The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.     

TNF skews monocyte differentiation from macrophages to dendritic cells

Monocytes represent a large pool of circulating precursors of APCs, both macrophages and dendritic cells (DCs). It is thus important to identify the mechanisms by which microenvironment regulates monocyte differentiation. We have previously shown that, upon contact with resting stromal cells such as fibroblasts, monocytes differentiate into macrophages in an IL-6/M-CSF-dependent fashion. Yet, in the inflamed tissue, monocytes need to yield DCs for the adaptive immunity to be induced. Inasmuch as TNF and IL-1 are present at the site of inflammation, we tested their capacity to modulate monocyte differentiation into either macrophages or DCs. TNF, but not IL-1, induce monocytes to become DCs despite the presence of fibroblasts. TNF-induced DCs contain Langerin-positive cells and are able to induce allogenic T cell proliferation. Then, TNF was found to decrease the expression and internalization of the M-CSF receptor, thus overriding the IL-6/M-CSF pathway. Thus, TNF facilitates the induction of adaptive immunity by promoting DC differentiation not only from CD34+ progenitors but also from CD14+ blood precursors.     

HLA-restricted lysis of herpes simplex virus-infected monocytes and macrophages mediated by CD4+ and CD8+ T lymphocytes

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.     

Dendritic cells generated either from CD34+ progenitor cells or from monocytes differ in their ability to activate antigen-specific CD8+ T cells.

Dendritic cells (DC) can be generated in vitro from monocytes (M-DC) or from CD34+ hemopoietic progenitor cells (CD34-DC) but their precursors are not equivalent cells, prompting a comparison of the functional capacities of these APC. Both types of DCs established from the same individuals using the same cytokines displayed a comparable phenotype of mature DC (CD1a+, CD83+, CD86+, CD4+, HLA-DR++, CD14-, CD15- ) and were equally potent stimulators of allogeneic T cell proliferation, being both more powerful than immature M-DCs. An autologous panel of APCs produced in HLA-A2+ individuals, including CD34-DC, M-DC, monocytes, and EBV-lymphoid cell line was comparatively evaluated for presentation of the Erb-B2 peptide E75 to a CTL line. After short exposures (5 h) to E75-loaded APCs, similar levels of intracellular IFN-gamma were induced in Ag-specific CD8+ T cells regardless of APC type. In sustained cultures (4-14 days), more Ag-specific T cells were obtained when peptide was presented on CD34-DC (p < 0.05) rather than on M-DC, EBV-lymphoid cell lines, or monocytes, and these effects were dose-dependent. Activated T cells expressed 4-1BB, and the presence of 4-1BB-Ig fusion protein partially blocked Ag-specific CD8+ cell activation after CD34-DC or M-DC presentation. Our results show that 34-DC have a preferential capacity to activate CD8+ T cells and that this property is not strictly correlated to their ability to induce allogeneic T cell proliferation but due to mechanisms that remain to be defined.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Differentiation to the CCR2+ inflammatory phenotype in vivo is a constitutive, time-limited property of blood monocytes and is independent of local inflammatory mediators

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2- monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80- monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.     

Differentiation of monocytic cell clones into CD8 alpha+ dendritic cells (DC) suggests that monocytes can be direct precursors for both CD8 alpha+ and CD8 alpha- DC in the mouse

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8alpha(+) DC had been designated as "lymphoid" DC, and CD8alpha(-) DC as "myeloid" DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8alpha(+) DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8alpha(+) DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8alpha(+) DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8alpha(+) DC lymphoma but with a monocytic phenotype (CD11c(low/-)D11b(high)CD8alpha(-)I-A(low)), can redifferentiate into CD8alpha(+) DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c(+)CD8alpha(+) DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c(+)CD8alpha(+) DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8alpha(+) DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.     

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

Efficient induction of peptide-specific cytotoxic T lymphocytes by LPS-activated spleen cells

Lipopolysaccharides of gram-negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages. We have investigated the use of LPS-activated spleen cells as antigen-presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides. Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS-activated spleen cells. Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells. The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B-cell blasts were largely responsible. This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T-cell repertoire contains reactive T cells.     

The Characteristics of Th1/Th2 Cytokine Receptors on Monocytes in Untreated Patients of Long Term Nonprogressor or Chronic HIV Infection

Monocytes/macrophages play crucial roles in immunity to microorganisms and are one of the important targets for human immunodeficiency virus (HIV) infection. The phenotypes and function of monocytes in HIV-infected patients were poorly determined. We herein detected the expression of Th1/Th2 cytokine receptors on monocyte subsets in the untreated HIV-infected patients of either long term nonprogressor (LTNP) or chronic infection (CHI). CD14+CD16- monocytes were significantly increased and CD14+CD16+ monocytes were reduced in patients of LTNP or CHI compared with healthy control. IL-6R expression on CD14+CD16- monocytes were decreased in patients of LTNP or CHI, whereas IL-4R and IL-10R expression on both CD14+CD16- and CD14+CD16+ monocyte subsets were increased in patients with LTNP or CHI, as determined by flow cytometry and real time PCR assays. The decreased IL-6R expression and enhanced IL-4R and IL-10R expression were also observed on CD4+ T cells of these patients, indicating that these changes in monocytes are not cell-specific. CD14+CD16- monocytes of HIV-infected patients produced less TNF-α and IL-1β but identical levels of IL-6, and IL-12 as the control after IFN-γ/LPS stimulation. However, in the presence of IL-4 or IL10, CD14+CD16- monocytes of HIV-infected patients produced more TNF-α, IL-6, IL-12 or Il-1β after IFN-γ/LPS stimulation than the healthy control, supporting the impaired IL-4R and IL-10R signal pathways in patients with LTNP and CHI. Therefore, our present study offered the basic information for the Th1/Th2 cytokine receptor expression and function on monocyte subsets in untreated HIV-infected individuals.     

Human circulating monocytes can express receptor activator of nuclear factor-kappaB ligand and differentiate into functional osteoclasts without exogenous stimulation

Osteoclast formation from mononuclear precursors is believed to require accessory cells expressing receptor activator of nuclear factor-kappaB ligand (RANKL). We recently identified a human cell population originated from circulating CD14(+) monocytes, called monocyte-derived multipotential cells (MOMCs), which can differentiate into several distinct mesenchymal cells, neuron and endothelial cells. This study was undertaken to examine whether MOMCs can differentiate into functional osteoclasts. MOMCs prepared from peripheral blood of healthy volunteers cultured on fibronectin for 7 days at high density (8 x 10(5) cells cm(-2)), but not at regular density (2 x 10(4) cells cm(-2)), resulted in the appearance of tartrate-resistant acid phosphatase-positive giant multi-nucleated cells forming actin ring without exogenous osteoclastogenic factors. A subset of these cells showed bone resorption capacity on dentine slices and expression of genes for cathepsin K and calcitonin receptor, characteristic of functional osteoclasts. Such osteoclast differentiation was not observed in high-density culture of circulating monocytes, macrophages or dendritic cells, or the high-density culture of MOMCs on type I collagen. Among cells of the monocyte lineage, untreated MOMCs exclusively showed gene and protein expression of RANKL. When osteoprotegerin/IgG1 Fc chimera was added to high-density MOMC cultures, osteoclast formation was completely inhibited by neutralizing the endogenous RANKL. These results indicate that human MOMCs derived from circulating monocytes can express RANKL and differentiate into functional osteoclasts without RANKL-expressing accessory cells.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

Recruitment of dendritic cells and macrophages during T cell-mediated synovial inflammation

Adoptive transfer of adjuvant-induced arthritis was used in this study to examine local macrophages and dendritic cells (DCs) during T cell-mediated synovial inflammation. We studied the influx of CD11b+CD11c+ putative myeloid DCs and other non-lymphoid CD45+ cells into synovium-rich tissues (SRTs) of the affected hind paws in response to a pulse of autoreactive thoracic duct cells. Cells were prepared from the SRTs using a collagenase perfusion-digestion technique, thus allowing enumeration and phenotypic analysis by flow cytometry. Numbers of CD45+ cells increased during the first 6 days, with increases in CD45+MHC (major histocompatibility complex) II+ monocyte-like cells from as early as day 3 after transfer. In contrast, typical MHC II(-) monocytes, mainly of the CD4(-) subset, did not increase until 12 to 14 days after cell transfer, coinciding with the main influx of polymorphonuclear cells. By day 14, CD45+MHC IIhi cells constituted approximately half of all CD45+ cells in SRT. Most of the MHC IIhi cells expressed CD11c and CD11b and represented putative myeloid DCs, whereas only approximately 20% were CD163+ macrophages. Less than 5% of the MHC IIhi cells in inflamed SRT were CD11b(-), setting a maximum for any influx of plasmacytoid DCs. Of the putative myeloid DCs, a third expressed CD4 and both the CD4+ and the CD4(-) subsets expressed the co-stimulatory molecule CD172a. Early accumulation of MHC IIhiCD11c+ monocyte-like cells during the early phase of T cell-mediated inflammation, relative to typical MHC II(-) blood monocytes, suggests that recruited monocytes differentiate rapidly toward the DC lineage at this stage in the disease process. However, it is possible also that the MHC IIhiCD11c+ cells originate from a specific subset of DC-like circulating mononuclear cells.     

CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8 T cells

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-alpha and IFN-beta) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7(-)/CD45RA(-) CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-gamma production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.     

Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.     

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.