An De Creus

“Alternatively Activated” Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2d) mouse bone marrow progenitors in IL-10 and TGF-β, then stimulated the cells with LPS. These “alternatively activated” (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

Low TLR4 Expression by Liver Dendritic Cells Correlates with Reduced Capacity to Activate Allogeneic T Cells in Response to Endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8α− (myeloid) and CD11c+CD8α+ (“lymphoid-related”) DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-γ) and Th2 (IL-4) responses. When higher LPS concentrations (≥100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.

Plasmacytoid Dendritic Cell Precursors Induce Allogeneic T‐Cell Hyporesponsiveness and Prolong Heart Graft Survival

Dendritic cell (DC) precursors were propagated from C57BL/10 (B10; H2b) mouse bone marrow in fms‐like tyrosine kinase 3 ligand. Cosignaling molecule (B7‐1/B7‐2 and B7‐H1) expression and stimulatory capacity of precursor (pre)‐plasmacytoid (p)DC (CD11c+B220+CD11b−CD19−) and classic myeloid DC (MDC) for allogeneic (C3H; H2k) T cells were compared. Unstimulated pre‐pDC exhibited very low levels of surface MHC class II and classic costimulatory molecules (B7‐1/B7‐2), whereas a minor population expressed B7‐H1 at levels higher than on MDC. The pre‐pDC were ineffective T‐cell stimulators and induced nonspecific hyporesponsiveness to rechallenge with donor alloantigens in vitro and in vivo. Following stimulation with CpG‐oligonucleotide (CpG‐ODN), B7 molecule expression was upregulated on pre‐pDC, however the ratio between coinhibitory (B7‐H1) and costimulatory (B7‐1/B7‐2) signals was much higher (five‐ to six‐fold) on pre‐pDC than MDC. Blockade of B7‐H1 expression on pDC increased their T‐cell allostimulatory capacity significantly. A single preoperative infusion of C3H hosts with pre‐pDC prolonged B10 heart graft survival significantly but nonspecifically compared with untreated mice (median survival times 22 vs. 9 days, respectively). Thus, pre‐pDC of donor origin have potential to regulate T‐cell responses to alloantigens and can prolong organ graft survival.

Expression of Ly49E and CD94/NKG2 on fetal and adult NK cells.

Murine NK cells express inhibitory receptors belonging to the Ly49 and CD94/NKG2 family. Ly49E and CD94 are the only NK cell receptor transcripts detectable in fetal NK cells. Still unproved is the surface expression of Ly49E on NK cells. Here we generated two novel mAbs, a mAb recognizing Ly49E with cross-reactivity to Ly49C, and a mAb against NKG2A/C/E. Ly49E was immunoprecipitated as a disulfide-linked homodimer with 46-kDa subunits. Removal of N-linked carbohydrates revealed a 31-kDa protein backbone. NKG2A was immunoprecipitated as a 38-kDa protein. Although the frequency of fetal NK cells expressing Ly49E was higher than 25%, it decreased drastically from 2 wk after birth. Phenotypic analysis showed that ∼90% of fetal NK cells and ∼50% of adult NK cells express high levels of CD94/NKG2. The remaining 50% of adult NK cells expressed low surface levels of CD94/NKG2. Expression of Ly49E and CD94/NKG2 was not restricted to NK cells, but was also observed on NK T and memory T cells. Functional analysis showed that sorted Ly49E+ and CD94/NKG2+ fetal NK cells could discriminate between MHC class I-positive and MHC class I-negative tumor cells. We also demonstrated that Ly49E becomes phosphorylated following pervanadate stimulation of fetal NK cells. The expression levels of Ly49E and CD94/NKG2 were similar in wild-type compared with β2-microglobulin−/− mice. In conclusion, generation of mAbs against Ly49E and NKG2 extended the phenotypic and functional characterization of NK cells.

Developmental and functional defects of thymic and epidermal V gamma 3 cells in IL-15-deficient and IFN regulatory factor-1-deficient mice.

In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine Vγ3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature Vγ3 cells were found in the fetal thymus of IL-15−/− mice, while IRF-1−/− mice displayed normal frequencies. Vγ3+ dendritic epidermal T cells (DETCs) were absent in IL-15−/− mice but present in IRF-1−/− mice. DETCs from IRF-1−/− mice displayed morphologically a less mature phenotype and showed different emergence kinetics during ontogeny. This corresponded with lower IL-15 mRNA levels in the skin epidermis. Comparable levels of IL-7 were found in the skin of WT and IL-15−/− mice. Adoptive transfer experiments of WT fetal thymocytes into IL-15−/− mice did not result in the development of Vγ3+ DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15−/− Vγ3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1−/− Vγ3 cells remained defective after stimulation with IL-15 in combination with IL-12. These data suggest that IL-15 is redundant for the development and/or survival of mature Vγ3 cells in the fetal thymus, whereas it is essential for the localization of Vγ3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of Vγ3 cells is suggested.

Expression of Inhibitory Receptors Ly49E and CD94/NKG2 on Fetal Thymic and Adult Epidermal TCR Vγ3 Lymphocytes

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR αβ lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vγ3+ cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rβ (CD122), and absence of IL-2Rα (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vγ3 T cells, the progeny of fetal thymic Vγ3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E+ or CD94/NKG2+ Vγ3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vγ3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1b-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vγ3 thymocytes were prevented from killing FcγR+ P815 target cells. These effects were most pronounced in the CD94/NKG2high subpopulation as compared with the CD94/NKG2low subpopulation of Vγ3 cells. Our data demonstrate that Vγ3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.

Ly49E expression points toward overlapping, but distinct, natural killer (NK) cell differentiation kinetics and potential of fetal versus adult lymphoid progenitors

Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E‐expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E‐positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up‐regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.

Heart, but not skin, allografts from donors lacking Flt3 ligand exhibit markedly prolonged survival time.

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L−/−) show severe reductions in both myeloid (CD11c+CD8α−) and lymphoid-related DC (CD11c+CD8α+) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c+ interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c+ cells that migrated from Flt3L−/− skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c+ cells. We examined the survival of Flt3L−/− heart or tail skin grafts (H2b) in allogeneic wt (BALB/c; H2d) recipients. The outcome of transplantation of BALB/c organs into Flt3L−/− recipients was also determined. Flt3L−/− mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L−/− heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L−/− hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L−/− hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L−/− recipients reflects presumably an intact role of the direct pathway of allorecognition.

Langerhans Cells That Have Matured In Vivo in the Absence of T Cells Are Fully Capable of Inducing a Helper CD4 as Well as a Cytotoxic CD8 Response

Langerhans cells (LCs) are immature dendritic cells (DCs) present in the skin epithelium. Upon Ag exposure, they migrate to the draining lymph nodes where they mature into potent stimulators of naive T cells. The aim of this study was to investigate the influence of T cells on LC migration and maturation. Therefore, the in vivo migration and maturation of LCs after sensitization with the hapten FITC was compared between C57BL/6 or BALB/c mice used as positive controls, and recombination activating gene (RAG) 1 knockout (−/−) mice or SCID mice used as T cell-deficient mice. Phenotypically, there was no difference between migrated LCs from RAG1−/− or SCID mice vs normal C57BL/6 or BALB/c mice: both populations of FITC+ cells had a dendritic morphology and a mature phenotype as they expressed high levels of MHC class II molecules and costimulatory molecules CD80, CD86, and CD54. Sorted migrated LCs of RAG1−/− or SCID mice were efficient stimulators of allogeneic T cells and Ag-specific CD4+ T cells. The same results were found if migrated LCs were fixed instead of irradiated, excluding the possibility that LCs derived from RAG1−/− or SCID mice would mature in the presence of T cells during the stimulation tests. Importantly, fixed migrated LCs of RAG1−/− mice were also efficient stimulators of cytotoxic CD8+ T cells. These data suggest that T cells are not required for full maturation of LCs.