Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.     

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.     

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.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Relationship between surface marker expression and encephalitogenic potency of BP-cultured lymphocytes

The relationship between surface marker expression and encephalitogenicity of lymphocytes from various lymphoid organs of Lewis rats was studied. The encephalitogenicities after culture with BP were spleen cells greater than lymph node cells much greater than thymus cells, in this descending order. The cells from every lymphoid organ proliferated significantly in response to BP. In spleen and lymph node cells, the expression of W3/25 and OX-3 molecules on T cells increased markedly after culture with BP, but the expression of OX-19 or OX-8 molecules did not change significantly. The up-regulations of W3/25 and OX-3 molecules were more pronounced in spleen cells than in lymph node cells. Thymus cells also showed a significant increase in the W3/25 molecule after the culture with BP. Therefore, T cells from all the lymphoid organs showed a selective up-regulation of the W3/25 molecule after culture with BP, and the degree of the up-regulation seems to correspond to the encephalitogenic potency in vivo. Since the W3/25 molecule apparently plays a direct role in the effector phase of experimental allergic encephalomyelitis (EAE) by enhancing BP-reactive T cell/antigen-presenting cell interaction in the central nervous system, the up-regulation on BP-cultured T cells may strengthen interaction with the class II major histocompatibility complex molecule on antigen-presenting cells, and therefore, contribute to the efficient transfer of EAE.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Synergy among rat T cells in the proliferative response to alloantigen.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

Immunosuppression and lymphoid hypoplasia associated with chronic graft versus host disease is dependent upon IFN-gamma production

We have previously shown that both IFN-gamma and IFN-beta are produced in vivo and in vitro by spleen cells obtained from mice experiencing a chronic form of graft vs host disease (GVHD). Further, we have shown that in vitro production of IFN-beta by spleen cells from GVHD mice may play a role in the suppressed in vitro mitogen responsiveness of these cells. This study was undertaken to investigate if treatment of such mice with mAb to IFN-gamma or IFN-beta could alter the immunosuppression or lymphoid hypoplasia associated with chronic GVHD. GVHD was induced across minor histocompatibilities by the i.v. injection of B10.D2 spleen cells into sublethally irradiated BALB/c mice. These mice were given daily injections for 20 days of one of the following: 1) mAb to IFN-gamma, 2) mAb to IFN-beta, or 3) control IgG. Histologic examination of these mice at 21 to 22 days post transplantation revealed that mice treated with mAb to IFN-beta or control IgG had dramatic hypoplasia of the thymus, spleen, and lymph nodes which was similar to untreated GVHD mice. Mice given mAb to IFN-gamma, however, had no lymphoid hypoplasia and had a near normal gross and histologic appearance of their thymus, spleen, and lymph node tissue when compared with syngeneic controls. In vitro mitogen-induced proliferative responses of spleen and lymph node cells obtained from GVHD mice or GVHD mice treated with mAb to IFN-beta were severely suppressed or absent. In contrast, spleen and lymph node cells from GVHD mice given mAb to IFN-gamma were capable of giving a significant in vitro proliferative response to Con A, PHA, and LPS. Further, natural suppressor cell activity and spontaneous production of IFN-beta, a characteristic of this form of GVHD, was absent in spleen cells obtained from GVHD mice treated with mAb to IFN-gamma. These results further identify the IFN as playing critical roles in the pathogenesis of GVHD.     

Cell cooperation in abolition of tolerance of xenogeneic tumor.

Thymectomized, lethally irradiated mice reconstituted with syngeneic bone marrow cells are tolerant to xenogeneic Yoshida ascites sarcoma (YAS). The tolerance was abolished by an injection of syngeneic normal spleen, thymus, or lymph node cells given simultaneously with YAS. Allogeneic and semiallogeneic spleen cells were ineffective. The YAS-rejecting mice produced specific anti-tumor antibodies. The serum of these mice transferred to tolerant T-cell-deficient mice protected the latter from inoculated YAS cells. These serum-protected mice were not able to resist the reinoculum of the tumor cells as the mice restored with lymphoid cells did. The latter mice rejected the YAS at the time when donor cells were practically absent in their lymphoid tissue. The low effective ratio of injected syngeneic lymphoid to tumor cells, efficiency of injected thymus cells, and other data led to the conclusion that transferred lymphoid cells did not act directly on tumor cells but through cooperation with host lymphoid cells. The cooperation of donor T- and host B-lymphocytes enabled the activation of the latter, and YAS cells were rejected.     

Peripheral tolerance induced in lymph nodes by syngeneic spleen cells inhibits generation of CTLs to hapten-altered self antigens but not to alloantigens.

The immunological tolerance that is induced in lymph nodes that have been exposed to syngeneic spleen cells has been examined. Development of cytotoxic T lymphocytes was used to assess the immunological status of the lymph node cells. The tolerance was studied from the viewpoint of its induction, its activation, and its specificity. We had already reported that injecting either T or B cells of splenic origin into a regional lymph node environment a week prior to immunization for CTL to hapten-altered self antigens prevents development of the CTL. Here, we confirm that syngeneic splenic cells but not lymph node cells will induce the suppression provided that spleen cells are not coupled with hapten. We now report that splenic cells that cannot replicate or synthesize and secrete protein are capable of inducing the suppression. The data suggest a preformed surface marker peculiar to spleen cells and perhaps on cells that traverse the thymus induces local tolerance that is mediated by suppressor cells. Triggering the induced suppressor T cells (previously identified as CD8-) was achieved by syngeneic spleen cells as well as by H-2-compatible, Mls-disparate spleen cells but not by syngeneic lymph node cells or apparently by allogeneic spleen cells. Furthermore, triggering suppression was achieved by hapten-coupled syngeneic spleen cells whereas such cells would not induce the suppression. Thus, activating the suppressor cells requires reexposure to splenic cells of the proper MHC haplotype, unaltered or coupled with either TNP or FITC. Once triggered, the suppression was manifested toward CTL generation against hapten-coupled syngeneic antigens on either spleen or lymph node cells but not against allogeneic antigens. Thus, the specificity of the tolerance was directed to altered self antigens despite its induction by unaltered spleen antigen. Furthermore, for suppression to be seen the spleen antigen was not required to be on the hapten-coupled syngeneic cells used for the CTL immunization. The relationship of the splenic cell "antigen" to hapten-altered self antigens and to other surface markers and its site of acquisition within the body and its significance for cell homing have become intriguing questions of importance. This information has been discussed from the viewpoint of its applicability to autoimmune diseases as well as to cessation of inflammatory reactions that may be mediated by lymph node cells.     

Property of class I H-2 alloantigen-reactive Lyt-2+ helper T cell subset. Abrogation of its proliferative and IL-2-producing capacities by intravenous injection of class I H-2-disparate allogeneic cells

The present study investigates the distinctiveness of Class I H-2 alloantigen-reactive Lyt-2+ helper/proliferative T cell subset in the aspect of tolerance induction. Primary mixed lymphocyte reactions (MLR) revealed that Lyt-2+ and L3T4+ T cell subsets from C57BL/6 (B6) mice were exclusively capable of responding to class I H-2 [B6-C-H-2bm1 (bm1)]- and class II H-2 [B6-C-H-2bm12 (bm12)]-alloantigens, respectively. Anti-bm12 MLR was not affected by i.v. injection of bm12 spleen cells into recipient B6 mice. In contrast, a single i.v. administration of bm1 spleen cells into B6 mice resulted in the abrogation of the capacity of recipient B6 spleen and lymph node cells to give anti-bm1 MLR. This suppression was bm1 alloantigen-specific, since lymphoid cells from B6 mice i.v. presensitized with bm1 cells exhibited comparable anti-bm12 primary MLR to that obtained by normal B6 lymphoid cells. Such tolerance was rapidly (24 h after the i.v. injection of bm1 cells) inducible and lasting for at shortest 3 wk. Addition of lymphoid cells from anti-bm1-tolerant B6 mice to cultures of normal B6 lymphoid cells did not suppress the proliferative responses of the latter cells, indicating that the tolerance is not due to the induction of suppressor cells but attributed to the elimination or functional impairment of anti-bm1 proliferative clones. The tolerance was also demonstrated by the failure of tolerant lymphoid cells to produce IL-2. It was, however, found that anti-bm1 CTL responses were generated by tolerant lymphoid cells which were unable to induce the anti-bm1 MLR nor to produce detectable level of IL-2. These results demonstrate that class I H-2 alloantigen-reactive Lyt-2+ Th cell subset exhibits a distinct property which is expressed by neither Lyt-2+ CTL directed to class I H-2 nor L3T4+ Th cells to class II H-2 alloantigens.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

The induction of peripheral T cell unresponsiveness in adult mice by monomeric human gamma-globulin

Monomeric human gamma-globulin (HGG), when injected into adult mice, induces a state of specific immunologic unresponsiveness to further challenge with immunogenic forms of HGG. In this report we have directly determined the role of the thymus in the induction of HGG tolerance and the proliferative responsiveness of T cells from normal and HGG-tolerant mice. Draining lymph node T cells were isolated from HGG-tolerized and -challenged mice, and tested for their proliferative response to HGG in vitro. T cells from untreated but challenged adult CBA/CaJ and A/J mice proliferate in response to HGG, whereas such mice given monomeric HGG before challenge fail to show an HGG-specific proliferative response. APC from tolerant or nontolerant mice were equally effective in the support of Ag-specific proliferation of primed T cells. The influence of the thymus gland on HGG-induced T cell unresponsiveness was assessed by determining whether thymectomized mice could be tolerized to HGG. The results suggest that the generation of T cell tolerance to HGG is independent of thymic function as assayed by both antibody production in vivo and T cell proliferation in vitro. Unresponsiveness of T cells from tolerant mice was not a result of the presence of CD8+ cells since removal of CD8+ cells from lymph node T cells did not alter unresponsiveness to HGG in vitro. Further, mixing tolerant T cells with normal HGG-primed T lymphocytes did not inhibit proliferation of the HGG-primed cells. The results of this investigation suggest that this mouse model of tolerance to HGG represents a thymus-independent unresponsiveness of mature peripheral T cells to a nonself-Ag. Understanding the regulation of tolerance to HGG may give additional insight into the mechanisms required for the maintenance and possibly the induction of tolerance to certain self-Ag in peripheral lymphoid organs.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing cells.     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

Identification of human peripheral T cell antigens.

A new type of differentiation antigens on human T cells was demonstrated by using a heterologous anti-human T cell serum (ATS). This type of antigen, referred to as human peripheral T cell antigen (HPTA), was found on peripheral T cells and medullary thymocytes, but not on cortical thymocytes and B cells. The percentage of ATS-reactive lymphocytes in human peripheral lymphoid organs was correlated with that of cells rosetting with sheep erythrocytes, but contrasted with the number of B cells defined by the presence of a complement (C) receptor or by rabbit anti-human B cell serum (ABS). ATS also reacted with T cells purified by nylon fiber column filtration but ABS did not. Chronic lymphocytic leukemia cells rosetted with either sheep erythrocytes or erythrocyte-antibody-complement complexes were lysed by ATS and ABS, respectively. Mitogenic responses of blood lymphocytes to phytohemagglutinin (PHA) and concanavalin-A (Con A) were abrogated by treating them with ATS and C, whereas ABS suppressed only their response to Con A. Although numerous thymus cells rosetted with SRBC, only 14% were reactive with ATS. Quantitative absorption studies demonstrated that HPTA content of the thymus cells was much lower than that of lymph node cells. Anatomical localization of ATS-reactive lymphocytes in human lymphoid organs studied by immunofluorescence indicated that they were present in the thymus-dependent paracortical areas of lymph node and in the medullary region of thymus. ABS, on the other hand, did not stain thymocytes but reacted selectively with the cells located in the lymphoid follicles of lymph node. These data, together with that from cell suspension studies, confirmed that HPTA were shared between medullary thymocytes and peripheral T cells.