CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : II. THE SOURCE OF HEMOLYSIN-FORMING CELLS IN IRRADIATED MICE GIVEN BONE MARROW AND THYMUS OR THORACIC DUCT LYMPHOCYTES

The number of discrete hemolytic foci and of hemolysin-forming cells arising in the spleens of heavily irradiated mice given sheep erythrocytes and either syngeneic thymus or bone marrow was not significantly greater than that detected in controls given antigen alone. Thoracic duct cells injected with sheep erythrocytes significantly increased the number of hemolytic foci and 10 million cells gave rise to over 1000 hemolysin-forming cells per spleen. A synergistic effect was observed when syngeneic thoracic duct cells were mixed with syngeneic marrow cells: the number of hemolysin-forming cells produced in this case was far greater than could be accounted for by summating the activities of either cell population given alone. The number of hemolytic foci produced by the mixed population was not however greater than that produced by an equivalent number of thoracic duct cells given without bone marrow. Thymus cells given together with syngeneic bone marrow enabled irradiated mice to produce hemolysin-forming cells but were much less effective than the same number of thoracic duct cells. Likewise syngeneic thymus cells were not as effective as thoracic duct cells in enabling thymectomized irradiated bone marrow-protected hosts to produce hemolysin-forming cells in response to sheep erythrocytes. Irradiated recipients of semiallogeneic thoracic duct cells produced hemolysin-forming cells of donor-type as shown by the use of anti-H2 sera. The identity of the hemolysin-forming cells in the spleens of irradiated mice receiving a mixed inoculum of semiallogeneic thoracic duct cells and syngeneic marrow was not determined because no synergistic effect was obtained in these recipients in contrast to the results in the syngeneic situation. Thymectomized irradiated mice protected with bone marrow for a period of 2 wk and injected with semiallogeneic thoracic duct cells together with sheep erythrocytes did however produce a far greater number of hemolysin-forming cells than irradiated mice receiving the same number of thoracic duct cells without bone marrow. Anti-H2 sera revealed that the antibody-forming cells arising in the spleens of these thymectomized irradiated hosts were derived, not from the injected thoracic duct cells, but from bone marrow. It is concluded that thoracic duct lymph contains a mixture of cell types: some are hemolysin-forming cell precursors and others are antigen-reactive cells which can interact with antigen and initiate the differentiation of hemolysin-forming cell precursors to antibody-forming cells. Bone marrow contains only precursors of hemolysin-forming cells and thymus contains only antigen-reactive cells but in a proportion that is far less than in thoracic duct lymph.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : III. CHROMOSOMAL MARKER ANALYSIS OF SINGLE ANTIBODY-FORMING CELLS IN RECONSTITUTED, IRRADIATED, OR THYMECTOMIZED MICE

Two new methods are described for making chromosomal spreads of single antibody-forming cells. The first depends on the controlled rupture of cells in small microdroplets through the use of a mild detergent and application of a mechanical stress on the cell. The second is a microadaptation of the conventional Ford technique. Both methods have a success rate of over 50%, though the quality of chromosomal spreads obtained is generally not as good as with conventional methods. These techniques have been applied to an analysis of cell to cell interaction in adoptive immune responses, using the full syngeneic transfer system provided by the use of CBA and CBA/T6T6 donor-recipient combinations. When neonatally thymectomized mice were restored to adequate immune responsiveness to sheep erythrocytes by injections of either thymus cells or thoracic duct lymphocytes, it was shown that all the actual dividing antibody-forming cells were not of donor but of host origin. When lethally irradiated mice were injected with chromosomally marked but syngeneic mixtures of thymus and bone marrow cells, a rather feeble adoptive immune response ensued; all the antibody-forming cells identified were of bone marrow origin. When mixtures of bone marrow cells and thoracic duct lymphocytes were used, immune restoration was much more effective, and over three-quarters of the antibody-forming mitotic figures carried the bone marrow donor chromosomal marker. The results were deemed to be consistent with the conclusions derived in the previous paper of this series, namely that thymus contains some, but a small number only of antigen-reactive cells (ARC), bone marrow contains antibody-forming cell precursors (AFCP) but no ARC, and thoracic duct lymph contains both ARC and AFCP with a probable predominance of the former. A vigorous immune response to sheep erythrocytes probably requires a collaboration between the two cell lineages, involving proliferation first of the ARC and then of the AFCP. The results stressed that the use of large numbers of pure thoracic duct lymphocytes in adoptive transfer work could lead to good adoptive immune responses, but that such results should not be construed as evidence against cell collaboration hypotheses. Some possible further uses of single cell chromosome techniques were briefly discussed.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : V. TARGET CELLS FOR TOLERANCE INDUCTION

Collaboration between thymus-derived lymphocytes, and nonthymus-derived antibody-forming cell precursors occurs during the immune response of mice to sheep erythrocytes (SRBC). The aim of the experiments reported here was to attempt to induce tolerance in each of the two cell populations to determine which cell type dictates the specificity of the response. Adult mice were rendered specifically tolerant to SRBC by treatment with one large dose of SRBC followed by cyclophosphamide. Attempts to restore to normal their anti-SRBC response by injecting lymphoid cells from various sources were unsuccessful. A slight increase in the response was, however, obtained in recipients of thymus or thoracic duct lymphocytes and a more substantial increase in recipients of spleen cells or of a mixture of thymus or thoracic duct cells and normal marrow or spleen cells from thymectomized donors. Thymus cells from tolerant mice were as effective as thymus cells from normal or cyclophosphamide-treated controls in enabling neonatally thymectomized recipients to respond to SRBC and in collaborating with normal marrow cells to allow a response to SRBC in irradiated mice. Tolerance was thus not achieved at the level of thelymphocyte population within the thymus, perhaps because of insufficient penetration of the thymus by the antigens concerned. By contrast, thoracic duct lymphocytes from tolerant mice failed to restore to normal the response of neonatally thymectomized recipients to SRBC. Tolerance is thus a property that can be linked specifically to thymus-derived cells as they exist in the mobile pool of recirculating lymphocytes outside the thymus. Thymus-derived cells are thus considered capable of recognizing and specifically reacting with antigenic determinants. Marrow cells from tolerant mice were as effective as marrow cells from cyclophosphamide-treated or normal controls in collaborating with normal thymus cells to allow a response to SRBC in irradiated recipients. When a mixture of thymus or thoracic duct cells and lymph node cells was given to irradiated mice, the response to SRBC was essentially the same whether the lymph node cells were derived from tolerant donors or from thymectomized irradiated, marrow-protected donors. Attempts to induce tolerance to SRBC in adult thymectomized, irradiated mice 3–4 wk after marrow protection, by treatment with SRBC and cyclophosphamide, were unsuccessful: after injection of thoracic duct cells, a vigorous response to SRBC occurred. The magnitude of the response was the same whether or not thymus cells had been given prior to the tolerization regime. The various experimental designs have thus failed to demonstrate specific tolerance in the nonthymus-derived lymphocyte population. Several alternative possibilities were discussed. Perhaps such a population does not contain cells capable of dictating the specificity of the response. This was considered unlikely. Alternatively, tolerance may have been achieved but soon masked by a rapid, thymus-independent, differentiation of marrow-derived lymphoid stem cells. On the other hand, tolerance may not have occurred simply because the induction of tolerance, like the induction of antibody formation, requires the collaboration of thymus-derived cells. Finally, tolerance in the nonthymus-derived cell population may never be achieved because the SRBC-cyclophosphamide regime specifically eliminates thymus-derived cells leaving the antibody-forming cell precursors intact but unable to react with antigen as there are no thymus-derived cells with which to interact.     

EFFECTS OF SHORT-TERM EPITHELIAL RETICULAR CELL AND WHOLE ORGAN THYMUS GRAFTS IN NEONATALLY THYMECTOMIZED MICE

Neonatally thymectomized mice were implanted with thymus grafts composed of epithelial reticular cells for periods of 7 and 14 days. Regardless of whether the grafts were placed immediately after thymectomy, or at 3 wk of age, there was little recovery of the lymphocyte depletion and impaired immunologic responsiveness, characteristically found in a neonatally thymectomized host. The findings were similar in animals studied at 2 months or 2 wk after graft removal. Many of the short-term remnant grafts were populated with lymphocytes and had attained the morphologic appearance of thymus by 14 days. A lesser degree of lymphocyte depletion and impaired responsiveness to SRBC occurred if thymectomy was delayed until 7 days of age, if remnant grafts were removed after 2 months, and if intact neonatal thymus was used for the short-term grafts. Complete normality was found in some of the animals in all of these groups. These observations suggest a direct role for mature thymus lymphocytes in reconstituting the neonatally thymectomized host and indicate that epithelial cell function is to direct the maturation of cells that ultimately behave as thymus lymphocytes.     

THE LYMPHOID TISSUES AND IMMUNE RESPONSES OF NEONATALLY THYMECTOMIZED MICE BEARING THYMUS TISSUE IN MILLIPORE DIFFUSION CHAMBERS

Neonatally thymectomized mice were implanted intraperitoneally at 7 days of age with Millipore diffusion chambers containing either embryonic or neonatal thymus tissue. Mice which received either empty diffusion chambers or no further treatment following neonatal thymectomy served as controls. In contrast to these controls, most of the mice implanted with thymus-filled chambers gained weight satisfactorily, did not develop a wasting syndrome, and had the capacity to produce serum antibodies in response to sheep erythrocytes and to reject allogeneic skin grafts. Lymphoid follicles were present in the lymph nodes, spleen, and intestinal tract of the implanted mice but most still showed some diminution in the population of lymphocytes in both blood and tissues. Control thymectomized mice had markedly depleted lymphoid tissues and low peripheral blood lymphocyte levels. The tissue recovered after 1 to 2 months from the diffusion chambers showed only epithelial-reticular cells but no lymphoid cells. It is suggested that a humoral factor produced by the thymus epithelial-reticular complex may be responsible for endowing lymphoid cells with immunological competence.     

STUDIES ON THYMUS FUNCTION : I. COOPERATIVE EFFECT OF THYMIC FUNCTION AND LYMPHOHEMOPOIETIC CELLS IN RESTORATION OF NEONATALLY THYMECTOMIZED MICE

Immunological restoration of 45-day old, neonatally thymectomized C3Hf mice by treatment with humoral thymic function (thymoma grafts, thymus or thymoma in diffusion chambers) ranges from 0 to 12% and is difficult to acheive. When small numbers (5–20 x 10⁶) of young adult lymphohemopoietic cells, ineffective by themselves, are given in association with humoral thymic function, a cooperative effect is observed and restoration ranges from 30 to 60%. With a particular cell dosage (20 x 10⁶), effectivity for cooperation with thymic function was the following in decreasing order: spleen, lymph nodes, thoracic duct cells, bone marrow, blood leukocytes, thymus, and Peyer's patch cells. Comparable results were obtained using spleen, thymus, and hemopoietic liver from newborn donors in association with thymic function. For similar cell dosages, newborn thymus cells were more effective than adult thymus in their cooperative effect with thymic function. Dispersed thymus cells in association with young adult bone marrow or newborn hemopoietic liver cells showed no synergism for the cooperative effect with thymic function in the present model. Using hemiallogeneic cells (F₁ hybrid into parent) it was possible to show that restoration was mediated by proliferative expansion of the injected cells. This was indicated by specific tolerance to tissues of the other parental strain and by cellular chimerism, especially of lymphoid tissues, as indicated by chromosome markers and absence of significant numbers of immunocompetent cells of host origin. A population of paritally differentiated cells of hemopoietic origin, termed postthymic, sensitive to humoral activity of the thymus and present in the lymphohemopoietic tissues of adult and newborn mice is postulated to explain our results. These cells are postthymic and thymus dependent in the sense that they already received thymic influence, probably through traffic, and are incapable of self-renewal in absence of the thymus. Sensitivity to humoral activity of the thymus is characterized by proliferative expansion and/or a differentiative process eventually leading to larger numbers of competent cells.     

IMMUNOCOMPETENCE OF SPLEEN CELLS FROM NEONATALLY THYMECTOMIZED MICE CONFERRED IN VITRO BY A SYNGENEIC THYMUS EXTRACT

Impaired immunological competence of spleen cells from neonatally thymectomized C57B1/6 young adult mice was apparent when these cells were tested in an in vitro graft-versus-host assay. Spleen cell inocula prepared from thymectomized mice did not induce enlargement of (C3H/eb x C57BI/6)F₁ newborn spleen explants, whereas the same number of cells from intact donors consistently initiated splenomegaly. Spleen enlargement was observed, however, when the explants were challenged by cells from thymectomized donors in the presence of syngeneic thymus extract, indicating that the spleen cells in suspension attained immunological competence under the influence of a non-cellular component of the thymus. Immunocompetence was also evident when the cells from thymectomized donors were first incubated with thymus extract for 1 hr and subsequently tested for reactivity. Cells from the same thymectomized donor mice exposed in parallel to extracts from syngeneic spleen or mesenteric lymph node at an equivalent protein concentration did not initiate a graft-versus-host response. These experiments demonstrate that immune reactivity in the graft-versus-host response involves activation of lymphoid cells by a humoral factor of the thymus acting directly upon these cells.     

ALLOGENEIC THYMUS GRAFTS AND THE RESTORATION OF IMMUNE FUNCTION IN IRRADIATED THYMECTOMIZED MICE

Irradiated and thymectomized CBA mice are markedly depressed in several immunological parameters (skin homograft rejection, graft-vs.-host activity and hemolytic plaque-forming cells of the spleen, hemolysin and hemagglutinin formation, and peripheral lymphocyte counts). In the present experiments the ability of homografts of neonatal thymus placed beneath the kidney capsule to restore immunological capacity of such animals was studied. Thymus homografts which share the same H-2 locus with the CBA mouse were permanently tolerated and immunological restoration was complete. Skin from the thymus donor was specifically retained, but third party skin with even minor (non-H-2) incompatibility was normally rejected and hemolytic plaque-forming cells of the spleen were restored. Thymus homografts which differ at the H-2 locus were promptly rejected and led to accelerated rejection of skin subsequently grafted from the thymus donor. With such H-2 incompatible thymus grafts, third party skin with minor histo-incompatibility was retained while there was slight to moderate restoration of rejection of skin with major (H-2) incompatibility. Graft-vs.-host activity was restored, but there was no return of plaque-forming spleen cells, hemolysins, hemagglutinins, or peripheral lymphocyte counts. In view of the cross-reactivity at the H-2 locus in CBA mice between thymus and third party skin donors, it was felt that restoration of skin rejection and graft-vs.-host activity could be adequately explained on the basis of immunization by the thymus graft and did not require the postulation of true immune restoration or a thymus hormone.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : VI. CONTRIBUTION OF THYMUS-DERIVED CELLS AND ANTIBODY-FORMING CELL PRECURSORS TO IMMUNOLOGICAL MEMORY

Collaboration between thymus-derived lymphocytes and nonthymus-derived antibody-forming cell precursors occurs in the primary antibody response of mice to heterologous erythrocytes and serum proteins. The purpose of the experiments reported here was to determine whether collaboration took place in an adoptive secondary antibody response. A chimeric population of lymphocytes was produced by reconstituting neonatally thymectomized CBA mice soon after birth with (CBA x C57BL)F₁ thymus lymphocytes. These mice could be effectively primed to fowl immunoglobulin G (FγG) and their thoracic duct lymphocytes adoptively transferred memory responses to irradiated mice. The activity of these cells was impaired markedly by preincubation with CBA anti-C57BL serum and to a lesser extent by anti-θ-serum. Reversal of this deficiency was obtained by adding T cells in the form of thoracic duct cells from normal CBA mice. Cells from FγG-primed mice were at least 10 times as effective as cells from normal mice or from CBA mice primed to horse erythrocytes. These results were considered to support the concept that memory resides in the T cell population and that collaboration between T and B cells is necessary for an optimal secondary antibody response. Poor antibody responses were obtained in irradiated mice given mixtures of thoracic duct cells from primed mice and of B cells from unprimed mice (in the form of spleen or thoracic duct cells from thymectomized donors). In contrast to the situation with T cells, the deficiency in the B cell population could not be reversed by adding B cells from unprimed mice. It was considered that memory resides in B cells as well as in T cells and that priming probably entails a change in the B cell population which is fundamentally different from that produced in the T cell population.     

STUDIES ON THE RECOVERY OF THE IMMUNE RESPONSE IN IRRADIATED MICE THYMECTOMIZED IN ADULT LIFE

Experiments performed on CBA mice thymectomized in adult life, exposed to lethal doses of irradiation and given tissue therapy are described. Marrow, foetal liver, or spleen cells from syngeneic donors could protect the mice against the lethal effects of irradiation. Between 30 and 70 days' postirradiation, however, marrow-treated, thymectomized irradiated mice showed evidence of trophic disturbances, such as failure to gain weight, in contrast to sham-operated, irradiated, marrow-treated controls. The immune responses of experimental and control mice were tested up to 150 days' postirradiation by challenging with sheep erythrocytes and allogeneic skin grafts. Sham-operated irradiated controls, whether protected with marrow, foetal liver, or spleen cells, produced normal immune responses when challenged at 28, 60, or 150 days after irradiation. Neither foetal liver cells nor marrow cells, in doses of up to 40 million cells per mouse, enabled thymectomized irradiated mice to recover normal immune functions. Spleen cells, from normal donors but not from neonatally thymectomized donors, restored immunological capacity in such mice. It is concluded that immunologically competent cells are present in the spleen of normal adult donors and can function in the absence of the thymus. Bone marrow, on the other hand, does not contain an adequate population of such cells but has lymphoid precursor cells, the descendants of which can become immunologically competent only in the presence of a functioning thymus mechanism.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : VIII. RADIOSENSITIVITY OF THYMUS-DERIVED LYMPHOCYTES

The helper function of carrier-primed T cells was found to be radiosensitive in vivo. The results could not be attributed to interference with the spleen-seeking properties of the irradiated cells. It is suggested that T cell division is essential for the induction of 7S antibody responses in vivo.     

THE EFFECTS OF THYMUS AND OTHER LYMPHOID ORGANS ENCLOSED IN MILLIPORE DIFFUSION CHAMBERS ON NEONATALLY THYMECTOMIZED MICE

When neonatally thymectomized CBA mice were implanted at 9 to 12 days of age with Millipore diffusion chambers (pore size, 0.1 µ) containing either syngeneic or allogeneic neonatal thymus, they were subsequently found to have the capacity to reject skin homografts and to form antibodies to sheep erythrocytes. In spite of displaying restored immune reactivity, thymectomized mice bearing thymus-filled diffusion chambers still had a lymphopenia and diminished numbers of small lymphocytes in their spleens, lymph nodes and Peyer's patches. Comparison of the lymphoid organs of these mice with those of the thymectomized control mice did not reveal any appreciable difference in the numbers of primary follicles or small lymphocytes. It is postulated that the thymus humoral factor induced immunological competence in lymphoid cells which had left the thymus prior to neonatal thymectomy. The paucity of circulating and tissue small lymphocytes in thymectomized animals, the immune reactivity of which was restored by thymus tissue in diffusion chambers, argues against the theory that the thymus humoral factor has a lymphocytosis-stimulating effect. There was no restoration of immune reactivity in those neonatally thymectomized mice which had been implanted with diffusion chambers containing neonatal or adult spleens, or adult lymph nodes. Thus, the competence-inducing factor is elaborated by the thymus but not by the spleen or lymph nodes. Allogeneic (C57Bl) neonatal thymus tissue, enclosed within diffusion chambers, had the capacity to restore the immune reactivity of totally thymectomized CBA mice, not only to skin homografts of a totally unrelated strain (Ak), but also to grafts isogeneic with the donor of the allogeneic thymus. Therefore, there is no strain barrier to the action of thymus humoral factor. To explain the apparent lack of full participation of thymus lymphocytes in immune reactions it is postulated that thymus lymphocytes are functionally immature in situ, and that they leave the thymus before attaining immunological competence. In the periphery, they undergo further maturation under the influence of the competence-inducing factor produced by the thymus.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : X. T-CELL-DEPENDENT SUPPRESSION IN TOLERANT MICE

Specific immunological tolerance was induced in CBA mice by a single injection of deaggregated fowl immunoglobulin G (FγG). The unresponsive state was stable on adoptive transfer and irreversible by pretreatment of tolerant cells with trypsin. Tolerant spleen cells could suppress the response of normal syngeneic recipients. They also suppressed the adoptive primary response of spleen cells to FγG in irradiated hosts. The inhibitory effect was on the indirect (7S) plaque-forming cell (PFC) response. Incubation of the tolerant cell population with anti-θ serum and complement reversed the suppressor effect. Furthermore, the addition of purified T cells from normal donors restored the capacity of the anti-θ serum-treated tolerant cells to transfer an adoptive response to FγG. The existence of FγG-reactive B cells was supported by the demonstration of normal numbers of antigen-binding cells in the spleen and thoracic duct lymph from tolerant animals. Moreover, the formation of caps by these cells implied that they could bind antigen normally. These experiments provided direct evidence for the existence of suppressor T cells in the tolerant population. Further evidence was derived from examination of the effect of antigen "suicide". Tolerant spleen cells were treated with radioactive FγG under conditions known to abrogate T-cell helper function. When these cells were transferred together with normal spleen cells into irradiated hosts, suppression of the primary adoptive response to FγG was no longer observed. Inhibition of an adoptive secondary response to FγG was obtained by transferring tolerant spleen cells with primed B cells provided high doses of tolerant cells were used. By contrast low doses exerted a helper rather than a suppressor effect in this system.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : IV. THE RESPONSE OF NORMAL AND IMMUNE RABBIT BONE MARROW AND LYMPHOID TISSUE LYMPHOCYTES TO ANTIGENS IN VITRO

Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VI. THE IMMUNE RESPONSE TO RED BLOOD CELLS IN IRRADIATED RABBITS AFTER ADMINISTRATION OF NORMAL, PRIMED, OR IMMUNE ALLOGENEIC RABBIT BONE MARROW CELLS

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.     

THE HOST CELL RESPONSE IN THE LOCAL GRAFT-VERSUS-HOST REACTION INDUCED IN THE KIDNEYS OF F1 RATS BY PARENTAL THORACIC DUCT LYMPHOCYTES

Radioautographs of infiltrative cells in the kidneys of (Lewis x BN)F₁ rats labeled with tritiated thymidine (TdR³H) before the subcapsular injection of parental (Lewis) thoracic duct lymphocytes (TDL) showed a predominantly host-proliferative response by 4 days after grafting. The immediate renal incorporation of TdR³H was used to measure the local graft-vs.-host (GVH) reactions. Substantial reactions could still be induced in the face of the considerable degree of leukopenia after 400 R whole body γ-irradiation. These results suggest that radioresistant cells are capable of carrying on the appropriate host activities and that the weakness of GVH reactions induced after higher doses of irradiation may be due to impairment of the mitotic mechanism of host cells. The importance of circulating leukocytes as a source of immunogenic stimulation was nevertheless substantiated by inducing local GVH reactions with Lew TDL in chimeric parental-type rats that had been repopulated with F₁ bone marrow. This result also emphasizes the nonspecific nature of tissue destruction in the renal GVH reaction in confirmation of Elkins. In this and other situations in which B cells were the predominant F₁ type elements available for interaction with parental-type TDL the reactions were nearly equivalent or equivalent to those in the appropriate controls. Typical local GVH reactions could be induced in heavily irradiated hosts by an inoculum of combined parental and F₁-type TDL in the apparent absence of mononuclear phagocytes. The possible relationship between the activation of host lymphocytes, the involvement of B cells, and the nonspecific nature of tissue damage in the renal GVH is discussed.     

ANTIGEN-BINDING CELLS IN MICE IMMUNE OR TOLERANT TO ESCHERICHIA COLI POLYSACCHARIDE

The number of PFC and of RFC was studied in mice which were unimmunized, immunized, or tolerant against lipopolysaccharide of E. coli 055:B5 origin. The number of PFC/10⁶ spleen cells increased from 0.5 in normal to 209 in immunized mice. The corresponding figures for RFC were 93 and 513 RFC/10⁶ spleen cells. In tolerant animals, which contained few or no PFC, the number of RFC was increased as compared to that found in unimmunized mice. The formation of rosettes was specific, since their formation was inhibited by soluble coli polysaccharide and by rabbit antisera against mouse immunoglobulins. The antigen-binding cells were not derived from thymus, neither in immune or tolerant mice, because they did not carry the theta antigen. It is suggested that the majority of antigen-binding cells present in tolerant animals are cells having receptors for the antigen of rather low affinity. The relevance of these findings for the induction of high and low zone tolerance is discussed.     

ANTIGEN-SPECIFIC SYNERGISM IN THE IMMUNE RESPONSE OF IRRADIATED MICE GIVEN MARROW CELLS AND PERITONEAL CAVITY CELLS OR EXTRACTS

A synergistic immune response to foreign erythrocytes may be induced in heavily irradiated mice injected with a mixture of isologous cells obtained from marrow and from the peritoneal cavity. Under appropriate conditions, homologous or heterologous peritoneal cavity cells, heat-killed cells, or cellfree extracts made from such cells are also effective. The activity of the peritoneal cavity cells or extracts is antigen-specific, in the sense that cells or extracts obtained from animals previously immunized with the test antigen produce much stronger synergistic effects than do cells from animals immunized with some other antigen; however, the peritoneal cavity cells or extracts are not immunogenic when tested in primed animals. The marrow cells, demonstrated to contain precursors of the antibody-forming cells produced during this synergistic immune response, also show a form of antigen-specificity.     

THE IMMUNE RESPONSE TO FOREIGN RED BLOOD CELLS AND THE PARTICIPATION OF SHORT-LIVED LYMPHOCYTES

The sequence of morphological changes in the rat spleen following SRBC injection associated with hemolysin production has been correlated with estimates of proliferative activity by splenic lymphatic tissue. Formation of new, reactive germinal centers containing macrophages which engulf nuclear debris is a prominent feature of the response. This is prevented by pretreatment of the animal with cortisol. Indirect evidence is provided that short-lived lymphocytes produced in germinal centers may be a necessary component in the induction of other cells to proliferate and differentiate into hemolysin-producing cells. The reasons are discussed for considering short-lived lymphocytes, such as those produced in the thymus, bone marrow, and germinal centers, as differing from long-lived lymphocytes capable of antibody synthesis.     

EXPERIMENTAL STUDIES UPON LYMPHOCYTES : II. THE ACTION OF IMMUNE SERA UPON LYMPHOCYTES AND SMALL THYMUS CELLS.

The work of previous investigators gives the impression that it is easy to produce sera which both in vitro and upon injection are leukotoxic. At the same time the specificity of these leukotoxic sera for the particular type of cell used as antigen, and even for leukocytes in general, has been doubtful. The methods used have made certain possible factors of error unavoidable. Even careful washing of an organ or suspension cannot render it wholly blood-free, so that it is not surprising that the sera should be moderately hemolytic and hemagglutinative. Pearce has shown that the injection of very small amounts of blood is sufficient to evoke the production of immune hemolysins. When such sera are injected the lesions, as Pearce states, may be due in part to the production of hemagglutinative thrombi, although this hardly seems to apply to the changes in lymphoid tissue described by Flexner. On the other hand, the lymphotoxic effect of hemolytic sera may be due to the lymphocytes injected with the red cells. Our own experiments indicate that the lymphotoxic and agglutinative factors are to a considerable degree distinct from the hemolytic and hemagglutinative ones, since they can be separated from one another by absorption. Further evidence is presented that the small thymus cells are biologically related to, if not identical with the lymphocytes derived from lymph glands.