Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

Tolerance to class II major histocompatibility complex molecules is maintained in the presence of endogenous, interleukin-2-producing, tolerogen-specific T lymphocytes

We have examined the requirement for clonal reductions of tolerogen-reactive lymphocytes in mice of the A strain background rendered neonatally tolerant of class II major histocompatibility complex molecules. Tolerogen-specific mixed lymphocyte reactivity of lymphocytes obtained from 130 adult, class II tolerant mice, bearing a healthy skin allograft, was examined. Lymphocytes obtained from 86 mice responded to the tolerogen, in vitro, with a positive mixed lymphocyte response (MLR) indicating that a large proportion (75%) of adult class II tolerant mice on the A strain background are not clonally deleted for tolerogen-reactive lymphocytes. In addition, lymphocytes from 29 mice were MLR-negative to the tolerogen, and lymphocytes from 15 mice demonstrated such high amounts of proliferation to syngeneic stimulators that their specific response to the tolerogen could not be determined. In view of the discordance between the in vivo and in vitro expressions of tolerance in the MLR-positive mice, lymphocytes from these mice were compared with normal lymphocytes by several assays. 1) Tolerogen-specific proliferative responses obtained from both normal and tolerant lymphocytes could be inhibited by the addition of monoclonal antibodies specific for the relevant class II antigens; 2) quantitative differences in the ability of normal, as compared with tolerant cells, to respond to the tolerogen in the MLR were not apparent; 3) no evidence of qualitative differences in the cell-surface phenotype of the proliferating cell was observed, (i.e., the cells were Thy-1+, L3T4+, Lyt-2-); and 4) lymphocytes from both normal and MLR-positive tolerant mice produced substantial amounts of interleukin-2 in response to the tolerogen. Thus, clonal deletion of helper cells is not required for tolerance to class II major histocompatibility complex antigens and we propose that tolerance may be maintained by either 1) in vivo suppression of the tolerogen-specific helper cells or 2) selective deletion or suppression of class II specific effector cells.     

The circulating life span, immunocompetence and 3H-uridine uptake of small lymphocytes from thymus-grafted, neonatally thymectomized rats.

By 7 weeks post-grafting, the number of small lymphocytes in the thoracic duct lymph (TDL) and blood of the thymus-grafted neonatally thymectomized adult rats had increased to 60% of the number of cells in sham controls, or 2-1/2 times thymectomized control values. This increasing consisted almost exclusively of long-lived, recirculating small lymphocytes and corresponded to a 60% recovery of cellular immunocompetence as measured by the mixed lymphocyte reaction (MLR). Associated with the return of cellular immunocompetence was an increased incorporation of 3H-uridine by the small lymphocytes. Cells from thymectomized animals grafted with lymph node fragments demonstrated no significant increase in lymphocyte numbers nor was there a return of immunocompetence as compared to thymectomized controls.     

Migratory patterns of B lymphocytes. IV. Effect of anti-Ig on follicular localization of radioactively labeled murine spleen and bone marrow cells.

A study was made of the localization of nylon-wool-adherent (AD) and nonadherent (NA) murine spleen cells in lymphoid tissue of irradiated syngeneic recipients. Cells were labeled in vitro with [³H]uridine or ⁵¹Cr and injected intravenously. Localization in recipient tissues was expressed as percent of injected radioactivity. NA and AD [³H]uridine labeled cells gave spleen to lymph node (S:LN) ratios of 1.0 and 2.7, respectively. After treatment of AD cells with rabbit anti-mouse Fab + C at 37 °C, localization in S decreased markedly.NA cells primarily localized in LN paracortex and splenic periarteriolar sheaths. Untreated and NRS-treated AD cells localized in lymphoid follicles, whereas anti-Fab-treated AD cells did not. When ⁵¹Cr-labeled AD cells were treated with anti-Fab at 4 °C without C, there was a transient decrease in splenic localization at 24 hr followed by a recovery to the normal pattern at 48 hr after transfer. [³H]uridine-labeled bone marrow (BM) cells showed less localization in lymphoid tissue than did S cells. Some BM cells were seen in LN follicles, particularly at 48 hr after transfer, but this localization was not affected by prior treatment with anti-Fab + C. The possible role of surface Ig in the determination of follicular localization of B lymphocytes is discussed.     

Suppressor cells in homograft tolerant rats.

If sufficient normal syngeneic lymphocytes to effect skin graft rejection are transferred to homograft tolerant rats, a prolonged period elapses before lymphoid cells from the recipient acquire normal levels of GvH responsiveness against tissues of which the donor was previously tolerant (Silvers and Billingham, 1970; Elkins, 1972; Miyamoto and McCullagh, 1974). Although the ability of lymphoid populations of such animals to mount GvH reactions can be demonstrated to reside in donor type cells during the weeks immediately after transfer, reactive cells are ultimately derived from the host itself (Elkins, 1973; Miyamoto and McCullagh, 1974). Not only are lymphoid cells from tolerant rats which have been injected recently with normal lymphocytes poorly responsive in a GvH assay, but they have been observed in some experiments to suppress the GvH activity of normal syngeneic lymphoid cells (Elkins, 1972; Atkins and Ford, 1972). It is not clear whether the cells mediating suppression of the normal lymphocytes were derived from the tolerant host itself or, alternatively, from the normal lymphocytes injected into it to terminate the tolerant state. The present experiments sought to delineate the origin of any suppressor cells within populations of lymphocytes collected from rats in which tolerance had recently been terminated. The indicate that suppression of the normal donor cells within such populations may be exerted by cells derived from the tolerant host.     

3H-uridine incorporation as a T lymphocyte indicator in the rat

Although about 70% of rat thoracic duct small lymphocytes labeled readily in vitro with ³H-uridine, only 3–38% of peritoneal exudate lymphocytes labeled. Since exudate cells are mostly B lymphocytes, ³H-uridine in concentrations used were presumed to label the T lymphocyte. Percentages of small lymphocytes that labeled in cell suspensions from various tissues were consistent with other estimates of T cells in those sources: 74.7% in thoracic duct, 70.2% in blood and 65.6% in spleen. When lymphopenia was induced by polyethylene ³²P strips applied to the spleen, a procedure that depletes mostly small recirculating lymphocytes, both labeled (T) and nonlabeled (B) cells were depleted in similar time sequence. Both cell types recovered at a similar rate after the spleen strips were removed. Induction of peritoneal inflammation by PPD in tubercle-bacilli immune rats caused an enhanced lymphocytic exudation but no increase in percentage of labeled (T) lymphocytes.The defect in ³H-uridine incorporation that characterizes the rat B lymphocyte seemed to be relatively specific for that RNA precurser; ³H-cytidine labeled the majority of lymphocytes in peritoneal exudate.     

Partial tolerance and immunity after adoptive abrogation of transplantation tolerance in the rat

Lewis rats were rendered hematopoietic and lymphoid cell chimeras by injection of (LBN)F₁ hybrid cells at birth or following treatment with cyclophosphamide in adult life. The establishment of transplantation tolerance was indicated by acceptance of (LBN)F₁ skin grafts and specific unresponsiveness in graft vs. host reaction (GvHR) and mixed lymphocyte interaction (MLI) in vitro. Tolerance was abolished by adoptively transferred Lewis lymphocytes, and the loss of chimerism and recovery of specific reactivity by blood lymphocytes were monitored independently by mixed lymphocyte cultures. Recovery of competence to initiate GvHR by splenic and lymph node cells was monitored by the local renal graft vs. host technique. Both techniques measure essentially the proliferative response of certain lymphocytes to foreign cellular AgB antigens, and both detected a prolonged, but gradually weakening, state of partial tolerance to the AgB factors to which tolerance had originally been induced. During this phase of partial tolerance the former chimera rejects skin and lymph node cell grafts from (LBN)F₁ donors with alacrity, but in some cases accepts (LBN)F₁ kidney grafts. Cytotoxic antibodies appear in the serum soon after allogeneic chimerism is terminated. These results are interpreted to indicate that a state of partial tolerance exists among the cells which proliferate in response to certain AgB antigens in GvHR and MLI in the formerly tolerant chimera, and that a state of transplantation immunity (possibly to other determinants) coexists with this partial tolerance.     

Analysis of neonatally induced tolerance of H-2 alloantigens

There is a considerable amount of evidence, confirmed and extended by our studies, in favor of clonal deletion of alloantigen-reactive cells in neonatally induced transplantation tolerance. We have demonstrated in adult mice bearing long-standing skin allografts that lymphocytes specifically reactive with the tolerated H-2 alloantigens are undetectable by mixed lymphocyte and graftversus-host reactions, and in cell-mediated lympholysis. In addition, lymphoid cells capable of suppressing the reactivity of syngeneic normal lymphocytes in these assays similarly escape detection. Moreover, putative precursors of T cells specific for the tolerated antigens cannot be activated polyclonally with concanavalin A (Con A), nor can they be identified among thymocytes ofH-2-tolerant mice. Since the tolerant state can be adoptively transferred with lymphohematopoietic cells to adult, syngeneic mice, we infer that transplantation tolerance is maintained by an active process that achieves specific clonal deletion at an early stage in the ontogeny of alloreactive T lymphocytes.     

Graft-versus-host response,measured by splenomegaly assay,by populations of allosensitized mouse lymphocytes

The graft-vs-host (G-v-H) reactivity of sensitized or nonsensitized mouse lymphoid cell populations was measured using a splenomegaly assay. Sensitized populations were obtained either from the local lymph nodes of alloimmunized animals or from the spleens of heavily irradiated mice previously infused iv with allogeneic lymphocytes (educated cells). Immunization of animals resulted in increased G-v-H responses of the cells in their local lymph nodes. This effect was more pronounced when the immunizing cells differed only at non-H-2 transplantation antigens than when H-2-disparate strain combinations were tested. There was no evidence of a changed doseresponse profile of lymphocytes obtained from immunized mice. The G-v-H reactivity of educated cell populations was complex. The slopes of the dose-response lines obtained for lymph node cells or thymic cells educated in an H-2-disparate strain were generally lower than those obtained for nonsensitized cells. This difference was particularly evident when testing educated thymocytes. By studying the G-v-H indices obtained in A/Sn × C57B1 hybrids after inoculation of nonsensitized C57B1 lymph node cells or specifically educated C57BL lymph node cells, it was observed that the latter cells were approximately 30 times more reactive when small cell inocula were compared. On the contrary, education of lymphocytes in H-2-compatible allogeneic hosts did not result in any increment of their G-v-H reactivity. The results indicate that different methods of sensitizing lymphocyte populations against alloantigens may lead to activation of different subclasses of T-cells which differ in their mode of antigen reactivity.     

Synthesis of ribosomal RNA in the lymphocytes with 21-chromosome trisomy]

Normal and 21-trisomic lymphocytes isolated from whole blood were cultivated with phytohemagglutinin (PHA) for 21 hours and labeled with 3H-uridine. Polysomes were then extracted, using magnesium precipitation. The samples of polysome rRNA were disc-electrophoresed, and the specific activity of the 28S-RNA was determined. In spite of the decreased RNA-synthetic response of the 21-trisomic lymphocytes (determined earlier) the specific activity of the rRNA in the aberrant cells failed to differ from the normal. A decrease in the number of the PHA-responsive cells in the 21-trisomic lymphoid population is supposed.     

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.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Adult murine lymph node cells respond blastogenically to a new differentiation antigen on isologous and autologous B lymphocytes.

Cells derived from lymph nodes (LN) of adult CBA mice respond blastogenically to mitomycin-treated autologous, as well as isologous spleen cells. This isogeneic LN-to-spleen (mixed lymphocyte culture) is best obtained when both responder and stimulator cells are derived from donors greater than 10 weeks of age. Responsive cells appear restricted to LN since they could not be detected in adult spleen, marrow, or thymus. LN cells do not require the presence of spleen in order to differentiate into responder cells since those derived from neonatally splenectomized mice are fully active. Stimulator cells appear in the spleen, bear Ig on their surfaces, and can be detected in spleens of irradiated, bone marrow-reconstituted mice. Experiments comparing the responsiveness of adult LN cells and that of neonatal T cells toward mitomycin C-treated lymphoid cells from a variety of sources suggest the presence of two iso-antigens on B lymphocytes. Since both antigens apparently are absent on precursor bone marrow cells and develop with time, they have been classified as murine differentiation antigens 1 and 2 (MDA-1, MDA-2). Whereas both appear in the spleen, only one, MDA-1, is also detectable by this methodology in LN. Both MDA-1 and MDA-2 activate neonatal T cells, but MDA-2 triggers only adult LN. Whereas MDA-2 developed in an x-irradiated, bone marrow-reconstituted spleen, MDA-1 did not over a 9-week interval.     

Thymus dependency of neonatal tolerance induction in the absence of detectable suppressor cells

Neonatal thymectomy prevents tolerance induction with bovine serum albumin (BSA) in Wistar Furth (WF) rats whose thymus-derived (T) cell deficit is reconstituted with adult nonadherent peripheral blood lymphocytes (PBL). Sham-thymectomized (STx) rats given PBL become tolerant. To establish whether the adult T cells become tolerant in STx rats, their carrier-reactivity was studied in a cooperative immune response following challenge with methylated BSA (mBSA). The results indicate that carrier-reactive cells, derived from PBL, do become tolerant of BSA in the presence, but not in the absence, of the thymus. To determine whether thymic function during tolerance induction is mediated by suppressor T cells, attempts were made to replace the thymus with various populations of thymocytes or lymphoid cells from neonatal or adult normal rats or neonatal BSA-injected rats. No cell population tried could substitute for the thymus during tolerance induction. In addition, it was found that BSA-tolerant rats with intact thymi do not contain either nonspecific suppressor cells whose activity can be boosted with mBSA or specific suppressor activity demonstrable on transfer to normal rats. Timed thymectomy experiments showed that the thymus is required for more than 2, but less than 5 to 7 days after tolerogen injection for significant tolerance induction. These results imply that the thymus itself is necessary for tolerance induction in a peripheral T-cell population and that its effect is not mediated by suppressor cells. It is suggested that peripheral T helper cells may periodically recirculate through the thymus, at least in young rats, and become tolerant of antigen complexed with Ia antigens in the thymic epithelium. Such a mechanism may be of great importance in the development of self-recognition.     

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

Summary Male Wistar rats were injected intravenously with 5-(³H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.R.B. was a fellow of the Alexander von Humboldt-Stiftung, on leave from the Department of Histology and Embryology, Institut of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

Cellular aspects of tolerance. VI. The effect of age on responsiveness and tolerance induceability of SJL mice

New born and 3-week-old SJL mice but not 8–12-week-old animals could be rendered tolerant to rabbit γ-globulin. Animals reconstituted with thymus cells from 12-week-old donors and bone marrow cells from 3-week-old donors showed resistance to tolerance induction. Animals reconstituted with bone marrow cells from 12-week-old animals and thymus cells from 3-week-old donors could be rendered tolerant. Earlier work has shown that tolerance could be induced in older animals, if they were deprived of competent accessory cells. It was suggested that a lesion in the thymus cell population is expressed through the mediation of accessory cells. The possibility of a relation between resistance to tolerance induction and lymphoid malignancies was discussed.     

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. II. Temporal and dose response relationships of tolerance induction in B lymphocytes of various differentiation states.

The induction of TNP-specific B lymphocyte tolerance by TNBS in sources representing various differentiation states was studied in an adoptive cell transfer system. An adoptive assay was utilized in which the delay of immunization with the T-independent antigen TNP-LPS resulted in an enhanced PFC response. TNBS induced tolerance in spleen cells which was independent of T cell activity, was dose dependent, and could be adoptively transferred. While bone marrow and spleen cells were susceptible to tolerogenesis after cell transfer, TNBS treatment of the donor induced unresponsiveness in splenocytes but not marrow cells. The tolerance dose response relationship and the effect of the temporal relationship between cell transfer and tolerogenesis were studied in B lymphocytes from various sources. Adult spleen cells were resistant to tolerance induction late in the adoptive response, and the tolerance induced by TNBS administration 1 hr after cell transfer was dose dependent. Athymic nude spleen cells and adult bone marrow cells displayed similar characteristics while fetal liver cells were somewhat more susceptible to the induction of unresponsiveness. Neonatal spleen cells were rendered tolerant at much lower doses and at any stage of the adoptive response. The hierarchy obtained in these studies in the order of decreasing resistance to tolerance induction is: adult normal and athymic nude spleen and adult bone marrow, fetal liver, and neonatal spleen. This variation in tolerogenesis appears to be due to the maturity of the cell types which may reflect differences in B lymphocyte sub-populations.     

The differentiation of T lymphocytes. Density characterisation of thymic and peripheral T cells responding in syngeneic and allogenic mixed lymphocyte reactions.

Equilibrium density separation on continuous albumin gradients was used to separate and characterise the T cells responding by proliferation to both syngeneic and allogeneic stimulating cells in the one-way mixed leucocyte reactions (MLR). In CBA mouse spleen both light and dense T cells were capable of responding in an allogeneic MLR. No T cells responding to stimulation be syngeneic B lymphocytes could be isolated from adult or 7-day CBA mouse spleen. In adult CBA mouse thymus, cells responding to allogeneic stimuli were enriched in the light density region, along with the low theta subpopulation. Self-reactive cells, responding with proliferation when cultured with syngeneic adult CBA splenic lymphocytes, and found in adult and 4-day CBA mouse thymus, were also enriched in the light density zones. However, in adult thymus syngeneic MLR reactivity was also found in the dense zones, and the density distribution profiles of total syngeneic MLR responding cells revealed a series of peaks extending over the whole density range. It was suggested that these syngeneic MLR responders undergo a complete maturation process, including progressive density increases, within the thymus gland. Such a sterile differentiation pathway could be a censorship process, leading to death of self-reactive cells within the thymus.