Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat

Summary The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme and immunohistochemical techniques at the light-microscopical level.The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes.Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla.The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found.Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.Abbreviations PLN popliteal lymph node - FDC follicular dendritic cell - IDC interdigitating cell - HEV high endothelial venule - TNP trinitrophenyl - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - GCPC germinal center precursor cell - sIg surface immunoglobulin - cIg cytoplasmic immunoglobulin     

Recruitment of memory B cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus

Successful recall Ab responses require recruitment of quiescent memory B cells to secondary lymphoid organs. However, the cellular dynamics of memory cells responding to local antigenic challenge at lymphoid sites distal from the initial Ag encounter are not well understood. We show in this study that memory B cells generated following s.c. immunization in one footpad generate secondary responses to soluble Ag given i.p. but not to Ag given s.c. in the contralateral footpad unless LPS is coadministered. Memory B cells do not express CD62L, and CD62L(-ve) cells cannot enter lymph nodes unless LPS-mediated inflammation is induced there. Functional TLR4 is required on the B cells, as well as on non-B cells, in the lymph node to achieve full recruitment. Furthermore, splenectomized mice fail to respond to such inflammatory s.c. challenge in contralateral footpads, unlike lymphadenectomized mice lacking the original draining lymph nodes. Splenectomized mice also fail to respond to i.p. challenge with soluble Ag. Together, these data indicate that, unlike the central memory pool of T cells, which circulates through resting lymph nodes, the majority of long-lived memory B cells are spleen resident and require inflammatory signals for mounting recall responses at distal challenge sites.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

Further studies on the tumor-specific suppressor cells induced by ultraviolet radiation.

Recent studies have suggested that the immunologic unresponsiveness of UV-irradiated mice against UV-induced fibrosarcomas might be due to the presence of suppressor lymphoid cells. In these experiments, we present additional evidence that suppressor lymphoid cells are present in lymph nodes and spleens of UV-irradiated mice and demonstrate that these cells are enriched after incubation on nylon wool columns, that they are inactive after treatment with anti-theta serum and complement, and that they are effective for at least 7 weeks after transfer to lethally x-irradiated mice. Splenectomy of UV-treated mice before tumor challenge did not restore their anti-tumor reactivity. The UV-induced suppressor cells appear to be specific for syngeneic UV-induced tumors, since they did not suppress the rejection of an allogeneic UV-induced tumor or two chemically induced syngeneic tumors.     

Multiple effector roles of lymphocytes in allograft immunity

Several classes of cytotoxic lymphoid cells were detected in the spleens of mice immunized with allogeneic lymphoma cells. Two types of radioresistant cells (retaining cytotoxicity after exposure to 1000 R x-rays) were detected 7 days after immunization with allogeneic cells, one a T-cell independent antibody-secreting cell type, the other T-cell dependent, directly cytotoxic and complement-independent in action. At 21 days after immunization only directly cytotoxic thymus dependent cells were found. These cells were radiosensitive (cytotoxicity ablated by 500 R x-rays), although after contact with specific antigen they were changed into a more radioresistant state. Sensitized lymphoid cells capable of rendering normal macrophages specifically cytotoxic to target cells were also found to be of T-cell origin. Immune cells which are directly cytotoxic and those which release the specific macrophage arming factor (SMAF) may differ, although both are thymus-derived and both respond biochemically to the specific antigen.     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.     

Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.     

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

Induction and mechanism of tolerance to bovine serum albumin in mice given total lymphoid irradiation (TLI).

BALB/c mice given total lymphoid irradiations (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.     

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.     

Expressed Salmonella antigens within macrophages enhance the proliferation of CD4+ and CD8+ T lymphocytes by means of bystander dendritic cells

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.     

Early lymphocytic responses to Heligmosomoides polygyrus infections in mice

Responses to parasite antigens were studied in three strains of mice, BALB/c, CBA and NIH, during the initial phases of a primary infection with the intestinal nematode Heligmosomoides polygyrus. Changes in the rate of in vivo cell division were analysed in mesenteric lymph nodes and spleens during the phases of larval maturation and adult establishment, and related to changes in organ size and cellularity. The nature of the proliferating cell populations was also investigated by flow cytometry, carried out on cell suspensions prepared at the time when larval development was complete. The variation in the ability of the strains of mice to become resistant to a challenge infection was manifest as only slight differences in their initial responses to infection. All three strains showed an increase in 125I-iododeoxyuridine incorporation in their mesenteric lymph nodes and spleen, and an increase in B cell frequency over that of T cells in the draining lymph nodes. Although lymph node weight in NIH mice continued to rise over a 4 week period, the majority of responses measured were short lived, peaking 10 to 14 days after infection. The low responder status of CBA mice was thus reflected in a transient and relatively small enlargement of lymphoid tissues, but their early proliferative responses to antigen were similar in scale to those of responder strains.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Cell-mediated immunity to male-strain histocompatibility alloantigens detected after natural insemination and systemic immunization in the female mouse using the cell-mediated microcytotoxicity test

Female cell-mediated immunity to allogeneic spermatozoa after repeated natural insemination, in the absence of pregnancy, was compared with that after systemic challenge using the cell-mediated microcytotoxicity test to measure cytotoxic cell alloreactivity. After multiple (3-6) inseminations the majority of females (11 out of 13) showed a significant degree of lymphocytotoxicity to male-strain histocompatibility alloantigens in the para-aortic lymph nodes, and to a lesser extent in the spleens, while a single insemination was usually not sufficient to evoke a specific cytotoxic cell response. This differed from the low and highly variable degree of female sensitization after multiple systemic challenge with allogeneic spermatozoa via the intraperitoneal route. By contrast, a single systemic challenge via the footpad proved to be the most highly consistent and effective route for eliciting cell-mediated immunity to male-strain histocompatibility alloantigens in all 9 female mice. This alloreactivity appeared to be directed at alloantigens other than the male-specific H-Y antigen. These findings show that the precise route of immunization is a major factor in the development of female cell-mediated immune responsiveness to allogeneic spermatozoa.     

Antigen-bearing Langerhans cells in skin,dermal lymphatics and in lymph nodes

Ferritin-challenged skin sites and draining lymph nodes were studied in normal guinea pigs and in guinea pigs which had been passively sensitized to ferritin or peroxidase by lymphoid cell transfer to ascertain whether Langerhans cells can bind antigen in skin and carry it to lymph nodes. After intradermal challenge with amounts of ferritin as small at 5 μg, ferritin-containing Langerhans cells were seen by electron microscopy in the marginal sinus and cortex of draining lymph nodes in ferritinscnsitized animals and, to an apparently lesser degree, in control animals. Lymph nodes from unchallenged normal guinea pigs contained rare Langerhans cells, none of which had ferritin. The findings indicate that Langerhans cells may pick up antigen in skin and from there circulate to draining lymph nodes, thus carrying out a function analogous to macrophages. In this way they may exhibit antigen to lymphocytes both in skin and in lymph nodes.     

Serum antibody response and nasal lymphoid tissue (NALT) structure in the absence of IL-4 or IFN-gamma

Immunization via the nasal route is effective for inducing not only mucosal immunity but also antibody (Ab) response in serum. Nasal lymphoid tissue (NALT) is important for induction of systemic immunity. It remains controversial which T effector cell response is important for serum Ab response after nasal immunization. We investigated serum Ab responses and NALT structures in interleukin (IL)-4 gene targeted (IL-4(-/-)) and interferon (IFN)-gamma gene targeted (IFN-gamma(-/-)) mice. Mice were immunized via nostrils with ovalbumin (OVA) and cholera toxin as adjuvant and serum Ab titers were measured 1 week after final antigen challenge. OVA-specific IgG titers in sera of IL-4(-/-) mice indicated a Th(1) type response, whereas titers in IFN-gamma(-/-) mice and wild-type mice indicated a Th(2) type response. Enhanced serum Ab responses were observed in IL-4(-/-) mice but not IFN-gamma(-/-) mice. OVA-specific Ab-forming cells were detected in the cervical draining lymph nodes but were rare or absent in and around the NALT of all strains of mice. Numbers of OVA-specific Ab-forming cells in cervical lymph nodes were significantly higher in IL-4(-/-) mice than in wild-type and IFN-gamma(-/-) mice. Germinal centers of lymphoid follicles were present in NALT of IL-4(-/-) and other mice. Immunohistochemistry for B and T cell markers revealed that NALT of all mice had approximately the same cellular compositions. Although the absence of IL-4 had no effect on NALT structure, IL-4 may suppress induction of serum Ab responses by nasal immunization.     

Factor producing lysis of thymus-derived cells in vitro by specific antigen.

Lymph node cells of immunized mice were lysed by specific antigen in vitro. These cells were not lysed by antigen after washing them four times with Hanks' solution. The factor lysing lymph node cells and thymus cells of normal mice in the presence of the specific antigen was found in the supernatant of the lymph node cell suspension of immunized mice. Bone marrow cells were also lysed by the factor and specific antigen only after preincubating the cells in extracts of thymus or lymph nodes of normal mice. When active supernatants were fractionated by gel filtration on Sephadex G-200, the active material eluted with molecules of approximately 20,000–45,000 MW. This factor was homogeneous on electrophoreses in polyacrylamide gels and detected in the prealbumin zone.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

A partial characterization of suppressor cells in the spleens of mice conditioned with fractionated total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) is a highly effective modality for inducing immunosuppression and transplantation tolerance. The cellular basis for this immunosuppression is not clear, although T cells have been implicated. To study further the effect of TLI on the immune system, we have examined the B cells and suppressor cells in the spleens from TLI-conditioned mice. Our results indicate that after TLI, the spleen is rapidly repopulated with many large, immature cells. The probable source of these cells is the shielded bone marrow (BM). The B cells from TLI-conditioned mice are transiently immature and hyporesponsive in vitro to a T-independent antigen. Spleen cells from TLI-conditioned mice nonspecifically suppress the in vitro T-independent anti-TNP response of normal B cells. The suppressor cells lack both B and T cell markers and adhere to Sephadex G-10. The suppressor cells in spleens from TLI-treated mice bear a number of similarities to those present in normal BM. When normal BM cells were analyzed by indirect immunofluorescence for the presence of the Mac-1 antigen, two populations of suppressor cells could be identified: one was Mac-1+ and the other was Mac-1-. These data are consistent with the possibility that a subpopulation of the suppressor cells found in normal BM and in the spleens from TLI-conditioned mice are immature cells of the monocytic/granulocytic lineage.