELATIONSHIP OF DONOR AGE TO IN VITRO PRODUCTION OF FOOT-AND-MOUTH DISEASE VIRUS BY MOUSE KIDNEY CELLS

Multiplication of foot-and-mouth disease virus (FMDV) was compared in kidney cells from 7- to 35-day-old mice representing various degrees of age resistance to this virus. Three types of cell preparations were used: primary monolayer cultures, suspensions of dispersed cells, and suspensions of minced tissue. Virus multiplication in the two types of cell suspensions was related to the age of the donors both in regard to time when multiplication first became evident and to the amount of virus produced. While adsorption rates were similar in the cells from all age groups, virus multiplication began earlier in cells from younger mice and more virus was produced by these cells than by cells from older animals. There was no significant difference in the virus growth rates in the primary monolayer cultures of cells. The results indicate that kidney cells from mice 7 to 35 days old vary in their ability to produce virus in relation to the degree of susceptibility of the cell donors. After propagation of the cells in primary monolayer cultures, however, this difference no longer exists probably because of cell selection under the cultural conditions.     

CYTOTOXICITY IN GRAFT-VERSUS-HOST REACTION : I. ROLE OF DONOR AND HOST SPLEEN CELLS

Under in vitro conditions spleen cells from nonirradiated F₁ hybrids, in which a (graft-vs.-host) (GVH) reaction had been induced with lymphoid cells of parental origin, lysed nonspecifically target cells, i.e., cells syngeneic or allogeneic to the parental genotypes. Furthermore, tumor cells exposed in vitro to spleen cells of F₁ hybrid mice undergoing GVH reaction had markedly decreased ability to grow in syngeneic recipients. Experiments involving inhibition of cytotoxicity with alloantisera indicated that this nonspecific effect was due to host cells. By contrast, spleen cells of lethally irradiated F₁ hybrids undergoing GVH reaction lysed specifically the target cells of the genotype against which the parental (donor) cells had been sensitized; this finding further supports the contribution of host cells to the nonspecific cytotoxic effects in GVH reaction. From these results it was deduced that the cytotoxic effects during GVH reaction involve at least two processes: (a) sensitization of the donor cells to the antigens of the recipient resulting in the activation of their potential to lyse specifically the recipient's cells, and (b) activation of the host's cells into a state of nonspecific cytotoxicity, as a consequence of the immunologically specific attack of the donor cells.     

DELAYED HYPERSENSITIVITY : I. EFFECT OF IN VITRO EXPOSURE OF CELLS TO ANTIGEN UPON LEUKOCYTIC TRANSFER OF DELAYED HYPERSENSITIVITY

Exposure to picryl guinea pig albumin with 3–6 picryl groups per mole failed to affect the ability of peritoneal exudate or peripheral blood leukocytes from sensitized donors to transfer delayed sensitivity to normal recipients. In contrast, conjugates containing 40–48 picryl groups per mole altered the ability of exposed leukocytes to transfer delayed sensitivity. Evidence is presented that highly conjugated guinea pig albumin is self-aggregating. Lightly conjugated albumin, previously heat-aggregated, also was effective in "desensitization." The properties of antigen size, cell association of antigen after exposure, and desensitization appear to be associated.     

GENETIC STUDY OF HUMAN CELLS IN VITRO : CARBOHYDRATE VARIANTS FROM CULTURES OF HELA AND CONJUNCTIVAL CELLS

The isolation of carbohydrate variants from cultures of HeLa and conjunctival cells was described. Factors inherent in the cell culture system, such as parent populations and dialyzed serums, have been shown to influence the outcome of variant isolations. Established stable variants incorporated significantly more pentoses or lactate into various cell fractions than the parent cultures. Besides their abilities to propagate continuously in the selecting environments, the variants multiplied slower, were more susceptible to sub-zero preservation and the cytotoxic effect of D-2-deoxyglucose, showed lower cloning efficiencies and were less susceptible to the deleterious effect of glucose oxidase. The ribose variants also differed from the parent cultures in morphological appearance such as formation of multinucleated cells and ring-shaped colonies. They converted more ribose into other component sugars of mucopolysaccharides than the parent cultures. Preliminary analyses of the mucopolysaccharides extracted from the ribose variants and parent cultures showed large difference in their carbohydrate (Molisch-positive materials) and DNA ratios. Evidence suggests that a sequence of interrelated events from genetic selection to primitive morphogenesis has been established.     

STUDIES ON ORGANOGENESIS : I. THE ABILITY OF ISOLATED BLOOD CELLS TO FORM ORGANIZED VESSELS IN VITRO

1. Isolated blood cells, when placed for incubation in a plasma substratum, are capable of constructing highly organized, tubular processes that project from the explanted cell mass into the surrounding medium. 2. The tubular structures have fibrillar walls that may be covered, eventually, by a membranous layer of leukocytes. Their lumina contain blood cells suspended in fluid. 3. The formation of the tubules is initiated by the red cells. The leukocytes, and more particularly the thrombocytes, are responsible for the construction of the walls. 4. The phenomenon occurs only in the presence of plasma, the coagulation of which has been slightly delayed. Once the surrounding medium has become firmly coagulated, no further change occurs either in the length of the tubules or in their diameter. 5. The development of the tubules is not suppressed by substances that enhance cellular activity unless they induce, at the same time, the immediate coagulation of the medium. The addition of embryo tissue juice prevents their formation by producing early coagulation. 6. The phenomenon as a whole is dependent upon the physicochemical nature of the medium, the character and thickness of the explanted fragment, and the physical and physiological peculiarities of the cells that comprise it. It is the expression of various physicochemical and physiological events that have occurred in a definite order, or sequence.     

CELLULAR IMMUNITY IN VITRO : I. IMMUNOLOGICALLY MEDIATED ENHANCEMENT OF MACROPHAGE BACTERICIDAL CAPACITY

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.     

SURFACE ANTIGENS OF IMMUNOCOMPETENT CELLS : III. IN VITRO STUDIES OF THE ROLE OF B AND T CELLS IN IMMUNOLOGICAL MEMORY

The effect of preincubation with anti-θ or anti-mouse immunoglobulin (Ig) and complement (C') on immune responsiveness of spleen cells from BALB/c mice immunized with sheep erythrocytes (SE) was investigated. Both treatments greatly depressed the remaining ability to produce a secondary response to SE in vitro. Normal BALB/c spleen cells were far less effective in reconstituting the responses of such depleted cell populations than were much smaller numbers of untreated immune spleen cells. Thymus-derived cell (T cell) memory appeared early after immunization and showed specificity for the immunizing antigens. Recombination of anti-Ig-treated with anti-θ-treated immune spleen cells resulted in virtually complete reconstitution of responsiveness. The presence of immunological memory in T cells and the nature of their surface receptors are discussed.     

COMPARATIVE STUDIES IN ROUS SARCOMA WITH VIRUS, TUMOR CELLS AND CHICK EMBRYO CELLS TRANSFORMED IN VITRO BY VIRUS : III. MALIGNANCY IN VIVO OF CELLS TRANSFORMED IN VITRO BY VIRUS

Chick embryo fibroblasts infected with Rous sarcoma virus in vitro are rendered malignant for such cells produce typical Rous sarcomas when injected into susceptible chicks since the tumors produced predominantly retain the sex chromatin patterns of the donor cells when such cells are injected into a recipient of the opposite sex. However, examination of the sex chromatin of cells at the periphery of the tumor shows presence of recipient cells though the bulk of the tumor is clearly of donor cell origin. Such tumors grow and cause death of the recipient. Injection of RSV induces tumors of the sex of the recipient as also does the injection of transformed cells rendered incapable of multiplication by x-rays. Following their injection into susceptible chicks, the cells transformed in vitro by virus behave in the same manner as tumor cells obtained from tumors induced by virus in vivo and cultivated in the same conditions in vitro. When such tumors induced by transformed cells are serially transferred in recipients of the opposite sex, they gradually convert to the sex of the recipient indicating that the tumors are not indefinitely transplantable. These chick embryo fibroblasts transformed in vitro show the same neo-plastic properties as tumor cells when they are introduced into the cheek pouch of the hamster or the eye of the guinea pig.     

THE PRESERVATION OF LIVING RED BLOOD CELLS IN VITRO : II. THE TRANSFUSION OF KEPT CELLS.

In order to determine the availability for functional uses of red cells kept in vitro by our methods, transfusion experiments have been carried out with rabbits by which a large part of their blood was replaced with kept rabbit cells suspended in Locke''s solution. It has been found that erythrocytes preserved in mixtures of blood, sodium citrate, saccharose, and water for 14 days, and used to replace normal blood, will remain in circulation and function so well that the animal shows no disturbance, and the blood count, hemoglobin, and percentage of reticulated red cells remain unvaried. Cells kept for longer periods, though intact and apparently unchanged when transfused, soon leave the circulation. Animals in which this disappearance of cells is taking place on a large scale, remain healthy save for the progressing anemia. The experiments prove that, in the exsanguinated rabbit at least, transfusions of cells kept for a long time in vitro may be used to replace the blood lost, and that when the cells have been kept too long but are still intact they are disposed of without harm. The indications are that kept human cells could be profitably employed in the same way.