Effect of age on the capacity of the bone marrow and the spleen cells to generate B lymphocytes

The effect of age on the regeneration of the B cell population was studied by cell transfer methods, using the allotype-congenic mouse strains BALB/c (Igha) and C.B-17 (Ighb) as donors of old and young bone marrow (BM) and spleen cells, and C.AL-20 (Igho) as recipients. This design allowed us to identify the origin of the sIgD+ B cells present in the recipients. It was found that in a simple cell transfer, BM cells or spleen cells of aged donors could reconstitute the peripheral B cell population of irradiated, thymectomized recipients essentially as effectively as could BM or spleen cells from young donors. However, when BM cells from aged donors and from young donors were mixed and were used to reconstitute a single recipient, the cells from the aged donor were less efficient than were the cells from the young donor. We found that sIgD+ B cells of young donor origin predominated in the peripheral B cell population of the recipient at 3 to 6 wk after cell transfer. In the BM of the recipients, however, there was no difference in the incidence of sIgD+ B cells derived from the young and the old donors. When recipients were reconstituted with a mixture of spleen cells from old and young mice, the sIgD+ cells of young donor allotype showed a tendency to predominate in the peripheral B cell population, although this predominance was not statistically significant. Under such competitive conditions, the spleen cells of aged donors were less efficient than the BM of aged donors in reconstituting the sIgD+ B cell population of the recipient's BM, but were more efficient in reconstituting the splenic sIgD+ cells. Thus, a subtle defect in the B cell precursor population of the BM and the spleen of aged mice has been demonstrated. The role of T cells in the generation of sIgD+ cells was also analyzed.     

Cell lines from old immunodeficient donors give normal responses in young recipients.

Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/wf1 mice.

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF₁ mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF₁ mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF₁ mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF₁ recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF₁ mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.     

Evidence for a B lymphocyte defect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.     

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. I. Impairment of mitogen responses and suppressor phenomena

In our laboratory, we have developed a murine model to examine GVHD across minor histocompatibility antigens. In our model, GVHD is induced by injecting B10.D2 spleen cells into irradiated BALB/c recipients. Seven to 10 days after irradiation and injection of cells, there are significant changes in cell function in the recipient spleens. In the B10.D2----BALB/c (600 rad) model, recipient spleen cells are profoundly unresponsive to Con A and LPS stimulation but show increased B cell activity measured by Staphylococcus aureus protein A plaque-forming activity. Spleen cells from such GVH mice profoundly suppress the mitogenic responses of normal BALB/c or B10.D2 spleen cells to Con A and LPS. The degree of impairment of the mitogenic response and the ability to suppress normal cells is proportional to the dose of cells used to induce GVH reactions. Both the inability to respond to mitogens and the capacity to suppress are also related to the dose of irradiation given to the recipients. In addition, immunosuppression across minor histocompatibility antigens shows an unevenhandedness. If we inject parental B10.D2 or BALB/c cells into F1 recipients (P----F1), there is greater inhibition of mitogenic responses when B10.D2 parental cells are given than when BALB/c cells are given to the irradiated F1 recipients. These experiments show that significant immunosuppression occurs during GVH reactions across minor histocompatibility barriers. The degree of suppression varies according to the dose of cells used to induce GVH, the dose of irradiation to the recipient and the "strength" of the GVH recognition system. Such experiments provide models for GVH disease seen in humans who receive treatment for leukemia or other diseases that involves recipient irradiation and infusion of HLA-identical bone marrow.     

Effects of tobacco glycoprotein (TGP) on the immune system. III. The effect of aging on the mitogenic response of human peripheral blood lymphocytes to TGP

We have shown that the mitogenic response of human peripheral blood lymphocytes (PBL) to tobacco glycoprotein (TGP), a glycoprotein rich in rutin or rutinlike polyphenol moieties, which is isolated from cured tobacco leaves, does not decrease with age. In contrast, the proliferative response to lipopolysaccharide (LPS) tends to decline with age, and a significant decrease is observed in the mitogenic response to rutin-bovine serum albumin (R-BSA). LPS and R-BSA are similar in some aspects to TGP, the former in that TGP and LPS are both T-independent B-cell mitogens for mice and are both highly negatively charged, and the latter in that covalently bound polyphenol groups are present on both R-BSA and TGP. Although the kinetics of the mitogenic responses to these three mitogens are similar (T. Francus, R. F. Klein, L. Staiano-Coico, G. W. Siskind, and C. G. Becker, Effects of tobacco glycoprotein (TGP) on the immune system. II. TGP is a mitogen for human peripheral blood lymphocytes. Submitted for publication.), multiple regression analyses show no correlation in the mitogenic responses of PBL from young donors to TGP and LPS, to TGP and R-BSA, or to LPS and R-BSA. In contrast, there are significant correlations between the proliferative responses to these mitogens by PBL from old donors. The results suggest that only a small subpopulation of the cells which are stimulated by R-BSA and LPS are not altered with age, and these are most likely the cells that are stimulated by the three mitogens studied.     

B cell development and regulation after T cell-depleted marrow transplantation

Antibody secreting B lymphocytes from immunized donors can be adoptively transferred after T cell-depleted marrow transplantation to produce protective levels of antibody in the recipient. We have investigated whether these transferred lymphocytes remain subject to continued clonal selection and subsequently became memory B cells even in the initial absence of T cells. Twenty-eight donor/recipient pairs were randomized pretransplant to be immunized or not with tetanus toxoid (TT). The recipients were then vaccinated with TT at 3, 6, and 12 mo posttransplant, and the anti-TT antibody response (IgG and IgM) was measured. Only when both donor and recipient were immunized pretransplant could the recipient respond to antigen challenge within the first year posttransplant. Examination of the spectrotype pattern of the recipient anti-TT antibody shows that selection of B cell clones continues, so that T cell depletion does not prevent the appearance of oligoclonal antibody responses. However, because the spectrotype pattern of the recipient did not match the donors, B cell regulatory mechanisms in donor and recipient are nonidentical. These data contrast with observations made in recipients of non-T cell-depleted marrow and serve to illustrate the role of T lymphocytes in the induction and regulation of secondary antibody responses in man. The results also suggest that optimal humoral responses to any antigen after T cell depletion can only occur when both donor and recipient are immunized pretransplant, a prediction borne out by studies on the influence of donor cytomegalovirus status on the severity of cytomegalovirus infection in the recipient.     

Time-dependent enhancement of lymphocyte activation by mitogens after exposure to isolation or water scheduling

The effects of isolation and water scheduling on mitogen induced lymphocyte proliferation were investigated. Isolated rats were animals which had been raised in group-housed conditions and then transferred to individual cages with ad lib access to water for a 1 or 2 week period. Water scheduled rats were maintained in group housing (5 rats per cage) with ad lib access to food but with access to water for a single 30 minute session each day. Responses of these groups were compared to those of animals which had been continuously group-housed with ad lib access to food and water. No differences in lymphocyte responses to phytohemagglutinin (PHA) were found 1 week after exposure to isolation. However, after 2 weeks, splenic and blood T lymphocytes from isolated animals demonstrated an increased proliferative response to suboptimum and maximum concentrations of PHA. Splenic B lymphocyte responses to lipopolysaccharide (LPS) from isolated animals were also increased by 2- to 3-fold compared to group-housed controls. Two weeks of exposure of animals to daily water scheduling similarly increased the splenic lymphocyte proliferation. This increased responsiveness to PHA was not accompanied by a significant change in the sensitivity of the lymphocytes to PHA, in the total number of white blood cells, or the proportion of splenic T or T helper lymphocytes. Our results show that the increase in lymphocyte proliferation is time-dependent, requires greater than 1 week of exposure to isolation and is due to factors other than changes in sensitivity to mitogen or T lymphocyte number.     

Cell-cell cooperation in lymphocyte colony formation: studies in human allogeneic marrow transplantation

The pace of restoration of phytohemagglutinin- (PHA) stimulated lymphocyte colony-forming response was investigated in 50 bone marrow transplant (BMT) recipients (49 allogeneic, 1 syngeneic) studied on 86 occasions. On the majority of occasions (28 of 48), patients studied early (3 to 8 wk) after BMT failed to make detectable lymphocyte colonies, whereas patients studied late (greater than 6 mo) after BMT displayed responses comparable to those of healthy adult volunteers. The sequential study of 10 of the 15 patients with positive lymphocyte colony-forming responses early after BMT revealed that this response is short-lived. The results of recipient donor mixing experiments and experiments involving the addition of the lymphokine interleukin 2 (IL 2) to recipient cells indicated that lymphocytes obtained from unresponsive recipients evaluated early after BMT can be driven to colony formation, and suggest that the major limiting component in such subjects is functional T help.     

An instructive role of donor macrophages in mixed chimeras in the induction of recipient CD4(+)Foxp3(+) Treg cells

The immune regulatory function of macrophages (M?s) in mixed chimeras has not been determined. In the present study, with a multi-lineage B6-to-BALB/c mixed chimeric model, we examined the ability of donor-derived splenic M?s in the induction of regulatory T cells (Treg). B6 splenic M?s from mixed chimeras induced significantly less cell proliferation, more IL-10 and TGF-β, and less IL-2 and IFN-γ productions of CD4(+) T cells from BALB/c mice than naive B6 M?s did, whereas they showed similar stimulatory activity to the third part C3H CD4(+) T cells. Importantly, highly purified donor F4/80(+)CD11c(-) M?s efficiently induced recipient CD4(+)Foxp3(+) Treg cells from CD4(+)CD25(-)Foxp3(-) T cells. Furthermore, donor M?s of mixed chimeras produced more IL-10 and less IFN-γ than those of naive mice when cultured with BALB/c but not the third party C3H CD4(+) T cells. Induction of recipient CD4(+) Treg cells by donor M?s was significantly blocked by anti-IL-10, but not by anti-TGF-β mAb. Therefore, donor M?s have the ability to induce recipient CD4(+)Foxp3(+) Treg cells in a donor antigen-specific manner, at least partially, via an IL-10-dependent pathway. This study for the first time showed that, in mixed allogeneic chimeras, donor M?s could be specifically tolerant to recipients and gained the ability to induce recipient but not the third party Foxp3(+) Treg cells. Whether this approach is involved in transplant immune tolerance needs to be determined.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Infectious nickel tolerance: a reciprocal interplay of tolerogenic APCs and T suppressor cells that is driven by immunization

Previously, we reported that tolerance to nickel, induced by oral administration of Ni(2+) ions, can be adoptively transferred to naive mice with only 10(2) splenic T cells. Here we show that 10(2) T cell-depleted spleen cells (i.e., APCs) from orally tolerized donors can also transfer nickel tolerance. This cannot be explained by simple passive transfer of the tolerogen. The APCs from orally tolerized donors displayed a reduced allostimulatory capacity, a tolerogenic phenotype, and an increased expression of CD38 on B cells. In fact, it was B cells among the APCs that carried the thrust of tolerogenicity. Through serial adoptive transfers with Ly5.1(+) donors and two successive sets of Ly5.2(+) recipients, we demonstrated that nickel tolerance was infectiously spread from donor to host cells. After the transfer of either T cells or APCs from orally tolerized donors, the spread of tolerance to the opposite cell type of the recipients (i.e., APCs and T cells, respectively) required recipient immunization with NiCl(2)/H(2)O(2). For the spread of tolerance from a given donor cell type, T cell or APC, to the homologous host cell type, the respective opposite cell type in the host was required as intermediate. We conclude that T suppressor cells and tolerogenic APCs induced by oral administration of nickel are part of a positive feedback loop that can enhance and maintain tolerance when activated by Ag associated with a danger signal. Under these conditions, APCs and T suppressor effector cells infectiously spread the tolerance to naive T cells and APCs, respectively.     

Lymphotoxin-alpha-dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation

Lymphotoxin alpha-deficient (LTalpha-/-) mice show dramatically reduced IgG responses after either primary or secondary immunizations with sheep red blood cells (SRBC). When splenocytes from SRBC-primed wild-type donor mice were infused into irradiated naive wild-type recipient mice, they generated a robust memory IgG response, but not when infused into LTalpha-/- recipients, indicating that the microenvironment that develops in LTalpha-/- mice is incompetent to support the activation of this memory response. When irradiated wild-type mice were reconstituted with splenocytes from primed LTalpha-/- donors and then challenged with the same immunizing Ag, no memory response was observed, indicating further that memory cells could not be generated in the LTalpha-/- environment. To address which lymphocyte subsets were impaired in the LTalpha-/- mice, we performed reconstitution experiments using a hapten/carrier system and T cells and B cells from different primed donors. There was no detectable defect in either the generation or expression of memory T cells from LTalpha-/- donors. In contrast, B cells were not primed for memory in the microenvironment of LTalpha-/- mice. Additionally, primed wild-type memory B cells could not express a memory IgG response in the LTalpha-/- microenvironment. Thus, splenic white pulp structure, which depends on the expression of LTalpha for its development and maintenance, is needed to support the generation of memory B cells and to permit existing memory B cells to express an isotype switched memory Ig response following antigenic challenge.     

T and B lymphocyte populations of the spleen and thymus in normal and immunosuppressed BALB/c mice.

The antituinor agent 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) has been studied in order to determine its effect on thymic and splenic T and B lymphocytes in normal and immunosuppressed BALB/c mice. Utilizing indirect immunofluorescence and lymphocyte proliferation studies we detected an initial reduction of splenic T and B cells as a result of the administration of an optimal dose, 30 mg/kg, of BCNU. The population dynamics of the thymic lymphocytes are totally different in their mitogenic reactivity than that of the splenic lymphocytes. An initial decline in the PHA and LPS-sensitive splenic lymphocytes of BCNU-treated mice was temporary. However, there was no return to normal levels detected for the Con A-sensitive splenic lymphocytes. On the other hand, the PHA-sensitive thymic lymphocytes of BCNU-treated mice not only failed to repopulate but were totally depleted by the tenth day.     

Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO)

The relative roles of donor and host T lymphocytes and the T cell activation requirements in adoptive transfer of experimental autoimmune orchitis (EAO) in (C57BL/6 x A/J)F1 mice were investigated in order to gain an understanding of the pathogenesis of this disease. Depletion of T cell subsets in recipients by adult thymectomy and treatment with monoclonal antibodies against CD4 or CD8 had no effect on the incidence of EAO following adoptive transfer of activated T cells from donors immunized with testis homogenate (TH) and adjuvants. In contrast, such depletion of CD4+ T cells inhibited development of EAO in actively immunized mice. Thus, CD4+ cells are required for induction of EAO, but donor CD4+ cells are sufficient by themselves without a comparable contribution from the recipient. Adoptive transfer of EAO required that donor splenic and lymph node T cells be activated in vitro before transfer. We found that exposure to antigen (TH) for as little as 4 hr allowed EAO to occur in 25% of recipients, and by 24 hr the cells were fully competent to induce disease. Proliferation of the cells could not be measured until 2 days later. In serial double-transfer experiments, it was found that the cells must be cultured with TH before each transfer in order for the secondary recipients to develop EAO. However, it was not necessary for the transferred T cells to "see" antigen in vivo in the primary recipients, since transfer to castrated primary recipients had no effect on EAO incidence in secondary recipients. Lymphocytes isolated from diseased testes of immunized donors were competent to transfer EAO without activation in vitro, suggesting that, unlike spleen and lymph node cells, these orchitic lymphocytes were already capable of trafficking to the testis.     

Induction of autogamy by transfer of macronuclear karyoplasm in Paramecium tetraurelia

Macronuclear karyoplasm was transplanted from pre-autogamous donor cells (clonal age, 22 fissions) into the macronucleus of young recipient cells (2 fissions after autogamy occurred) by means of microinjection. A reciprocal experiment was carried out by injecting karyoplasm from young clonal age donors into pre-autogamous recipients. In the case of karyoplasm transfer from pre-autogamous donors to young recipients, autogamy occurred early in 67% of injected cells, whereas reciprocal injections had no influence on the onset of autogamy, and all of the injected cells underwent autogamy. Such results indicate a distinct role of pre-autogamous cells of macronucleus in the induction of autogamy.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.