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A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model
- Carina A Bäuerlein1, 2, 3,
- Simone S Riedel1, 2, 3,
- Jeanette Baker1, 2, 3, 4,
- Christian Brede1, 2, 3,
- Ana-Laura Jordán Garrote1, 2, 3,
- Martin Chopra1, 2,
- Miriam Ritz1, 2,
- Georg F Beilhack5,
- Stephan Schulz6,
- Robert Zeiser7,
- Paul G Schlegel3, 8,
- Hermann Einsele1, 3,
- Robert S Negrin4 and
- Andreas Beilhack1, 2, 3Email author
© Bäuerlein et al.; licensee BioMed Central Ltd. 2013
Received: 3 November 2012
Accepted: 19 April 2013
Published: 21 May 2013
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.
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.
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.
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.
Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment option for many malignant diseases due to the beneficial immunological graft-versus-leukemia (GVL) effect . However, (acute) graft-versus-host disease ((a)GVHD) continues to be a major complication after allo-HCT. This syndrome is caused by donor T cells that attack the intestinal tract, liver, and skin . Early diagnosis remains challenging and to date mainly relies on clinical symptoms and histopathology. When clinical symptoms are readily detected, response to therapy is rarely beneficial to the patient. Therefore, a predictive test to identify patients at risk before the visible onset of aGVHD is highly desirable.
A detailed understanding of the pathophysiological processes including the kinetics of alloreactive T cell priming, migration, and effector mechanisms after allo-HCT may provide clues to identify patients that are likely to develop aGVHD. Recently, in vivo bioluminescence imaging (BLI) of T cells that carried the firefly luciferase (luc+) gene in a major histocompatibility complex (MHC) mismatched mouse model helped define a distinct aGVHD initiation and effector phase . Donor T cells first migrated into secondary lymphoid organs (SLOs), where they were activated by interacting with host antigen presenting cells (APCs). This interaction led to the upregulation of certain homing receptors which the cells used for target tissue infiltration . The subsequent effector phase, taking place in peripheral aGVHD target tissues, started 3 days later when alloreactive T cells left the SLOs and migrated via the peripheral blood (PB) to their respective target organs, mainly the gastrointestinal tract (GIT), the liver, and the skin [4–6].
In the present work, we asked whether it is possible to detect alloreactive donor T cells in the PB early after allo-HCT in a clinically relevant minor histocompatibility antigen (miHAg) mismatch mouse model. We characterized donor T cells according to the expression of two well-described homing receptors, α4β7 integrin and P-selectin ligand. Based on these observations we succeeded in effectively treating mice with rapamycin on first alloreactive cell detection in the PB to timely prevent aGVHD exacerbation.
All experiments were performed according to the German regulations for animal experimentation and approved by the Regierung von Unterfranken as the responsible authority (Permit Number 55.2-2531.01-30/09).
All HCT experiments were performed with sex-matched 8 to 12-week-old mice. BALB/c (H-2d, CD90.2) and C57Bl/6 (H-2b, CD90.2) mice obtained from Charles River (Sulzfeld, Germany); BALB/b (H-2b, CD90.2) and congenic C57Bl/6 (H-2b, CD45.1) obtained from Jackson Laboratories (Bar Harbor, ME, USA). The luciferase-expressing (luc+) transgenic FVB-L2G85 line  was backcrossed over 12 generations onto C57Bl/6 (H-2b, CD90.1) background. Mice were housed in a pathogen-free facility at the Center for Experimental Molecular Medicine (ZEMM), Würzburg, Germany.
Flow cytometry analysis
Cells were stained with the following antibodies (clones): anti-CD8α (53-6.7), anti-CD4 (RM4-5), anti-CD90.1 (H1S51), anti-CD45.1 (A20), anti-LPAM-1/α4β7 (DATK32) from Biolegend (Uithoorn, The Netherlands) and eBioscience (Frankfurt, Germany), P-selectin ligand-IgG fusion protein (Becton Dickinson (BD), Heidelberg, Germany), anti-human-IgGκ-FITC (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Dead cells were excluded with propidium iodide (PI) or 4′,6-diamidino-2-phenylindole (DAPI) staining. Flow cytometry was performed on a FACS-Canto II (Becton Dickinson), and data was analyzed with FlowJo Software version 8 (Treestar, Ashland, OR, USA). Gates were set using the fluorescence minus one-gating strategy . Anti-mouse or anti-rat/hamster CompBeads (BD) were used for compensation controls.
Recipient mice (BALB/c, BALB/b, and C57Bl/6) were myeloablatively irradiated (BALB/c, BALB/b: 8 Gy; C57Bl/6: 9 Gy) with an electron linear accelerator (Mevatron Primus, Siemens, Germany). Bone marrow (BM) cells from wild-type (WT) C57Bl/6 donor mice were flushed from femur and tibia bones with phosphate-buffered saline (PBS; PAN, Aidenbach, Germany). T cells were isolated from spleens of luc + C57Bl/6-L2G85 donor mice and red blood cells were lysed. Splenic CD3+ single cell suspensions were enriched using the Dynal Mouse T cell Negative Isolation Kit (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. Cell purity of the CD3+ was confirmed by post-enrichment fluorescence-activated cell sorting (FACS) analysis (>90%) For hematopoietic reconstitution, all recipient mice were injected intravenously with 5 × 106 C57Bl/6 WT BM cells. To induce aGVHD, BALB/c recipients (MHC major mismatch model) and syngeneic C57Bl/6 recipients were coinjected intravenously with 1.2 × 106 enriched CD3+/luc + and 5 × 106 BM cells, while BALB/b recipients (miHAg mismatch model) were coinjected intravenously with 5 × 106 CD3+/luc + T cells and 5 × 106 BM cells within 3 h after irradiation. Transplanted mice were monitored daily for survival, weight change, and clinical GVHD symptoms according to Cooke et al. .
For in vivo studies, rapamycin (Wyeth, Reading, UK) was dissolved in carboxymethylcellulose sodium salt (C-5013; Sigma-Aldrich, Munich, Germany) and polysorbate 80 (P-8074, Sigma-Aldrich) to a final concentration of 1.5 mg/kg body weight (BW)  and injected intraperitoneally daily from day +6 to day +15 in a final volume of 100 μL.
In vivobioluminescence imaging
Mice were anesthetized and coinjected intraperitoneally with 80 mg/kg BW ketamine (Pfizer, Berlin, Germany) and 16 mg/kg BW xylazine (CP Pharma, Burgdorf, Germany) together with d-luciferin (Biosynth AG, Staad, Switzerland) at a dose of 150 μg/g BW. Three or six mice per group per day were imaged for analysis. Images were captured as previously described [5, 7] using an IVIS Spectrum charge-coupled device (CCD) imaging system (Caliper-Xenogen, Alameda, CA, USA). Imaging data were analyzed and quantified with Living Image Software 3.1 (Caliper-Xenogen).
Peripheral blood samples
Mice were bled daily (three mice per group) via the tail vein and erythrocytes were lysed for FACS analysis. In addition, 25 μL PB from each mouse was added to 100 μL PBS/EDTA (1 mM) for white blood cell counts using a Sysmex XT-2000i (Horgen, Switzerland).
Immunofluorescence staining and immunohistochemistry
Organs were embedded in optimal cutting temperature (OCT) compound (Sakura, Zoeterwoude, The Netherlands) and cut into 5-μm thick sections. Slides were kept at -20°C until staining. After air drying and acetone fixation (10 minutes at room temperature), sections were incubated with blocking solution for 15 minutes (PBS + 1% fetal calf serum (FCS)) prior to staining with the following antibodies for 1 h: CD8α-Alexa488 (clone 53-6.7, Biolegend), CD90.1-APC (clone HIS51, eBioscience), CD4-biotin (clone RM 4-5, Biolegend), all diluted 1:100 in PBS. Streptavidin-Alexa 546 (1:200 in PBS) was used as secondary antibody for 30 minutes. Nuclei were stained with Hoechst for 3 minutes, diluted 1:1,000 in PBS. Washing steps after antibody incubation and Hoechst staining were performed in 1 × PBS (three times, 2 minutes each). Fluorescence microscopic evaluation was performed on a Carl Zeiss AxioImager Z1. For immunohistochemistry, air-dried slides were incubated with 1% H2O2 for 10 minutes before blocking with avidin-biotin (Avidin/Biotin Blocking Kit, Vector, Linaris GmBH, Wertheim, Germany) for 15 minutes each, followed by a 15-minute block with 1% FCS. CD90.1-biotin antibody conjugate (clone HIS51) was used for donor T cell staining for 1 h followed by the ABC method (Vector) according to the manufacturer’s protocol.
For histological analysis organs were fixed in PBS containing 4% paraformaldehyde (PFA) (Roth, Karlsruhe, Germany). Representative PFA fixed samples of GIT, liver and skin of each group were embedded in paraffin, cut into 3-μm thick sections, and stained with hematoxylin and eosin (H&E). GVHD scoring was performed according to Lerner et al.  based on tissue damage. The scoring system categorized 0 as normal, 1 for mild, 2 for moderate, and 3 for severe tissue damage caused by donor T cells. All slides were examined by an experienced unbiased pathologist (SS).
Statistical analysis was performed with the non-parametric Mann-Whitney test using GraphPad InStat 3 software (GraphPad, La Jolla, CA, USA) where appropriate. Measurements are expressed as the mean ± standard error of mean (SEM). Analysis was performed using GraphPad Prism 5 software. Data reaching statistical significance (P ≤0.05) is indicated with an asterisk (*).
Results and discussion
In line with our results, two clinical retrospective studies indicate a correlation between homing receptor upregulation and the subsequent development of aGVHD symptoms. Chen and colleagues found at least tenfold upregulation of α4β7 integrin on naïve as well as memory T cell subsets before the onset of clinically apparent intestinal GVHD compared to cutaneous or no aGVHD in a case-controlled study including 59 patients . Another study with 33 patients found an association of cutaneous lymphocyte antigen (CLA, a P-selectin glycoprotein ligand-1)-upregulation on PB T cells and the onset of severe aGVHD . Furthermore, in a recent clinical study Chen et al. could correlate the detection of α4β7+ memory T cells with the appearance of intestinal aGVHD in patients . However, these studies did not address how soon before aGVHD onset and for how long these T cell surface receptors could be detected.
Different functional studies in murine models across major or minor MHC mismatches confirmed that α4β7 or ß7 negative donor T cells cause less aGVHD morbidity and mortality compared to WT T cells mainly due to reduced homing to the intestinal tract while still exhibiting the GVT effect [12, 17]. Likewise, ameliorated aGVHD was observed in P-selectin-deficient mice, which underscores the importance of the interaction between P-selectin and its ligands that are highly expressed on migrating effector T cells . In our experiments we also found significantly higher α4β7 integrin and P-selectin ligand expression on migrating allogeneic T cells than in syngeneic controls. The expression of these molecules on PB donor T cells correlates with the identified cell migration phase and strongly associates with aGVHD induction in allo-HCT suggesting their potential usefulness as predictive markers. Recently, research efforts have focused on the identification of reliable predictive markers to identify patients at risk before aGVHD onset with some potential biomarkers being investigated thoroughly [18, 19]. Different combinations of these biomarkers have been used to predict treatment response and survival of aGVHD patients, but a predictive marker panel to reliably identify patients at risk for aGVHD is still lacking.
We propose that the identified migration phase of alloreactive T cells may serve as a potential diagnostic window for early and timely targeted therapeutic interventions. In addition to the described surface markers, α4β7 integrin and P-selectin ligand, combinations with other markers appear attractive. Further investigations of suitable surface receptor combinations to precisely identify alloreactive PB T cells could be complemented by detecting polypeptides in the urine of patients as early indicators for aGVHD. Kaiser et al. identified two peptides of the leukotriene A4 hydrolase and of serum albumin as possible biomarkers . Furthermore, tumor necrosis factor receptor 1 (TNFR1), interleukin 2 receptor alpha (IL-2Ra), IL-8, and hepatocyte growth factor (HGF) as plasma biomarkers relevant for aGVHD effectively discriminated between patients with and without aGVHD  and elafin functioned as a biomarker for skin aGVHD patients . Different combinations of these biomarkers have been used to predict treatment response and survival of aGVHD patients [18, 19]. We envision combining the described homing receptor panel with these biomarkers in order to develop a reliable predictive test to identify patients at risk before disease onset.
Encouraging for the clinical allo-HCT setting, the directed, preemptive rapamycin treatment starting immediately at the first detection of alloreactive donor T cells significantly improved aGVHD symptoms and granted survival in all miHAg allo-HCT recipients. Similarly, preemptive treatment with other immunomodulators and, particularly, drugs altering T cell trafficking  appear highly attractive, but may exert their biggest benefit if individuals at high risk for aGVHD can be identified in a timely manner before the first clinical symptoms appear.
In the present study, we demonstrate in a clinically relevant miHAg allo-HCT mouse model that it is feasible to detect migrating alloreactive donor T cells for an extended time period of 2 weeks and, importantly, before aGVHD symptoms become apparent. As the prediction of aGVHD is highly relevant to the clinical outcome, it may be beneficial to closely monitor the kinetics of T cell engraftment, expansion, and homing receptor expression in allo-HCT patients. Our preclinical findings may have implications for the development of a predictive blood test identifying patients at risk for aGVHD and thereby giving physicians the chance for a timely and targeted therapeutic intervention.
We thank Dr Franz Schwab, Katharina Mattenheimer, and Carolin Kiesel for expert technical assistance and lab management and the members of the Beilhack lab and Tor B. Stuge (University of Tromsø, Norway) for helpful discussions. CAB was funded by the Deutsche José Carreras Leukämie Stiftung (DJCLS F 11/05). This work was supported by a grant from the Wilhelm-Sander-Stiftung (2007.081.1, to AB), the Deutsche Forschungsgemeinschaft grant Z2 TR52 (to AB) and the Interdisciplinary Center for Clinical Research (IZKF) Würzburg (to AB) and in part by a grant from BayImmuNet (D2-F5121.7.1/13/8 to PGS). This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing.
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