The Role of Host Dendritic Cells during the Effector Phase of Intestinal Graft-versus-Host Disease

Monocytes can be functionally divided in two subsets, both capable to differentiate into dendritic cells (DCs): CX3CR1loCCR2+ classical monocytes, actively recruited to the sites of inflammation and direct precursors of inflammatory DCs; and CX3CR1hiCCR2− non-classical monocytes, characterized by CX3CR1-dependent recruitment to non-inflamed tissues. Yet, the function of non-classical monocyte-derived DCs (nc-mo-DCs), and the factors, which trigger their recruitment and DC differentiation, have not been clearly defined to date. Here we show that in situ differentiated nc-moDCs mediate immunosuppression in the context of intestinal graft-versus-host disease (GVHD). Employing multi-color confocal microscopy we observed a dramatic loss of steady state host-type CD103+ DC subset immediately after transplantation, followed by an enrichment of immune-regulatory CD11b+ nc-moDCs. Parabiosis experiments revealed that tissue-resident non-classical CX3CR1+ monocytes differentiated in situ into intestinal CD11b+ nc-moDCs after allogeneic hematopoietic cell transplantation (allo-HCT). Differentiation of this intestinal DC subset depended on CSF-1 but not on Flt3L, thus defining the precursors as monocytes and not pre-DCs. Importantly, CX3CR1 but not CCR2 was required for this DC subset differentiation, hence defining the precursors as non-classical monocytes. In addition, we identify PD-L1 expression by CX3CR1+ nc-moDCs as the major mechanism they employ to suppress alloreactive T cells during acute intestinal GVHD. All together, we demonstrate that host nc-moDCs surprisingly mediate immunosuppression in the context of murine intestinal GVHD – as opposed to classical “inflammatory” monocyte-derived dendritic cells (mo-DCs) – via coinhibitory signaling. This thorough study unravels for the first time a biological function of a - so far only in vitro and phenotypically described - DC subset. Our identification of this beneficial immunoregulatory DC subset points towards alternate future strategies in underpinning molecular pathways to foster their function. We describe an unexpected mechanism of nc-moDCs in allo-HCT and intestinal GVHD, which might also be important for autoimmune disorders or infections of the gastrointestinal tract

A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model

Background Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. Methods Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. Results We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. Conclusions Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention

Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1

Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome

Tumor detection by <i>in vivo</i> BLI correlates to M315 IgA serum measurement by ELISA.

<p>(<b>A</b>) M315 serum levels of melphalan treated (n = 5) and vehicle control (n = 5) on day 14 of treatment and simultaneous BLI measurement of the same mice. The treatment schedule is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052398#pone-0052398-g003" target="_blank">Figure 3A</a>. Measurement of idiotype specific M315 IgA significantly differed between the groups (two-tailed p value 0.0171) as it did with BLI (ventral+dorsal signal) (two-tailed p value 0.0221). (<b>B</b>) Scheme indicating time points for BLI measurement and serum collection for data presented in (C–G). (<b>C–G</b>) Idiotype specific M315 IgA serum levels of 5 untreated mice constantly increased over time correlating with progressing tumor burden as measured with BLI (ventral+dorsal signal). Of note, BLI measurements provided signals in early disease stages starting from day +9, whereas M315 IgA levels were not detectable at this time point. The left y-axis displays BLI signal intensity (black circles, solid lines); the right y-axis displays serum M315 IgA (red triangles, dashed lines).</p

Detection of residing MOPC-315.BM luc⁺ cells and <i>in vivo</i> monitoring of reduced myeloma progression due to melphalan treatment.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. On day +19 after inoculation, tumors were established in all mice and readily detected by BLI. Then treatment was started ( = day 0 of treatment). Mice received 5 mg/kg melphalan (n = 9, two independent experiments) or mock treatment (vehicle control, n = 13, three independent experiments) intraperitoneally. One control group of MOPC-315.BM luc⁺ tumor bearing mice did not receive any treatment (untreated control, n = 14, three independent experiments). (<b>A</b>) Schematic study design, indicating treatment time points in respect to time after MM injection and end of experiment. Red arrows pinpoint melphalan treatment. (<b>B</b>) BLI images of two representative mice per group at selected time points in ventral (left) and dorsal (right) view. (<b>C</b>) Quantification of bioluminescence signal intensities over time from ventral or dorsal. Signals at day +19 were set as starting point and the following measurements were calculated as fold change of this initial signal intensity. Mice were treated at time points as indicated by arrows. Significant difference between melphalan treated mice vs vehicle control or vs untreated animals starting on day 10 of treatment for both, ventral (untreated vs melphalan p<0.0001, vehicle vs melphalan p = 0.0032) and dorsal (untreated vs melphalan p = 0.0006, vehicle vs melphalan p = 0.0024). (<b>D</b>) Quantification of skeletal tumor foci in untreated, vehicle control and melphalan treated mice on day 0 and 14 of drug treatment.</p

Engraftment and growth dynamics of MOPC-315.BM luc⁺ myeloma cells in vivo.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. Tumor growth and spread was regularly monitored by BLI. (<b>A</b>) BLI images of one representative mouse at indicated time points after MM injection from ventral (top) and dorsal (bottom) view. Additional emerging tumor foci over time are marked with black or white arrows. (<b>B</b>) Number of skeletal spots per mouse on days +11 (n = 51), 19 (n = 56) and 33 (n = 25) and (<b>C</b>) percentage of mice presenting signals from liver and spleen. (<b>D</b>) Quantification of single tumor foci over time as marked in (A): 1 and 2 = BM compartment of femur/tibia, 3 = spleen, 4 = BM compartment of shoulder. (<b>E</b>) Absolute signal quantification by whole body BLI from ventral and dorsal views. (<b>F</b>) Representative osteolytic lesion in the neck of femur 42 days after MM injection. Corticalis is marked as c which is destroyed (arrow) by MOPC-315.BM luc⁺ cells marked with T. Original magnification 40X, scale bar is 200 µm. Insert: original magnification 200X, scale bar is 100 µm.</p

Flow cytometric measurement of surface receptors associated with BM homing and infiltration of myeloma cells.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. (<b>A</b>) 42 days after MM injection mice showed high BLI signals from hematopoietic compartments such as femur/tibia and spleen. Shown are two representative mice from ventral and dorsal view immediately before cells from BM and spleens were harvested for flow cytometry. (<b>B–D</b>) Besides BM and spleen derived MM cells, we also analysed MOPC-315.BM luc+ cells from culture. Dead cells were excluded by propidium iodide staining and MOPC cells identified as CD138⁺CD4⁺ double positive cells. (<b>B</b>) α4β1 integrin positive MOPC-315.BM luc⁺ cells were identified by flow cytometry as α4⁺ (CD49d⁺) and α4β7⁻. Representative FACS plots and the corresponding graph are shown, stating the frequency within CD138⁺CD4⁺ MOPC-315.BM luc⁺ cells expressing α4β1. For CXCR4 (<b>C</b>) and CD44 (<b>D</b>) representative histograms for each organ and cell line, including unstained fluorescence minus one (FMO) sample are displayed. Corresponding graphs state the fold difference in mean fluorescence intensity (MFI) related to the unstained FMO sample. BM and spleen: two independent experiments, n = 10, cells from cell culture: n = 4 for CXCR4 and CD44, n = 3 for α4β1. * indicates p<0.05 and ** indicates p<0.01 as determined by Kruskal-Wallis test with Dunn post test. (<b>E</b>) MOPC-315.BM luc⁺ cells were sorted for CXCR4^low and CXCR4^high expression. After 2 days in cell culture sorted cells regained the original CXCR4 expression level of the cell line. (<b>F</b>) 5×10⁵ sorted cells were i.v. injected into 4 female BALB/c mice each and BLI from ventral, lateral and dorsal was performed 10 days later. Sorted CXCR4^low as well as CXCR4^high CXCR4 cells readily homed to the BM compartment as well as to the spleen. (<b>G</b>) After BLI the mice were sacrificed, cells from left and right femur/tibia (separately) and the spleen extracted, and percentage as well as absolute numbers for CD138⁺CD4⁺ MM cells determined. From these values a ratio of spleen/BM was calculated to determine the homing capacity of the sorted populations. (<b>H</b>) Comparison of CXCR4 expression levels of sorted CXCR4^low and CXCR4^high cells immediately before injection and MM cells from BM and spleen after 10 days <i>in vivo</i> revealed a dynamic CXCR4 regulation.</p

A T-Cell Surface Marker Panel Predicts Murine Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8$^+$ T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions

The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations