Macrophage-restricted interleukin-10 receptor deficiency, but not IL-10 deficiency, causes severe spontaneous colitis.

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1(hi) macrophages as a decisive factor that determines gut health or inflammation.

Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches.

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.

Lack of conventional dendritic cells is compatible with normal development and T cell homeostasis, but causes myeloid proliferative syndrome.

Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c(hi) dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.

Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8(+) T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Organ-dependent in vivo priming of naive CD4+, but not CD8+, T cells by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4(+) T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4(+) T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8(+) T cell priming. Our results establish PDCs as a bona fide DC subset that initiates unique CD4(+) Th cell-dominated primary immune responses.

Coupled pre-mRNA and mRNA dynamics unveil operational strategies underlying transcriptional responses to stimuli.

Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude.

CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival.

CX(3)CR1 is a chemokine receptor with a single ligand, the membrane-tethered chemokine CX(3)CL1 (fractalkine). All blood monocytes express CX(3)CR1, but its levels differ between the main 2 subsets, with human CD16(+) and murine Gr1(low) monocytes being CX(3)CR1(hi). Here, we report that absence of either CX(3)CR1 or CX(3)CL1 results in a significant reduction of Gr1(low) blood monocyte levels under both steady-state and inflammatory conditions. Introduction of a Bcl2 transgene restored the wild-type phenotype, suggesting that the CX(3)C axis provides an essential survival signal. Supporting this notion, we show that CX(3)CL1 specifically rescues cultured human monocytes from induced cell death. Human CX(3)CR1 gene polymorphisms are risk factors for atherosclerosis and mice deficient for the CX(3)C receptor or ligand are relatively protected from atherosclerosis development. However, the mechanistic role of CX(3)CR1 in atherogenesis remains unclear. Here, we show that enforced survival of monocytes and plaque-resident phagocytes, including foam cells, restored atherogenesis in CX(3)CR1-deficent mice. The fact that CX(3)CL1-CX(3)CR1 interactions confer an essential survival signal, whose absence leads to increased death of monocytes and/or foam cells, might provide a mechanistic explanation for the role of the CX(3)C chemokine family in atherogenesis.

Large-scale preparation of human anti-third-party veto cytotoxic T lymphocytes depleted of graft-versus-host reactivity: a new source for graft facilitating cells in bone marrow transplantation.

Induction of donor type chimerism in mildly prepared hosts without graft-versus-host disease (GvHD) is a most desirable goal in bone morrow transplantation. We have recently demonstrated in a mouse model that donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. We extend this approach to human cells, by preparing CTLs in two major steps: primary culture in the absence of interleukin 2, leading to death by neglect of antihost clones, and addition of interleukin 2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones. CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard (51)Cr release assay. We conclude that human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells.

CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells.

The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8+ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8+ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Ralpha). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8+ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11chigh dendritic cells. Our results suggest a dual role of CD11chigh dendritic cells as unique APC and cytokine-presenting cells.

Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-alpha.

Previous studies suggest that cells within the CD34(+) hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti-IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti-tumor necrosis factor-alpha (anti-TNF-alpha) and not by anti-transforming growth factor-beta (anti-TGF-beta), suggesting a potential role for TNF-alpha in the regulatory activity of CD34+ cells.

Eradication of B-CLL by autologous and allogeneic host nonreactive anti-third-party CTLs.

Establishment of cell lines capable of killing leukemia cells, in the absence of alloreactivity against normal host cells, represents a most desirable goal in bone marrow transplantation (BMT) and cancer immunotherapy. By using a human --> mouse chimeric model, we demonstrate that allogeneic anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity are endowed with a potent anti-B-cell chronic lymphocytic leukemia (B-CLL) reactivity. Likewise, CTL preparations generated from autologous T cells of the same patients with B-CLL exhibited comparable leukemia eradication, suggesting that the reactivity of allogeneic anti-third-party CTLs is not mediated by residual antihost clones. This specificity was also exhibited in vitro, and annexin staining revealed that B-CLL killing is mediated by apoptosis. While the CTLs killing of third-party cells could be blocked by anti-CD3 antibody, the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a T-cell receptor (TCR)-independent cytotoxicity. The role of cell contact leading to apoptosis of B-CLL cells is shown in transwell plates and by anti-lymphocyte function-associated antigen-1 (LFA-1)-blocking antibody. Up-regulation of CD54 and the subsequent apoptosis of B-CLL cells depend on the initial LFA-1/ICAM-1 (intercellular adhesion molecule 1) interaction. Taken together, these results suggest that allogeneic or autologous host nonreactive anti-third-party CTLs may represent a new therapeutic approach for patients with B-CLL.

Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34(+) cells.

Stem cell-dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34(+) population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34(+) cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34(+)CD33(-) cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34(+)CD33(+) and CD34(-)CD33(+). These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34(+) cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in "minitransplants," without any risk of graft-versus-host disease.