In vitro responses of rat lymphocytes following adult thymectomy. II. Increased inhibition by splenic adherent cells of responses to phytohemagglutinin

Adult thymectomy in rats results in a marked fall of spleen cell responsiveness to PHA over a period of days to weeks. Examination of dose-response curves showed that, with high PHA dose or ≥ 10⁶ cells/ml, there is a profound inhibition of the response with spleen cells from thymectomized animals compared with cells of matched sham-operated controls. However, when adherent cells are removed from the cell suspensions, the remaining nonadherent cells give an almost linear dose-response relationship with increasing PHA similar to that exhibited by the nonadherent cells of the controls or sometimes a slightly decreased response. Similarly, when increasing numbers of spleen cells from these animals are cultured (with admixed thymocytes to make a constant total of 2 × 10⁶ cells/ml) with PHA, the linear portion of the doseresponse curve can be extrapolated to give a similar value for the maximal potential response, which again is the same as or somewhat less than the corresponding value for sham-operated controls. A difference in inhibitory capacity is also shown in mixtures of the two spleen cell populations with LNC or with purified spleen cells. It is concluded that adult thymectomy results in increased “suppressor” activity in the spleen within a few days and may reduce slightly the number of T lymphocytes in the spleen reactive with PHA.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

The cell-mediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo.

The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10² up to 5 × 10⁴ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a ⁵¹Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.     

Cellular events in tolerance. V. Detection, isolation and fate of lymphoid cells which bind fluoresccinated antigen in vivo.

The binding of tolerogen to specific receptors of lymphocytes and the subsequent fate of such cells was directly studied in Lewis rats injected with fluorescein-labeled sheep gamma globulin (F-SGG). This tolerogen produced unresponsiveness both in SGG-specific T cells (carrier tolerance) and F-specific antibody-forming cell precursors. The former (T-cell tolerance) was still significant more than 60 days after tolerogen whereas tolerance in the latter (B-cell tolerance) had waned by that time.Cells which have bound the tolerogen (antigen-binding cells, ABC) in vivo were detectable by direct immunofluorescence of washed spleen cell suspensions from rats injected with F-SGG up to 7 days previously. These cells were isolated using antifluorescein affinity columns, and shown to contain immunocompetent precursors for F- and SGG specific responses.The frequency of such ABC was between 30 and 80 per 10⁵ spleen, lymph node or bone marrow cells; no ABC were detected in the thymus. Both Ig positive and Ig negative cells were found to be ABC; Ig negative ABC usually showed a “capped” fluorescent pattern whereas Ig positive ABC generally were “spotted.”By 10 days after injection, ABC were not detectable in the spleen, lymph nodes, thymus or bone marrow of tolerant rats. Furthermore, reinjection of F-SGG after this time did not label any cells. This suggests that antigen-binding cells are not present at this time or that such cells, if available, lack receptors. In contrast, rats previously injected with a lower non-tolerogenic dose of F-SGG or an immunogenic form (F-SGG on bentonite) possessed cells at these later times which could be labeled with F-SGG. Thus, ABC remain detectable following immunogen or a subtolerogeic dose of F-SGG, but disappear in tolerant rats.By approximately 40 days after initial high dose tolerogen injection (when B cell tolerance has started to wane), cells capable of binding a second dose of F-SGG again became detectable. It is suggested that high doses of F-SGG are bound by specific lymphocytes (identifiable as ABC) and that these cells either fail to regenerate new receptors or die. As tolerance begins to wane, either new receptors or new cells are generated.     

Further studies on the biological properties of Friend virus-induced leukemic cells differentiating along the erythrocytic pathway

When Friend virus-induced leukemic cell lines were injected into irradiated hosts after the second radiation dose, the colony-forming unit (CFU) in the recipient spleens per 10⁴ cells was found to be 7-fold higher than the CFU obtained when the second radiation dose had been given shortly after the inoculation of the cells. Serial passage of the cells from the spleen colonies to irradiated hosts resulted in a marked increase of the CFU value, indicating that this cell population was capable of both self-replication and erythroid differentiation. The “f” fraction, which indicates the percentage of the inoculated cells that reach the spleen in the irradiated recipients, was found to be approximately 15%. If the highest CFU value obtained from serial colony-to-colony passages is corrected by this factor, a final cloning efficiency of about 18% is demonstrated. Neither induced plethora nor the administration of erythropoietin (1 u/mouse/for 2 days) appeared to affect the spleen colony-forming ability of the leukemic cells. Erythroid differentiation is not detectable in the transplantable subcutaneous tumors which were used to initiate the tissue culture lines and which also are capable of inducing erythroid spleen clones in irradiated recipients. This lends support to the theory of the influence of “microenvironmental factors” on the fate of stem cells with potential for differentiation.     

Effector cells for antibody-dependent cell mediated cytotoxicity. I. Increased cytotoxicity after priming with BCG-SS.

Intraperitoneal injection of an aqueous extract of Bacillus Calmette-Guérin (BCG-SS) is shown to increase the cytotoxicity of murine spleen cells which mediate antibody-dependent cellular Cytotoxicity (ADCC). Three to forty-four days after in vivo stimulation with BCG-SS, spleen cells were tested for their ability to lyse antibody-coated chicken red blood cells in a ⁵¹Cr release assay. Significantly increased lysis compared to non-BCG-SS primed litter mates was observed from 3 days through 3 weeks after priming. Aqueous extracts of other bacteria including Listcria, Brucella, Salmonella, Staphlococcus and Eschcrichia did not elicit the same cytotoxic response. Separation of BCG-SS stimulated spleen cells on columns of G-10 Sephadex showed increased cytotoxicity in both the adherent (presumptive macrophage and polymorph) and nonadherent (presumptive K lymphocyte) populations. The possible relationship of these results to BCG-mediated anti-tumor effects is discussed.     

Natural killer cell activity in mouse aggregation chimeras

The activity of natural killer (NK) cells in spleen against syngeneic and allogeneic tumor cells was studied by the use of tetraparental mouse chimeras. Chimeras were produced by aggregation of early embryos of histoincompatible mouse strains of “high” and “low” NK cell activity. NK activities of spleen cells were assayed in vitro by the ⁵¹Cr-release method. Coat color distribution and isozymal analysis (glucose-phosphate isomerase) of several lymphoid organs (thymus, lymph nodes, and bone marrow) revealed a predominant share of the “high”-NK-reactive genotype in the chimeras. However, the cellular NK activity against two target cell lines differing in their susceptibility to lysis was significantly lower in chimeras than in the “high”-reactive strain. Addition of “low”-NK spleen cells or of NH₄Cl-inactivated “high”-NK spleen cells to “high”-NK spleen cells inhibited their cytolytic activity. Possible mechanisms of the suppression of the cytolytic capacity of NK cells in chimeras are discussed.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Lipopolysaccharide-Induced Mediators Assisting the Proliferative Response of C3H/HeJ Thymocytes to Concanavalin A

³H-Thymidine uptake of thymocytes from LPS-responder Balb/c mice in the presence of a submitogenic dose (0.5 μg/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 μg/ml), while the uptake of thymocytes from LPS-nonresponder C3H/HeJ was not enhanced by LPS. However, “endotoxin soups,” which were prepared from the supernatants of LPS-responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of ³H-thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup-producer cells and responder cells to con A was different, the soups were still effective. The active substance in the “endotoxin soups” was eluted through a Sepharose CL-4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.     

Studies of the immunological capacity of germ-free mouse radiation chimeras. II. Thymus cell function in a humoral immune response to sheep erythrocytes

Experiments described here were undertaken to determine the reason for the depressed humoral immune response in germ-free mouse allogeneic radiation chimeras. Indirect immunofluorescence using the theta (θ) antigen as a marker demonstrated that about 10% of the nucleated cells in the spleen of both allogeneic and syngeneic chimeras bear the θ antigen. One type of in vivo cell transfer assay employed to determine the capacity for “helper” function of thymocytes revealed that allogeneic chimera thymocytes were only 7–18% as efficient in “helper” function as normal thymocytes. A second type of in vivo cell transfer assay demonstrated that the presence of intact normal thymic stroma had no effect on the “helper” inefficiency of thymocytes obtained from allogeneic radiation chimeras.     

Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

Membrane defects in the tolerant B cell. II. Mechanism of capping failure and reversal by antigen exposure.

The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10^?4M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.     

Cross-circulation and cell distribution kinetics in parabiotic mice

Blood‐borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross‐circulation. Here, we investigated the cross‐circulation and cell distribution kinetics of C57/B6 GFP⁺/wild‐type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a “parabiotic barrier” based on cell size or surface characterstics; all peripheral blood cell subpopulations in this study reached equilibrium within 14 days. Whole blood fluorescence analysis indicated that the mean exchange flow rate was 16 µl/h or 0.66% of the circulating blood volume per hour. Studies of peripheral lymphoid organs indicated differential cell distribution kinetics. Some subpopulations, such as CD8⁺ and CD11c⁺, equilibrated in both lymph nodes and spleen indicating a residence time <28 days; in contrast, other lymphocyte subpopulations, such as B220⁺ and CD4⁺ cells, had not yet reached equilibrium at 28 days. We conclude that parabiosis can provide important insights into defining tissue distribution, residence times, and recirculating pools using fluorochrome markers of cell lineage. J. Cell. Physiol. 227: 821–828, 2012. © 2011 Wiley Periodicals, Inc.     

Response of Mouse T and B Lymphocytes to Sheep Erythrocytes

THE primary immune response of mice to sheep red blood cells (SRBC) involves two types of lymphocytes: the antibody-forming cell series, whose precursors are found in bone marrow (B cells) and cooperating or “helper” cells of uncertain function whose precursors are found in the thymus (T cells)¹. Within 24 h of an intravenous injection of SRBC, an increase of haemolytic antibody plaque-forming cells (p.f.c.) occurs in the spleen, reaching a peak 5 days later. Part, but not all, of this increase is due to division among the B cells². T cells in the spleen also undergo a wave of mitosis, detectable from the second to the fifth day³.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Participation of three lymphoid cell types in the in vitro activation of cell-mediated immunity to a syngeneic gross virus-induced lymphoma in rats.

W/Fu rats inoculated with the syngeneic Gross-virus induced lymphoma, (C58NT)D, had transient lymphocyte-mediated specific cytotoxicity against the tumor cells at 7–15 days after tumor injection. Spleen cells 40 days after immunization (spleen 40) were unreactive by a direct 4-hr ⁵¹Cr release assay, but activity appeared after in vitro culture of these spleen eclls by themselves for 18–24 hr. The nature of the cells involved in the activation of cytotoxicity and the characteristics of the effector cells themselves were studied. Significant differences were seen in the cell types involved in the activation phase and the effector phase. Activation appeared to require the cooperation of three cell types. Induction of activity was lost by treatment of cells with ATS plus complement, by passage over an EAC-column, or by treatment with carbonyl iron. Thus, T cells, CRL and macrophages were necessary for full activation of cytotoxicity in spleen 40. In contrast, after activation, only CRL seemed to be required for cytotoxicity, and treatment wih ATS or carbonyl iron had little effect. The effector cell detected after in vitro activation was quite distinct from that seen in the direct cytotoxicity assay with spleen cells at 10 days after tumor cell inoculation. The early, direct cytotoxic reactivity was dependent on T cells, being eliminated by treatment with ATS and complement but not by EAC columns or carbonyl iron. It appears therefore that the in vitro activation is a separate mechanism for cytotoxicity against tumor cells, rather than a simple recovery of T cells from in vivo inhibition.