Depletion Of Antigen-presenting Cells Research Articles

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Impact of Graft-Resident Leucocytes on Treg Mediated Skin Graft Survival.

The importance and exact role of graft-resident leucocytes (also referred to as passenger leucocytes) in transplantation is controversial as these cells have been reported to either initiate or retard graft rejection. T cell activation to allografts is mediated via recognition of intact or processed donor MHC molecules on antigen-presenting cells (APC) as well as through interaction with donor-derived extracellular vesicles. Reduction of graft-resident leucocytes before transplantation is a well-known approach for prolonging organ survival without interfering with the recipient’s immune system. As previously shown by our group, injecting mice with IL-2/anti-IL-2 complexes (IL-2cplx) to augment expansion of CD4 T regulatory cells (Tregs) induces tolerance towards islet allografts, and also to skin allografts when IL-2cplx treatment is supplemented with rapamycin and a short-term treatment of anti-IL-6. In this study, we investigated the mechanisms by which graft-resident leucocytes impact graft survival by studying the combined effects of IL-2cplx-mediated Treg expansion and passenger leucocyte depletion. For the latter, effective depletion of APC and T cells within the graft was induced by prior total body irradiation (TBI) of the graft donor. Surprisingly, substantial depletion of donor-derived leucocytes by TBI did not prolong graft survival in naïve mice, although it did result in augmented recipient leucocyte graft infiltration, presumably through irradiation-induced nonspecific inflammation. Notably, treatment with the IL-2cplx protocol prevented early inflammation of irradiated grafts, which correlated with an influx of Tregs into the grafts. This finding suggested there might be a synergistic effect of Treg expansion and graft-resident leucocyte depletion. In support of this idea, significant prolongation of skin graft survival was achieved if we combined graft-resident leucocyte depletion with the IL-2cplx protocol; this finding correlated along with a progressive shift in the composition of T cells subsets in the grafts towards a more tolerogenic environment. Donor-specific humoral responses remained unchanged, indicating minor importance of graft-resident leucocytes in anti-donor antibody development. These results demonstrate the importance of donor-derived leucocytes as well as Tregs in allograft survival, which might give rise to new clinical approaches.

Antitumor Efficacy of Radiation plus Immunotherapy Depends upon Dendritic Cell Activation of Effector CD8+ T Cells.

Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I-restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell-dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621-30. ©2016 AACR.

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development.

Autoreactive T cell trafficking into pancreatic islets during type 1 diabetes is more permissive as infiltration increases and requires CD11c+ APCs. (CAM4P.153)

Abstract Type 1 diabetes (T1D) is a largely T cell mediated autoimmune disease that destroys the β-cells of the pancreatic islets. While initiation of disease requires T cell activation in the pancreatic lymph node, T cells must enter the islets to destroy β-cells. To investigate mechanisms required for T cell entry into the islets we established an intra-vital 2-photon pancreas imaging technique in the non-obese diabetic mouse. We determined that extravasation into the pancreatic islets is an extended process compared to lymph node entry, averaging 47 minutes from T cell arrest to completion of extravasation in islets with advanced T cell infiltration. Entry was more restricted at earlier stages of infiltration, with a duration of extravasation longer than our observation period of 2 hours. T cell entry into the islets was dependent on CD11c+ antigen presenting cells (APCs) in the islets. T cells transferred prior to CD11c+ APC depletion were able to enter the islets; while CD11c+ APC depletion prevented further entry. However, antigen was not required for entry of either activated or naïve T cells, suggesting a role for CD11c+ APCs other than antigen presentation. These studies demonstrate that T cell entry requires the presence of CD11c+ APCs within the islets, and as islet infiltration increases T cell extravasation becomes less restricted. Overall, CD11c+ APCs may be a therapeutic target for treatment of T1D by preventing T cell entry into remaining or transplanted islets.

Two Photon Intravital Microscopy Reveals CD4+ T Cells Making Cognate Interactions With Tissue Dendritic Cells In Skin Graft-Versus-Host-Disease

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs (SLO), we have consistently observed MHCII+ donor-derived tissue APCs (t-APCs), including tissue dendritic cells (t-DCs) in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells. We hypothesize that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease locally. We could not use knockout or APC depletion approaches to study T cell: t-APCs interactions as they impact APCs systemically and might therefore affect T cell stimulation in SLO. We therefore utilized two-photon intravital microscopy to analyze interactions between fluorescent donor CD4+ T cells and t-DCs in skin. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) CD11c-YFP transgenic (Tg) Bone Marrow (BM) with B6 RFP Tg CD4 cells and nonfluorescent B6 CD8 cells. We imaged ear skin in GVHD mice 4 weeks later. In general CD4 cells co-localized with DCs. We observed CD4+ T cells that were highly motile and only in transient contact with DCs and others that made stable contact with DCs. To determine how much TCR: MHCII interactions drive sustained CD4+ T cell: DC interactions and arrest CD4+ T cell motility, mice were imaged and then injected with an MHCII blocking antibody (Ab; Y3P) with continued imaging of the same regions. After injection, T cell mean speed significantly increased and the proportion of T cells in stable contact with DCs decreased, indicating that transient disruption of TCR: MHCII is sufficient to restore motility to some T cells. In a second approach to assess the specificity of CD4+ T cell: t-DC interactions we transplanted 129 mice with B6 RFP+ CD4 cells, nonfluorescent CD8 cells and a mix of CD11c-YFP MHCII-/- and RFP wt BM or a mix of CD11c-YFP wt and RFP Tg MHCII-/- BM. We are currently comparing the motility of CD4 cells that make contact with MHCII+ as compared to MHCII- DCs, with the prediction that contact times will be shorter with the latter. Our data suggest that CD4+ T cells make cognate interactions with t-DCs in skin and we hypothesize that these interactions promote GVHD locally. Because the graft-versus-leukemia effect occurs primarily in bone marrow and secondary lymphoid tissues, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. Disclosures:No relevant conflicts of interest to declare.

Development of a Novel Fusion Toxin Reagent for Targeting Antigen Presenting Cells In Vivo: DT390-CTLA-4

Targeted cell therapies are possible through the generation of chimeric recombinant proteins that combine a toxin, such as Diptheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the chimeric protein to the cell of interest, the DT is internalized which results in protein synthesis inhibition and subsequent cell death. The Cytotoxic T lymphocyte antigen-4 (CTLA-4), also known as CD152, is expressed on T helper cells and functions to down-regulate T cells via binding to CD80/CD86 (B7 complex) on antigen presenting cells (APC). Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80/CD86, has potential for induction of peripheral tolerance in autoimmune disease and organ transplantation. Currently, CTLA-4-Ig has proven to be an effective reagent for blocking these interactions in vivo. In our lab, we have a unique large animal model, the MGH MHC-defined miniature swine, for studying immune regulation and transplantation tolerance. We hypothesized that linking DT to CTLA-4 may provide a novel approach for in vivo depletion of APCs. We have expressed and purified recombinant soluble porcine CTLA-4 both with and without N-glycosylation in Yeast Pichia Pastoris for in vivo use in our large animal model. The glycosylated and non-N-glycosylated versions of this recombinant protein competitively inhibit the binding of an anti-CD80 antibody to a CD80 expressing porcine B cell lymphoma cell line (13271) with equal affinity (KD = 13nM). When linked to a truncated diphtheria toxin the non-N-glycosylated version of this recombinant protein proved to efficiently inhibit protein synthesis in target cells after 18 hours and significant cell death was observed after 52 hours in vitro. We are currently analyzing the effects of this DT-linked non-N-glycosylated version of recombinant CTLA-4 in vivo.

Conjunctival antigen presenting cells: Sentinels for ocular surface inflammation

AbstractPurpose To evaluate the role of conjunctival antigen presenting cells (APC) in experimental dry eye disease (EDE) and in the immune rejection of corneal allografts.Methods Ocular surface APC were depleted by subconjunctival injection of liposomes containing clodronate. APC depletion was confirmed by immunohistochemistry using anti‐CD11c (dendritic cells) and anti‐Iba1 (macrophages) antibodies. EDE was induced by exposing mice to desiccating stress (DS) for 5 days and pathogenic CD4+ T cells were to T cell‐deficient nude mice. Experiments were also performed in a murine model of keratoplasty.Results Injection of clodronate liposomes produced 65% and 86% reductions in conjunctival macrophages and DC respectively. Depletion of APC prior to DS inhibited the generation of autoreactive CD4+ T cells and prevented EDE. APC deletion in nude mice prior to their receiving EDE‐producing CD4+ T cells significantly mitigated EDE. APC depletion prior to keratoplasty completely prevented corneal allograft rejection. However, depleting APC in nude mice prior to transferring CD4+ T cells did not affect corneal allograft rejection indicating that primed CD4+ T cells do not require secondary activation by APC to mediate graft rejection.Conclusion Ocular surface APC play an important role in the generation of autoreactive pathogenic CD4+ T cells that mediate EDE and for secondary activation of these cells at sites of ocular surface inflammation. Ocular surface APC are also crucial for the induction of corneal allograft rejection, but secondary activation of previously primed allospecific CD4+ T cells by resident ocular surface APC is not required. Targeting ocular surface APC may be a facile therapeutic modality for managing ocular surface inflammation.

In this issue

In this issue

Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice

There is ongoing debate regarding the initiation of psoriatic plaque as primarily arising from an anomaly in epidermal keratinocytes (KCs) or from abnormalities in immunocytes that secondarily activate otherwise normal KCs. In mice engineered to overexpress the angiopoietin receptor Tie2 in KCs, skin spontaneously develops the characteristic clinical, histological and immune cell phenotypes of psoriasis which can be reversed with either transgene repression or ciclosporin administration, suggesting key roles for both KCs and T cells in mediating the skin disease in this murine model. To determine if antigen-presenting cells (APCs) and macrophages alone are sufficient to sustain psoriasiform inflammation in the KC-Tie2 murine model of psoriasis. Clodronate liposomes were intradermally injected into involved dorsal skin of KC-Tie2 or control animals once a week for 6weeks and acanthosis, angiogenesis, immune cell infiltration and cytokine production were quantitated using immunohistochemistry and interactive image analyses, enzyme-linked immunosorbent assay (ELISAs) and quantitative real-time polymerase chain reaction (qRT-PCR). Clodronate liposome injection eliminated CD11c+, F4/80+ and CD11b+ cells in the skin and returned CD8+ T-cell numbers to control mouse levels. APC depletion in KC-Tie2 mouse skin resulted in resolution of the acanthotic skin phenotype, decreased dermal angiogenesis, and a return to control mouse levels for interleukin (IL)-1α, IL-6, IL-23 and tumour necrosis factor (TNF)-α expression and modest reductions in interferon-γ and IL-17. These findings suggest a critical role for APCs and myeloid cell-derived IL-23 and TNF-α and underscore the importance of Th1 and Th17 T cells in maintaining the psoriasiform skin phenotype in the KC-Tie2 mouse model.

Depletion of Blood Antigen-Presenting Cells by IMMU-114: Therapeutic Potential Against Graft-Versus-Host Disease

AbstractAbstract 2544 Background:Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Since antigen-presenting cells (APCs) play a critical role in initiating GVHD, selective depletion of host APCs before or during preparative conditioning may yield an improved approach for prevention of GVHD compared to the prevailing therapies, which often compromise the pre-existing T-cell immunity against opportunistic pathogens. IMMU-114 (Immunomedics, Inc.) is an anti-human HLA-DR humanized IgG4 monoclonal antibody (mAb) with a point mutation (Ser241Pro) in the hinge region of the γ4 sequence to avoid the formation of half molecules, and has direct cytotoxicity against a variety of HLA-DR-expressing hematological malignancies. In this study, we investigated the effects of IMMU-114 on the subsets of human blood APCs, and evaluated its effects on the proliferation of alloreactive T cells and preexisting anti-viral memory T-cell immunity in allogeneic mixed leukocyte cultures. Methods:We assessed the effects of IMMU-114 on T cells and all subsets of APCs in human peripheral blood mononuclear cells, including dendritic cells (DCs), B cells, and monocytes. In order to evaluate the therapeutic potential of IMMU-114 against GVHD, we tested the effect of IMMU-114 on the proliferation of alloreactive T cells in allogeneic mixed leukocyte cultures; In order to evaluate the direct effect of IMMU-114 on T cells, we tested the effect of IMMU-114 on the proliferation of already proliferated T cells, and to evaluate the impact of IMMU-114 on preexisting T-cell immunity, we measured the effect of IMMU-114 on HLA-A2-restricted, CMV-specific CD8+ memory T cells by pentamer assay. Results:IMMU-114 depleted almost all subsets of APCs, including dendritic cells (DCs), B cells, and monocytes from human peripheral blood mononuclear cells without affecting the survival of T cells. Specifically, the myeloid DCs type 1 (mDC1) were depleted by 59.2% (P = .0022), myeloid DCs type 2 (mDC2) by 84.8% (P<.0001), plasmacytoid DCs (pDCs) by 52.2% (P = .1927), B cells by 86.2% (P<.0001), and monocytes by 74.7% (P = .01139). In addition, IMMU-114 suppressed the proliferation of alloreactive T cells in mixed leukocyte cultures, yet had no effect on the proliferation of already proliferated T cells. These results suggest that IMMU-114 has therapeutic potential against GVHD, which may be through the depletion of APCs rather than a direct effect on T cells. Importantly, the result demonstrated that CMV-specific, CD8+ memory T cells were preserved in IMMU-114-treated allogeneic mixed leukocyte cultures, suggesting that IMMU-114 may not affect the preexisting anti-viral T-cell immunity, which is essential for immunological control of opportunistic infections following stem cell transplantation. Conclusion:These results indicate that IMMU-114 may be a promising novel agent for prevention and treatment of GVHD through the depletion of APCs. It also supports the investigation of IMMU-114 as a novel component of conditioning regimens for preventing GVHD without alteration of preexisting anti-viral immunity, which would have a distinct advantage over currently available agents for GVHD prevention. (Supported in part by NIH grant PO1-CA103985.) Disclosures:Chang:Immunomedics, Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Recipient B Cells Are Not Required for Graft-Versus-Host Disease Induction

Recipient antigen presenting cells (APCs) are required for CD8-mediated graft-versus-host disease (GVHD), and have an important and nonredundant role in CD4-mediated GVHD in mouse major histocompatibility complex-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs-dendritic cells, macrophages, and B cells-are not well defined. If recipient B cells are important APCs they could be depleted with rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses, and consequently, B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient because of prior rituximab. We therefore studied the role of recipient B cells in major histocompatibility complex-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4- and CD8-dependent models, B cell-deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD, and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.

Tissue Damage and Lymphoma Derived Chemoattractive Signals Impact Regulatory T Cell Trafficking After Allogeneic Hematopoietic Cell Transplantation.

Abstract 3564Poster Board III-501Regulatory T cells (Treg) have been shown previously to reduce graft-versus-host disease (GvHD) but allow for graft-versus-leukemia (GvL) effects. It is still unclear why the adoptive Treg transfer does not paralyze every immune response to the same extent. In that context Treg recruitment and localization may account for differential immune regulation. Therefore, we studied the impact of lymphoma and inflammation derived chemoattractive signals on Treg recruitment. Luciferase transgenic Treg accumulated in B cell lymphoma (BCL) tissue after allogeneic hematopoietic cell transplantation (alloHCT) when no conventional T cells were given. Microarray based analysis of the BCL tissue revealed increased expression of the CXC chemokine ligands (CXCL) 9, 10, 12 and CCL22. In vivo antibody based neutralization or interference with receptor signaling identified CCL22 and CXCL12 as critical chemokines for Treg attraction towards BCL tissue after alloHCT. Conversely, the CXCL9/10-CXCR3 axis was not critical for Treg recruitment. Depletion of antigen presenting cells (APC) from BCL tissue abrogated CCL22/CXCL12 secretion. CD11c-DTR recipients displayed reduced Treg recruitment towards BCL, indicative for a major role of host APC in the process of Treg recruitment. Strong local inflammatory stimuli caused by subcutaneous complete Freund's adjuvant injection caused local Treg accumulation and reduced recruitment to the BCL. Infusion of conventional T cells that caused subacute GvHD related inflammation redirected Treg towards secondary lymphoid organs and abdominal GvHD target organs.In conclusion our study demonstrates the following novel findings: First Treg are recruited towards BCL infiltrated tissues after alloHCT and this process is dependent on CCL22 and CXCL12 production. Secondly, BCL infiltrating host type APC are the major source for CCL22 and CXCL12 and their targeted deletion impacts Treg recruitment towards BCL tissue. Thirdly, strong local or systemic inflammation can redirect Treg trafficking in the presence of BCL and affect the accumulation of this cell population at the tumor site which may explain why Treg preferentially regulate GvHD reactions post allografting. Disclosures:No relevant conflicts of interest to declare.

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen-presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome-encapsulating clodronate (LP-CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP-CL showed a significantly reduced type 1 helper T cell cytokine up-regulation than control-LP-treated recipients. In the LT model, LP-CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP-CL-treated-BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control-LP-treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP-CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control-LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance.

Persistent Recipient Antigen-Presenting Cells in Human Hematopoietic Stem Cell Transplantation: Identification of a New Dermal Subset That Outlives Epidermal Langerhans Cells.

Graft versus host disease (GVHD), a potentially lethal complication of hematopoietic stem cell transplantation (HSCT), is initiated by recipient antigen presenting cells (APC) priming donor T cells. The skin, a commonly affected organ, contains diverse APC including epidermal Langerhans cells (LC) and heterogeneous dermal dendritic cells (DC) whose phenotype, turnover and contribution to GVHD is not well described. The persistence of LC in mice and to some extent humans, is well known, yet the importance of LC relative to dermal DC in the lichenoid dermal infiltration of GVHD remains unproven. In this study we have:1.analyzed the depletion of dermal APC by conditioning, demonstrating a resistant DR+ CD14+ subset;2.identified a DR+ CD14+ fXIIIa+ dermal cell that remains recipient in origin for up to 1 year post HSCT and may therefore contribute to the sensitization of donor T cells and promotion of GVHD.From 2003 to 2007 we obtained 22 pairs of 2mm skin biopsies pre and post conditioning (high dose BuCy or CyTBI and reduced intensity Flu/Mel) and 85 biopsies at day 40, 100 and 365 post HSCT from patients with sex-mismatched donors. Pre and post conditioning samples were digested with dispase and collagenase followed by single step B–D Trucount analysis with CD45, HLA-DR, CD14 and CD1a antibodies. Chimerism was determined on cytospins of spontaneously migrated dermal APC and collagenase digested dermal cells using sequential four-colour confocal microscopy and X/Y fluorescence in situ hybridization. We find 3 populations of CD45+ DR+ dermal cells: CD14+ CD1a- fXIIIa-; CD14+ CD1a- fXIIIa+; CD1a+ CD14- fXIIIa-. Characterization of these cells in vitro shows that CD14+ cells are more adherent and phagocytic than CD1a+ cells and that the fXIIIa+ component is in addition, heavily granulated with ingested melanin. CD1a+ APC are more sensitive to depletion by conditioning (201 pre /97 post; mean cells per mm2; P 0.03) compared with CD14+ cells (210 pre /213 post; cells per mm2; P 0.86). Both are reduced in HSCT patients compared with controls (706 CD14+ and 468 CD1a+ cells per mm2; both P 0.01), in contrast to LC that are relatively well preserved in HSCT patients. CD1a+ cells are equally sensitive to depletion by both high dose and reduced intensity HSCT. Chimerism analysis at 40, 100 and 365 days post HSCT has shown regimen-dependent but nearly complete donor LC engraftment by 100 days. In dermal cells, two strikingly different patterns of engraftment are seen. CD1a+ and CD14+ fXIIIa- cells, obtained either by migration or digestion, engraft rapidly in all patients, ahead of LC, reaching medians of 99% and 100% donor, respectively, at day 40. In contrast, CD14+ fXIIIa+ cells, which are obtained only from digested dermis, are very slow to engraft in the absence of GHVD, reaching only 63% median donor after high dose and 10% median donor chimerism after reduced regimens, at 1 year. Acute GVHD promotes engraftment with 100% donor chimerism seen in nearly all patients at day 100. Preliminary data indicate that these cells are not actively synthesizing DNA, suggesting a different means of survival compared with persistent recipient LC. In conclusion, we have identified a new subset of human dermal APC with protracted survival after high dose and reduced intensity HSCT, sensitivity to GVHD and potential importance in promoting donor T cell reactivity to host antigens.

Characterization of Antigen-Presenting Cells in Fresh and Cultured Human Corneas Using Novel Dendritic Cell Markers

Adult healthy human corneas bear a distinctive number of antigen-presenting cells (APCs) important for the fate of a graft. The purpose of this study was to differentiate between Langerhans cells (LCs) and other dendritic cells (DCs) and between mature and immature APCs in fresh and cultured human corneas using specific markers. Immunofluorescence double staining was performed for Langerin/CD207, CD1a, DC-SIGN/CD209, DC-LAMP/CD208, CD45, CD11c, CD11b and HLA-DR. Langerin(+)/CD1a(+)/HLA-DR(+) LCs (approximately 100 cells/mm(2) in fresh corneas) were found in the limbal and peripheral regions of corneal epithelium and the anterior stroma up to 83 days of culture. All these cells coexpressed CD45 and CD11c. DC-SIGN(+)/CD45(+) DCs (approximately 150 cells/mm(2) in fresh corneas) were detected mainly peripherally and in the anterior stroma, even in long-term cultured corneas. Most of these cells were HLA-DR(-). Few mature DCs (DC-LAMP(+)/HLA-DR(+)) were found in fresh and cultured corneas. Macrophages (CD11c(-)/CD11b(+)) were seen in the peripheral, paracentral, and even central regions of the posterior stroma. This is the first demonstration that human corneas harbor populations of Langerin(+)/CD1a(+)/HLA-DR(+) LCs and DC-SIGN(+) DCs in a distribution pattern similar to that in the skin. Few APCs are in a mature state (DC-LAMP(+)). Given the reduced but not complete depletion of APCs during organ culture, these grafts still bear a potential risk for rejection.

The Fate of Human Langerhans Cells in Hematopoietic Stem Cell Transplantation.

The fate of Langerhans cells (LC) and other antigen-presenting cells (APC) in haematopoietic stem cell transplantation (HSCT), their depletion by conditioning regimens, reconstitution and chimerism are important factors in understanding graft versus host disease and the outcome of transplantation. Hitherto untested predictions in humans state that depletion of recipient LC may prevent acute graft versus host disease (GVHD), the acquisition of donor chimerism in LC may drive the evolution of clinical GVHD from acute to chronic and the persistence of recipient LC may explain late acute GVHD phenomena and donor lymphocyte infusion toxicity.Between April 2003 and June 2005 we obtained 184 skin biopsies from 76 patients at 4 UK centres. Using confocal microscopy of intact epidermal sheets to image up to 2000 LC at a time, we find that full intensity transplant (FIT) conditioning with TBI-based regimens or busulfan and cyclophosphamide depletes 55% of LC at day 0 (n 14; p 0.001). In contrast, fludarabine-based reduced intensity transplant (RIT) depletes only 9% of LC (n 23; p 0.061). A minimum LC density is reached at 14-21 days in both regimes with little difference at the nadir, suggesting that the effect on density at day 0 is primarily kinetic. Rapid recovery occurs by 40 days in the absence of acute cutaneous GVHD but is delayed beyond 100 days in the presence of GVHD (n 50; p 0.006)We assessed LC chimerism in sex-mismatched transplants by allowing single cells to migrate from small epidermal sheets in vitro and applying a two-step Giemsa/FISH assay. At 40 days median LC donor chimerism is 97% after FIT and 36.5% after RIT (n 11; p 0.004). At 100 days median donor chimerism is 100% after FIT and 97.5% after RIT (n 28; p 0.133.). The majority of transplants - 16/28 (57%) - achieve 100% LC donor chimerism at day 100 and all are at least 90%. Attainment of full donor chimerism at 100 days shows only a trend with FIT (p 0.133), and with complete myeloid chimerism in the blood (p 0.080) but is strongly associated with prior acute cutaneous GVHD (p 0.002). At one year post transplant it is possible to detect rare recipient cells in a minority of transplants (12/1746 interphase nuclei - less than 1%), confirming that LC may regenerate in the skin under quiescent conditions.In conclusion we find that: 1) recipient LC survive conditioning, especially after RIT, suggesting that targeted APC depletion might have potential as a novel means of preventing acute GVHD; 2) LC turnover is accelerated by full conditioning, complete myeloid chimerism and acute GVHD; 3) the majority of LC are donor-derived in all transplants at 100 days, consistent with the hypothesis that donor engraftment of APC drives the transition of acute to chronic GVHD. 4) there is little evidence using these conditioning regimens that acute GVHD or DLI-toxicity occurring after 100 days is related to the persistence of recipient APC.

413. Development and Characterization of Lentiviral Vectors for Hemophilia Gene Therapy

Monogenic inherited diseases can potentially be cured by gene therapy, which has already proven to be successful in a number of preclinical and clinical studies. A major target for gene therapy is the liver, since many proteins are synthesized in hepatocytes and secreted proteins can readily be delivered into the bloodstream. Lentiviral vectors are attractive vectors for hepatic gene delivery since they stably integrate into the target cell genome and thus can potentially give rise to life-long expression of the therapeutic protein (VandenDriessche et al., Blood 100:813–822, 2002). Lentiviral vectors do not express any potentially immunogenic viral genes and induce only minimal and transient liver toxicity and no pro-inflammatory cytokines (IL-6). However, intravenous delivery of a lentiviral vector pseudotyped with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G), resulted in efficient transduction of antigen presenting cells (APCs), primarily in liver and spleen, and transgene expression in APCs when a ubiquitously expressed cytomegalovirus (CMV) immediate early promoter was used. This increased the risk of developing a humoral immune response directed against the transgene product. Obviously, to obtain long-term expression, such an immune reaction has to be avoided. Different strategies are being pursued to direct expression of the transgene exclusively in hepatocytes and to prevent gene expression in APCs. We have therefore generated lentiviral vectors containing different hepatocyte-specific expression cassettes, which were titered by an optimized Q-PCR approach. Expression of coagulation factor IX using a human alpha1-antitrypsin promoter in conjunction with an ApoE enhancer repeat and a matrix attachment region (MAR) resulted in therapeutic FIX expression levels in vivo that were as high as when a CMV promoter were used. An alternative strategy to redirect expression to hepatocytes is to increase hepatocyte-specific transduction, while reducing vector uptake by APCs. This could potentially be achieved by pseudotyping the lentiviral vectors with alternative envelope proteins. Another potential advantage of reducing vector uptake by APCs, is that it may reduce the total vector dose necessary to reach a certain expression level. In a separate study with high-capacity adenoviral (HC-Ad) vectors, 10-fold higher expression levels were obtained after temporary depletion of macrophages (Chuah et al., Blood 101:1734–1743, 2003). However, the effect of transient APC depletion on transgene expression levels was not as dramatic following lentiviral gene transfer, suggesting that lentiviral vectors result in reduced APC transduction compared to when HC-Ad vectors are used. To further improve the safety profile of lentiviral vectors, purification of vector preparations is being optimized by ion-exchange chromatography. In summary, lentiviral vectors for hepatic gene delivery are being improved further at multiple levels by (i) designing more potent and hepatocyte-specific expression cassettes; (ii) developing alternative pseudotypes and (iii) implementing improved vector purification methods.

Green tea polyphenol (−)-epigallocatechin-3-gallate treatment to mouse skin prevents UVB-induced infiltration of leukocytes, depletion of antigen-presenting cells, and oxidative stress

Ultraviolet (UV) radiation-induced infiltrating leukocytes, depletion of antigen-presenting cells, and oxidative stress in the skin play an important role in the induction of immune suppression and photocarcinogenesis. Earlier we have shown that topical application of polyphenols from green tea or its major chemopreventive constituent (-)-epigallocatechin-3-gallate (EGCG) prevents UV-B-induced immunosuppression in mice. To define the mechanism of prevention, we found that topical application of EGCG (3 mg/mouse/3 cm(2) of skin area) to C3H/HeN mice before a single dose of UV-B (90 mJ/cm(2)) exposure inhibited UV-B-induced infiltration of leukocytes, specifically the CD11b+ cell type, and myeloperoxidase activity, a marker of tissue infiltration of leukocytes. EGCG treatment was also found to prevent UV-B-induced depletion in the number of antigen-presenting cells when immunohistochemically detected as class II MHC+ Ia+ cells. UV-B-induced infiltrating cell production of H2O2 and nitric oxide (NO) was determined as a marker of oxidative stress. We found that pretreatment of EGCG decreased the number of UV-B-induced increases in H2O2-producing cells and inducible nitric oxide synthase-expressing cells and the production of H2O2 and NO in both epidermis and dermis at a UV-B-irradiated site. Together, these data suggest that prevention of UV-B-induced infiltrating leukocytes, antigen-presenting cells, and oxidative stress by EGCG treatment of mouse skin may be associated with the prevention of UV-B-induced immunosuppression and photocarcinogenesis.

Bone marrow transplant conditioning intensified with liposomal clodronate to eliminate residual host antigen presenting cells fails to ameliorate GVHD and increases PERI-BMT mortality.

Graft versus host disease (GVHD) mediated by allogeneic donor T cells may be initiated and/or exacerbated by residual host antigen presenting cells (APC) which survive the transplant conditioning regimen. We examined whether the depletion of hepatic and splenic APC could reduce the severity of hepatic GVHD after bone marrow transplantation (BMT). Recipient mice were depleted for hepatic and splenic phagocytic APCs by i.v. injection of clodronate- (dichloromethylene diphosphonate) containing liposomes before fully allogeneic or MHC-matched, minor Ag-mismatched BMT. Severity of hepatic GVHD was scored on histological sections 2, 3, 4, or 9 weeks after BMT. No differences in the severity of GVHD were observed between APC-depleted mice and control mice. APC-depleted mice had increased peritransplant mortality due to sepsis. Bacterial clearance assays showed that APC-depleted mice were unable to efficiently clear bacteria, although nondepleted, transplanted mice were able to clear bacteria as quickly as naive control mice. Residual host phagocytic APC do not appear to play a role in the induction of GVHD after BMT. They are, however, essential for prevention of sepsis in the transplant host.