Different Subpopulations of Developing Thymocytes are Associated with Adherent (Macrophage) or Nonadherent (Dendritic) Thymic Rosettes

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8+CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROSassociated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity

Dendritic cell subsets in the intestinal lamina propria: ontogeny and function

The intestinal mucosa is exposed to large amounts of foreign antigen (Ag) derived from commensal bacteria, dietary Ags, and intestinal pathogens. Dendritic cells (DCs) are believed to be involved in the induction of tolerance to harmless Ags and in mounting protective immune responses to pathogens and, as such, to play key roles in regulating intestinal immune homeostasis. The characterization of classical DCs (cDCs) in the intestinal lamina propria has been under intense investigation in recent years but the use of markers (including CD11c, CD11b, MHC class II), which are also expressed by intestinal MΦs, has led to some controversy regarding their definition. Here we review recent studies that help to distinguish cDCs subsets from monocyte-derived cells in the intestinal mucosa. We address the phenotype and ontogeny of these cDC subsets and highlight recent findings indicating that these subsets play distinct roles in the regulation of mucosal immune responses in vivo

Characterization of Thymic Nurse-Cell Lymphocytes, Using an Improved Procedure for Nurse-Cell Isolation

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4+8+3low thymocytes, about 12% of apparently mature CD4+8-3high and CD4-8+3high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells

Early passenger leukocyte migration and acute immune reactions in the rat recipient spleen during liver engraftment: With particular emphasis on donor major histocompatibility complex class II⁺ cells

After a short course of tacrolimus, Lewis rat liver allografts induce donor-specific nonreactivity in Brown Norway recipients that is immunosuppression-independent after 28 days. To clarify the role of donor major histocompatibility complex (MHC) class II+ cells, we investigated the migration to the recipient splenic T- and B-cell compartments of different subsets of Lewis MHC class II+ passenger leukocytes. The rise and decline of immune activation were monitored in the hepatic allograft and in the host spleen by analyses of BrdU+ (proliferating) leukocytes, TUNEL+ (apoptotic) cells, apoptosis-associated molecules, TH1/TH2 cytokine profiles, and histoimmunocytochemical examination of graft and splenic tissues. Serial flow cytometry studies during the 28-day period of drug-assisted "hepatic tolerogenesis" showed that migratory MHC class II+ cells accounted for less than half of the donor cells in the host spleen. The class II+ cells consisted mostly of B cells that homed to splenic B-cell follicles with only a sparse representation of dendritic cells that were exclusively found in the splenic periarteriolar lymphoid sheath. In parallel studies, transplantation of the less tolerogenic heart produced a diminutive version of the same events, but with far fewer donor cells in the host spleen, evidence of sustained immune activation, and the development of chronic rejection by 100 days. The data are consistent with the paradigm that migration of donor leukocytes is the prime determinant of variable tolerance induction induced by transplantation of the liver and other organs, but without regard for donor MHC class II+ expression

Donor and recipient leukocytes in organ allografts of recipients with variable donor-specific tolerance: With particular reference to chronic rejection

We have attributed organ engraftment to clonal exhaustion-deletion of host-versus-graft and graft-versus-host reactions that are reciprocally induced and governed by migratory donor and recipient leukocytes. The so-called donor passenger leukocytes that migrate from the allograft into the recipients have been thoroughly studied (chimerism), but not the donor leukocytes that remain in, or return to, the transplanted organ. Therefore, using flow cytometry we determined the percentage and lineages of donor leukocytes in cell suspensions prepared from Lewis (LEW) cardiac allografts to 100 days posttransplantation. The LEW hearts were transplanted to naive untreated Brown Norway (BN) recipients (group 2), to naive BN recipients treated with a 28-day or continuous course of tacrolimus (TAC) (groups 3 and 4), and to drug-free BN recipients pretolerized by earlier bone marrow cell (BMC) or orthotopic LEW liver transplantation (groups 5 and 6). The findings in the heart cell suspensions were correlated with the results from parallel histopathologic-immunocytochemical studies and other studies of the grafts and of host tissues. Although the LEW heart allografts were rejected in 9.6 days by the unmodified recipients of group 2, all beat for 100 days in the recipients of groups 3 through 6. Nevertheless, all of the long-surviving cardiac allografts (but not the isografts in group 1) were the targets of an immune reaction at 5 days, reflected by dramatic increases in the ratio of leukocytes to nonleukocyte nucleated cells from normal values of 1:5-1:6 to 1:1-5:1 and by manifold other evidence of a major inflammatory event. The acute changes returned to baseline by 100 days in the chronic rejection (CR) free hearts of groups 4 and 6, but not in the CR-afflicted hearts of short-course TAC group 3 or the less-severely damaged hearts of the BMC-prime group 5. The freedom from CR in groups 4 and 6 was associated with a large donor contribution to the intracardiac leukocyte population at 5 days (28.6% and 22% in the respective groups) and at 100 days (30.5% in group 4 and 8.4% in group 6) compared with 2% and 1.2% at 100 days in the CR-blighted allografts of the partially tolerant animals of groups 3 and 5. Whether large or small, the donor leukocyte fraction always included a subset of class II leukocytes that had histopathologic features of dendritic cells. These class II+ cells were of mixed myeloid (CD11-b/c+) and lymphoid lineages; their migration was markedly inhibited by TAC and accelerated by donor-specific priming and TAC discontinuance. Although a large donor leukocyte population and a normal leukocyte/nonleukocyte cell ratio were associated with freedom from CR, these findings and the lineage profile of the intracardiac leukocytes were not associated with tolerance in the animals of groups 3 and 4 under active TAC treatment. The findings in this study, singly and in their entirety, are compatible with our previously proposed leukocyte migration-localization paradigm of organ allograft acceptance and tolerance

Striking augmentation of hematopoietic cell chimerism in noncytoablated allogeneic bone marrow recipients by flt3 ligand and tacrolimus

The influence of granulocyte-macrophage colony-stimulating factor (GM- CSF) and the recently identified hematopoietic stem-progenitor cell mobilizing factor flt3 ligand (FL) on donor leukocyte microchimerism in noncytodepleted recipients of allogeneic bone marrow (BM) was compared. B10 mice (H2b) given 50 x 106 allogeneic (B10.BR [H2(k)]) BM cells also received either GM-CSF (4 μg/day s.c.), FL (10 μg/day i.p.), or no cytokine, with or without concomitant tacrolimus (formerly FK506; 2 mg/kg) from day 0. Chimerism was quantitated in the spleen 7 days after transplantation by both polymerase chain reaction (donor DNA [major histocompatibility complex class II; I-E(k)]) and immunohistochemical (donor [I-E(k+)] cell) analyses. Whereas GM-CSF alone significantly augmented (fivefold) the level of donor DNA in recipients' spleens, FL alone caused a significant (60%) reduction. Donor DNA was increased 10-fold by tacrolimus alone, whereas coadministration of GM-CSF and tacrolimus resulted in a greater than additive effect (28-fold increase). A much more striking effect was observed with FL + tacrolimus (>125-fold increase in donor DNA compared with BM alone). These findings were reflected in the relative numbers of donor major histocompatibility complex class II+ cells (many resembling dendritic cells) detected in spleens, although quantitative differences among the groups were less pronounced. Evaluation of cytotoxic T lymphocyte generation by BM recipients' spleen cells revealed that FL alone augmented antidonor immunity and that this was reversed by tacrolimus. Thus, although FL may potentiate antidonor reactivity in nonimmunosuppressed, allogeneic BM recipients, it exhibits potent chimerism-enhancing activity when coadministered with recipient immunosuppressive therapy. This property of FL may offer considerable potential for the augmentation of microchimerism, with therapeutic implications for organ allograft survival and tolerance induction

Mouse Plasmacytoid Cells: Long-lived Cells, Heterogeneous in Surface Phenotype and Function, that Differentiate Into CD8+ Dendritic Cells Only after Microbial Stimulus

The CD45RAhiCD11cint plasmacytoid predendritic cells (p-preDCs) of mouse lymphoid organs, when stimulated in culture with CpG or influenza virus, produce large amounts of type I interferons and transform without division into CD8+CD205− DCs. P-preDCs express CIRE, the murine equivalent of DC-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN). P-preDCs are divisible by CD4 expression into two subgroups differing in turnover rate and in response to Staphylococcus aureus. The kinetics of bromodeoxyuridine labeling and the results of transfer to normal recipient mice indicate that CD4− p-preDCs are the immediate precursors of CD4+ p-preDCs. Similar experiments indicate that p-preDCs are normally long lived and are not the precursors of the short-lived steady-state conventional DCs. However, in line with the culture studies on transfer to influenza virus-stimulated mice the p-preDCs transform into CD8+CD205− DCs, distinct from conventional CD8+CD205+ DCs. Hence as well as activating preexistant DCs, microbial infection induces a wave of production of a new DC subtype. The functional implications of this shift in the DC network remain to be determined