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The in vitro generation of memory cells reactive to transplantation antigens is described. Blast cells, obtained from rat lymphocytes sensitized on xenogeneic or allogeneic fibroblast monolayers, reverted to secondary small lymphocytes after transfer from the foreign sensitizing to syngeneic monolayers. These secondary small lymphocytes had a limited in vitro life span of 4–6 wk. They manifested properties of memory cells: upon re-exposure to fibroblasts of the sensitizing phenotype, the secondary lymphocytes adhered to the fibroblast monolayer and transformed into blast cells with cytotoxic activity. The response of secondary lymphocytes was rapid, compared to that of normal lymphocytes, and directed specifically against the primary sensitizing antigens.

The in vivo intrasplenic cloning of haemopoietic cells was used as an experimental system for the study of some aspects concerned with the regulation of erythropoiesis. Experiments on the effects of polycythemia on the formation of erythroid clones demonstrated that the "feedback" suppression of such clones was inhibited if the polycythemic animals were kept under hypoxic conditions. Overloading with oxygen is, thus, an essential factor in the polycythemia-induced suppression of erythroid clones. Polycythemic animals which were transferred to hypoxic conditions for only 48 hours before the termination of the experiment showed the formation of erythroid clones. Analysis of clone formation following such short exposures to hypoxia suggests that the endogenous erythropoietin which had been functioning in this situation acted not upon the single clone-forming cell, but rather on small cell populations which derived from the clone-forming cells in the absence of erythropoietin. There is, therefore, an early, erythropoietin-independent phase of replication of the clone-forming cells. The application of erythopoietin only during this "early" phase of replication resulted in the formation of erythroid clones, which, following the discontinuation of erythropoietin, disappeared from the spleen. Similar results were obtained when the action of endogenous erythropoietin was terminated by subjecting animals to polycythemic conditions a few days after the injection of bone marrow. It appears that in both these cases the reduction in the number of spleen colonies following discontinuation of erythropoietin activity is due to the total maturation of the erythroid colonies, ending in the evacuation from the spleen of the mature red blood cells. The differentiation of the cloned cells is, thus, causally related to the cessation of the replicating effect of erythropoietin.

Primary antibody response against the dinitrophenyl group has been elicited in vitro after the stimulation of normal mouse spleen explants with 2,4-dinitrophenyl (DNP)-hemocyanin or α-DNP-poly-L-lysine (PLL). Antibodies were detected in the culture medium by the inactivation of DNP-T4 phage. The specificity of the reaction was manifested by the lack of the capacity of the medium to inactivate the unmodified bacteriophage and by the inhibition of the inactivation of DNP-T4 with DNP-lysine.

Polycythemic x-irradiated female mice, injected with 12-day fetal liver cells, showed 81% suppression of erythroid spleen colonies as compared with nonpolycythemic recipients. On the other hand, in male recipients only 16% suppression was observed. Hence, androgenic hormones seem to play a role in regulating erythropoiesis of explanted fetal stem cells. To test this, we examined the effect of testosterone injected into polycythemic female recipients on the production of erythroid colonies from fetal liver cells. Testosterone was found to alleviate the suppressive effect of polycythemia. Antierythropoietin prevented the appearance of erythroid colonies in testosterone-treated animals. Thus, testosterone seems to act by increasing the levels of, or susceptibility to erythropoietin. Under similar conditions, testosterone did not trigger the formation of erythroid colonies from bone marrow cells in polycythemic recipients. Hence, fetal cells can be induced to form erythroid colonies by doses of erythropoietin that are too low to induce erythroid colonies in bone marrow cells.

Two series of experiments were performed, utilizing a modification of the hemolysin plaque technique which registers 19S antibody, in an attempt to determine the frequency of cells capable of simultaneously producing antibody to two non-cross-reacting antigens. Mice were immunized i.v. with rabbit and camel RBC and their spleens assayed for cells producing antibody against both antigens. 16,904 cells producing antibody of one or the other specificity, from 26 mice, were counted. Not one cell was detected which produced antibody of two specificities. Rabbits were immunized intradermally with HSA to which polyalanyl and p-azobenzenearsonate groups were chemically attached. The individual haptens, polyalanyl, and p-azobenzenearsonate groups were coupled to separate aliquots of SRBC, and the lymph nodes of immunized rabbits were assayed for cells releasing antibody against both haptens. In a study of 11 rabbits, after counting 27,845 cells producing antibody, we detected no "double" plaques.

An in vitro cell-mediated immune response to pokeweed mitogen (PWM) is described. Rat lymphocytes were stimulated by PWM, by phytohemagglutinin (PHA), and by concanavalin A (ConA). In the presence of PWM only a fraction of the lymphocytes underwent blastogenesis. This was in contrast to the apparent total blastogenesis obtained in response to PHA or ConA. When blast cells derived from each of the mitogens were plated on rat fibroblast monolayer in the absence of mitogen they differentiated into a distinct type of lymphocyte termed "secondary lymphocyte." Addition of mitogens to cultures of these lymphocytes resulted in a retransformation to blast cells. The secondary lymphocytes were tested for their ability to effect lysis in the presence of each of the three mitogens. In. the presence of PWM, lysis of fibroblasts produced by PWM-lymphocytes was considerably more efficient than lysis obtained by ConA- or PHA-lymphocytes. No difference in effect on target fibroblasts was obtained when the three types of secondary lymphocytes were tested in the presence of either PHA or ConA. The stimulating action of PWM on lymphocytes was shown to be immunologically specific. No such specificity was found in the case of PHA or ConA. The results are interpreted to indicate that PWM combines with cell membranes and acts on the lymphocytes as a "transplantation antigen." Lymphocytes capable of responding to "PWM-transplantation antigen" transform to blast cells capable of specifically lysing PWM-conjugated fibroblasts. In the absence of the mitogen, PWM-induced blast cells differentiate to lymphocytes hypersensitive to PWM.

Autosensitization of rat or mouse lymphoid cells against syngeneic fibroblast antigens was induced in cell culture. Rat lymphoid cells autosensitized by this method were able to produce immunospecific lysis of syngeneic target fibroblasts in vitro or GvH reactions in newborn rats. Autosensitized mouse spleen cells mediated similar GvH reactions when injected into newborn mice. The nature of the system used to induce immunity in vitro appears to argue against the possibility that lymphocytes capable of reacting against self-antigens could arise by mutation in cell culture. Hence, it is likely that cells potentially reactive against self-antigens preexisted in the lymphoid cell donors. The ability of autosensitized cells to mediate immune reactions in vivo suggests that the immunogenic self-antigens present on sensitizing fibroblasts also were accessible in the intact animals. Loss of natural self-tolerance in vitro, therefore, can be explained most simply by the existence of lymphocytes which are reversibly tolerant to self. Hence, ontogenic elimination of potentially self-reactive cells may not be the only basis for natural tolerance. Regulatory mechanisms, such as antigen excess, may have to function in vivo to prevent differentiation of self-tolerant lymphocytes. These regulatory mechanisms appear to be annulled in the cell-culture system. The present system thus may offer a new approach to studies of tolerance and regulation of cellular immunity.

This paper reports a model system of cellular immunity in which allosensitization of mouse spleen cells is induced in vitro. Allosensitization was achieved by culturing spleen cells upon monolayers of allogeneic fibroblasts. The ability of the spleen cells to inhibit the growth of tumor allografts in vivo served as a functional assay of sensitization. We found that unsensitized spleen cells or spleen cells sensitized against unrelated fibroblast antigens had no inhibitory effect on the growth of allogeneic fibrosarcoma cells when they were injected together into irradiated recipients. In contrast, spleen cells which were specifically allosensitized in vitro were found to be highly effective in inhibiting the growth of an equal number of allogeneic tumor cells. Several times more spleen cells from mice sensitized in vivo were required to produce a similar immune effect. This confirms the findings of previous studies which indicate that sensitization in cell culture can promote the selection of specifically sensitized lymphocytes. Preincubating sensitizing fibroblasts with allo-antisera blocked the allosensitization of spleen cells. This suggests that antibodies binding to fibroblasts may inhibit the induction of sensitization by competing with lymphocytes for antigenic sites. Mouse spleen cells which were able to recognize and reject tumor allografts in vivo were unable to cause lysis of target fibroblasts in vitro. Such fibroblasts, however, were susceptible to lysis by rat lymphoid cells sensitized by a similar in vitro method. These findings indicate that the conditions required for lymphocyte-mediated lysis of target cells may not be directly related to the processes of antigen recognition and allograft rejection in vivo.

We studied the influence of glucocorticoids on the sensitization phase as well as on the cytolytic effector phase of an in vitro lymphocyte-mediated immune reaction. Lymphocytes obtained from the spleens or lymph nodes of unimmunized inbred rats were sensitized against foreign rat or mouse embryonic fibroblasts in cell culture. The capacity of the sensitized lymphocytes to produce a cytolytic effect was tested by transferring them to target fibroblast cultures. Injury to target fibroblasts was measured by release of radioactive 51Cr from previously labeled fibroblasts or by direct count of viable fibroblasts after incubation with sensitized lymphocytes. Various concentrations of water-soluble hydrocortisone or prednisolone were added to cell cultures during the 5 day sensitization phase and/or during the subsequent cytolytic effector phase and the influence of these hormones on the number and cytolytic capacity of the lymphocytes was measured. During the sensitization phase, the presence of glucocorticoid hormones, at concentrations of about 1 µg/ml, led to a profound decrease in the total number of recoverable lymphocytes. However, the per cent of large transformed lymphocytes was much greater in these treated cultures. The antigen-specific cytolytic capacity per cell of the glucocorticoid-treated lymphocytes, after the hormone was removed, was several times greater than that of lymphocytes sensitized in the absence of added hormones. Glucocorticoids influenced the effector phase of the reaction by inhibiting lymphocyte-mediated injury to target fibroblasts. The hormones, at concentrations of about 1 µg/ml, inhibited the cytolytic effect by about 50% without reducing the viability of the sensitized lymphocytes. Dose-dependent toxicity to lymphocytes and increasing inhibition of cytolytic effect appeared at higher concentrations of hormones. Thus, hydrocortisone and prednisolone, at concentrations of about 1 µg/ml, did not suppress the induction of sensitization, a process which they seem to facilitate in vitro. However, similar concentrations of these hormones appear to inhibit the cytolytic effector mechanism of sensitized lymphocytes. These findings may be relevant to the use of glucocorticoids as immunosuppressive agents in vivo.

The in vivo intrasplonic cloning method for bone marrow cells was applied to studios on the regulation of erythropoiesis. Experiments were made to test whether erythropoietin would reverse the specific suppression of erythroid clones, caused by polycythemia. It was found that the injection of anemic plasma (erythropoietin) elicited the re-formation of erythroid clones. The kinetics of the reversal is discussed in relation to the target cell for erythropoietin and its possible morphogenetic action.

The intrasplenic cloning method for bone marrow cells has been applied to produce populations of defined cell types, of bone marrow and spleen origin. The clones thus produced were found to consist of either erythroid, granuloid, or mixed (erythro- and granulo-) cell lines. No lymphoid elements were represented in the intrasplenic colonies. The immunological, competence of such “cloned” cell populations has been tested. No antibodies to red blood cell agglutinogens or to Shigella dys. antigen were formed by “cloned” cells, when tested after inoculalation to X-irradiated recipients. The application of Simonsen's test for the graft-versus-host response has shown that “cloned” cell populations of either bone marrow or spleen origin are not capable of forming a detectable anti-host response. In view of their immunological deficiency, their capacity to confer protection on lethally X-irradiated animals across genetic barriers has been studied. It was demonstrated that “cloned” cells of parental strain origin can confer protection on irradiated F1hybrid recipients. The protective effect obtained—in contrast to lethal secondary disease produced by bone marrow cells of the same genetic origin—was attributed to the immunological incompetence of the “cloned” haemopoietic cells. The chimeras thus produced regained immunological reactivity, as manifested by testing them 3 months following treatment. Whether the cellular basis of this restoration is of host or donor origin has not been demonstrated. The haemopoietic system, however, was found to have reverted to host type between the 2nd and 5th month following treatment. The possibility of avoiding the lethal secondary disease in allogeneic radiation chimeras by the application of “cloned” haemopoietic cells, is indicated and discussed.

This study reports a survey of subjective reactions of medical and nursing personnel to the delivery of abortion services. Staff and resident physicians described reported occasional depression, anxiety, and much obsessive ruminative thinking regarding their involvement with the performance of large numbers of abortions. One physician had to withdraw for a period of several months because of the stress related to his involvement with the program.Physician involvement, beyond the direct services in the clinic or the operating room, were perfunctory, despite the disclaimers that they treated these patients in the same way. Nursing personnel keenly felt this lack of leadership and suffered through considerable stress in attempting to resolve their ambivalence about participation in these procedures. They also reported some anxiety and depression, as well as a good deal of anger towards the patients for their sexual acting-out.