Contribution of dendritic cells to stimulation of the murine syngeneic mixed leukocyte reaction

We have studied the proliferative response of unprimed T cells to syngeneic dendritic cells (DC) (syngeneic mixed leukocyte rection [SMLR]) in cultures of mouse spleen and lymph node. T cells purified by passage over nylon wool contain few DC and exhibit little proliferative activity during several days of culture. Addition of small numbers of purified syngeneic DC induces substantial, dose-dependent, T cell-proliferative responses that peak at day 4-5. B cells purified on anti-Ig-coated plates do not respond to DC at all doses tested. DC cultures medium does not induce proliferation, and coculture of DC and T cells is required. Purified mouse B and T lymphocytes stimulate SMLR weakly if at all. Likewise, peritoneal and spleen macrophages are weak or inactive. Therefore, DC are potent and possibly unique primary cells for stimulating the SMLR in mice. sIg- spleen lymph node cells show extensive background proliferative responses in vitro, and fail to respond to small numbers of purified DC. If the sIg- cells are treated with anti-Ia and complement, or passed over nylon wool, DC are removed and proliferative activity falls. Proliferative activity is restored by adding back DC at levels similar to those present in sIg- cells (1-2%). Thus, DC-dependent, T cell proliferation probably occurs in all spleen and lymph node cultures. As expected from previous work (6), DC are also potent inducers of allogeneic MLR. On a per DC basis, the syngeneic response is 10 times weaker than the allogeneic MLR, and it is not accompanied by the development of cytotoxic lymphocytes. The magnitude of the SMLR was not altered by antigen priming, and DC maintained in isologous rather than fetal calf serum were active stimulators. Therefore, syngeneic stimulation appears to be an intrinsic property of DC, and modification by exogenous agents does not seem to be required. Coculture of DC and T cells results in the development of cell clusters that can be isolated and characterized directly. The clusters account for 10-20% of the viable cells in the culture, but contain \u3e80% of the responding T cells and stimulating DC by morphologic and surface-marker criteria. The efficient physical association of DC and responding T cells implies specific cell-cell recognition. We conclude that the SMLR reflects the ability of T cells, or some subpopulation of T cells, to interact with and proliferate in response to small numbers of DC

Studies of the cell surface of mouse dendritic cells and other leukocytes

[No abstract available

Studies of the cell surface of mouse dendritic cells and other leukocytes

Nussenzweig, M.C., Steinman, R.M., Unkeless, J.C., Witmer, M.D., Gutchinov, B., and Cohn, Z.A. Studies of the cell surface of mouse dendritic cells and other leukocytes. J. Exp. Med. 154: 168-187, 1981https://digitalcommons.rockefeller.edu/historical-scientific-reports/1006/thumbnail.jp

Tolerogenic dendritic cells

Dendritic cells (DCs) have several functions in innate and adaptive immunity. In addition, there is increasing evidence that DCs in situ induce antigen-specific unresponsiveness or tolerance in central lymphoid organs and in the periphery. In the thymus DCs generate tolerance by deleting self-reactive T cells. In peripheral lymphoid organs DCs also induce tolerance to antigens captured by receptors that mediate efficient uptake of proteins and dying cells. Uptake by these receptors leads to the constitutive presentation of antigens on major histocompatibility complex (MHC) class I and II products. In the steady state the targeting of DC antigen capture receptors with low doses of antigens leads to deletion of the corresponding T cells and unresponsiveness to antigenic rechallenge with strong adjuvants. In contrast, if a stimulus for DC maturation is coadministered with the antigen, the mice develop immunity, including interferon-γ-secreting effector T cells and memory T cells. There is also new evidence that DCs can contribute to the expansion and differentiation of T cells that regulate or suppress other immune T cells. One possibility is that distinct developmental stages and subsets of DCs and T cells can account for the different pathways to peripheral tolerance, such as deletion or suppression. We suggest that several clinical situations, including autoimmunity and certain infectious diseases, can be influenced by the antigen-specific tolerogenic role of DCs

Dendritic cells of the mouse: Identification and characterization

We have identified and characterized a distinctive population of dendritic cells (DCs) in mouse spleen, lymph nodes, thymus, and liver. Dendritic cells can adhere to tissue culture surfaces but otherwise differ considerably from macrophages, the other major class of adherent cell. Morphological differences are evident by phase contrast and electron microscopy, and by cytochemistry. Dendritic cells exhibit little or no binding and phagocytosis of opsonized particles. During culture, they retain their unusual morphological features and surface markers, but lose the capacity to adhere. All DCs express and synthesize Ia antigens for several days in vitro, whereas only a subpopulation of mouse macrophages expresses Ia in all organs we have studied. Thus, DCs can be distinguished from macrophages in several independent and stable traits. Highly enriched preparations of the 2 cell types have been obtained. Spleen DCs are derived from bone marrow and are present in nude mice. Dendritic cells do not proliferate, but exhibit a rapid turnover. Other features in their life history are not known. We are studying the contribution of DCs to several immune responses. In all organs we have studied, they are powerful stimulators of the primary mixed leukocyte reaction. B cells, T cells, and macrophages from these organs are weak or inactive. Dendritic cells are potent accessory cells in T cell proliferative responses to mitogens and tuberculin antigens. These dendritic cells and Langerhans cells may belong to a similar lineage, but to date, Birbeck granules, surface ATPase, and binding of opsonized erythrocytes have not been demonstrated in spleen dendritic cells. However, in functional assays, both DCs and Langerhans cells synthesize Ia antigens and contribute to transplantation reactions, accessory cell function, and the development of contact sensitivity

Contribution of dendritic cells to stimulation of the murine syngeneic mixed leukocyte reaction.

We have studied the proliferative response of unprimed T cells to syngeneic dendritic cells (DC) (syngeneic mixed leukocyte rection [SMLR]) in cultures of mouse spleen and lymph node. T cells purified by passage over nylon wool contain few DC and exhibit little proliferative activity during several days of culture. Addition of small numbers of purified syngeneic DC induces substantial, dose-dependent, T cell-proliferative responses that peak at day 4-5. B cells purified on anti-Ig-coated plates do not respond to DC at all doses tested. DC cultures medium does not induce proliferation, and coculture of DC and T cells is required. Purified mouse B and T lymphocytes stimulate SMLR weakly if at all. Likewise, peritoneal and spleen macrophages are weak or inactive. Therefore, DC are potent and possibly unique primary cells for stimulating the SMLR in mice. sIg- spleen lymph node cells show extensive background proliferative responses in vitro, and fail to respond to small numbers of purified DC. If the sIg- cells are treated with anti-Ia and complement, or passed over nylon wool, DC are removed and proliferative activity falls. Proliferative activity is restored by adding back DC at levels similar to those present in sIg- cells (1-2%). Thus, DC-dependent, T cell proliferation probably occurs in all spleen and lymph node cultures. As expected from previous work (6), DC are also potent inducers of allogeneic MLR. On a per DC basis, the syngeneic response is 10 times weaker than the allogeneic MLR, and it is not accompanied by the development of cytotoxic lymphocytes. The magnitude of the SMLR was not altered by antigen priming, and DC maintained in isologous rather than fetal calf serum were active stimulators. Therefore, syngeneic stimulation appears to be an intrinsic property of DC, and modification by exogenous agents does not seem to be required. Coculture of DC and T cells results in the development of cell clusters that can be isolated and characterized directly. The clusters account for 10-20% of the viable cells in the culture, but contain \u3e80% of the responding T cells and stimulating DC by morphologic and surface-marker criteria. The efficient physical association of DC and responding T cells implies specific cell-cell recognition. We conclude that the SMLR reflects the ability of T cells, or some subpopulation of T cells, to interact with and proliferate in response to small numbers of DC

A monoclonal antibody specific for mouse dendritic cells

Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33D1. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (MΦ) cell line, and mitogen-stimulated lymphoblasts. 33D1 Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and Ia- MΦ (Ia, I-region-associated antigen). Quantitative binding studies with 3H-labeled 33D1 Ab showed that DC had an average of 14,000 binding sites per cell. Binding to DC was inhibited with Fab fragment of 33D1 Ab but not with a panel of other monoclonal antibodies, including anti-Ia Ab. Adherence and flotation procedures that enriched for DC enriched for 3H-labeled 33D1 Ab binding in parallel. 33D1 antigen was not detectable on: MΦ from spleen, peritoneal cavity, and blood; three MΦ cell lines; lymphocytes; granulocytes; platelets; and erythroid cells. DC continued to express the 33D1 antigen after 4 days in culture, whereas MΦ and lymphocytes did not acquire it. Quantitative and autoradiographic studies confirmed that spleen and lymph node suspensions contain less than 1% DC. We conclude that 33D1 Ab detects a stable and specific DC antigen and can be used to monitor DC content in complex lymphoid mixtures

A monoclonal antibody specific for mouse dendritic cells

Nussenzweig, M.C., Steinman, R.M., Witmer, M.D., and Gutchinov, B. A monoclonal antibody specific for mouse dendritic cells. Proc. Natl. Acad. Sci. USA 79: 161-165, 1982https://digitalcommons.rockefeller.edu/historical-scientific-reports/1007/thumbnail.jp

Dendritic cells are accessory cells for the development of anti-trinitrophenyl cytotoxic T lymphocytes

This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (MΦ) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. MΦ added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test MΦ populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-A(b,d) antibody, clone B-21. MΦ that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of MΦ to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% MΦ. In contrast, 0.5% Ia-bearing MΦ from BCG-immune boosted mice inhibited \u3e80% of the DC-mediated CTL response. Addition of indomethacin reversed MΦ inhibition, and 10-9M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen MΦ. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing MΦ are weak or inactive; and (c) MΦ can inhibit DC-mediated response by an indomethacin-sensitive mechanism

Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated in activated B lymphocytes by activation-induced deaminase (AID). AID is thought to make lesions in DNA by deaminating cytidine residues in single-stranded DNA exposed by RNA polymerase during transcription. Although this must occur in the nucleus, AID is found primarily in the cytoplasm. Here we show that AID is actively excluded from the nucleus by an exportin CRM1-dependent pathway. The AID nuclear export signal (NES) is found at the carboxyl terminus of AID in a region that overlaps a sequence required for CSR but not SHM. We find that AID lacking a functional NES causes more hypermutation of a nonphysiologic target gene in transfected fibroblasts. However, the NES does not impact on the rate of mutation of immunoglobulin genes in B lymphocytes, suggesting that the AID NES does not limit AID activity in these cells