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Homing- und Chemokin-Rezeptoren können wichtige Informationen liefern über Aktivierungsstand und Organspezifität der einzelnen Zelle, aber auch über Homöostase und Gleichgewicht von T-Zellen untereinander. Mittels FACS-Analyse konnte in murinen Transplantationsmodellen durch die Hochregulation einzelner Homing-Rezeptoren auf T-Zellen die Entwicklung einer aGvHD vorhergesagt werden. Ob sich ein solches diagnostisches Fenster auch beim Menschen darstellen könnte, sollte in einer klinischen Pilotstudie untersucht werden. Zu diesem Zweck wurde der Expressionsverlauf von 19 verschiedenen Aktivierungsmarkern, Chemokin- und Homingrezeptoren auf T-Zell-Subpopulationen bei allogen transplantierten Patienten charakterisiert. Anschließend wurde versucht, eine Assoziation zwischen der Höhe der jeweiligen Rezeptorexpression und der späteren Entwicklung einer akuten Graft-versus-Host-Disease herzustellen. Die Expression der meisten untersuchten Rezeptoren nahm im Zeitverlauf ab und war insgesamt nur schwach ausgeprägt. Dabei zeigte sich insbesondere im frühen Verlauf nach SZT eine hohe relative Expression dieser Marker bei Patienten ohne aGvHD verglichen mit Patienten mit aGvHD im Verlauf. Insofern scheint eine hohe relative Expression dieser Rezeptoren kein Hinweis auf eine gesteigerte Alloreaktivität der jeweiligen T-Zellen zu sein. Gleichzeitig schien die absolute Anzahl an rezeptorpositiven Zellen eher positiv mit einer aGvHD korreliert zu sein. Die erhöhte Anzahl rezeptorpositiver Zellen errechnete sich allerdings aus der höheren absoluten Anzahl aller T-Zellen bei Patienten mit aGvHD. Damit war diese nicht rezeptorspezifisch, so dass sich Rezeptoren vom Expressionstyp 1 nicht eignen, um in der klinischen Routine eine aGvHD vorherzusagen. Einige Rezeptoren waren auf T-Helfer-Zellen auf mittlerem Niveau konstant exprimiert. Für manche von diesen zeigten sich unterschiedlich hohe relative Expressionsniveaus vor GvHD-Beginn bei Grad 2-4 verglichen mit Grad 1. Die absolute Anzahl rezeptorpositiver Zellen war bei allen Rezeptoren bei aGvHD Grad 2-4 höher als bei Grad 1. Die Expression einiger Marker war auf T-Zellen ausgeprägt, aber im Zeitverlauf fluktuierend. Für die Expression von beta 7 integrin allein und auch für dessen Koexpression zusammen mit CD 49d alpha 4 deuteten sich bei Patienten mit aGvHD Grad 2-4 höhere relative und absolute Werte an als für Patienten mit aGvHD Grad 1. Die Expression der übrigen hoch exprimierten Rezeptoren schien nicht vom Ausmaß der später einsetzenden aGvHD beeinflusst zu werden. Ob durch FACS-Analyse der Rezeptoren CLA, CCR 4, CD 45RA und/oder alpha 4 beta 7 allein oder in Kombination tatsächlich die alloreaktiven T-Zellen der aGvHD charakterisiert und quantifiziert werden können, muss an einem größeren Patientenkollektiv untersucht werden. Da zum jetzigen Zeitpunkt der prädiktive Wert dieser Marker weder postuliert noch ausgeschlossen werden kann, erscheint eine weitergehende Untersuchung sinnvoll. / Homing and chemokine receptors can provide important information about the activation and organ specifity of the cell itself as well as about homeostasis and equilibrium among t cells in general. In murine transplantion models, FACS analysis of t cells could detect the upregulation of homing receptors on t cells prior to the clinical onset of aGvHD. We tried to invesigate if such a diagnostic window could also be found in a clinical setting. Therefore we characterized the expression of 19 homing and chemokine receptors on t cells subsets in allogeneicly transplantated patients. We then looked for any association between the amount of the very receptor expression and the later development of aGvHD. The expression of most studied receptors was decreasing over the course of time and overall low. Especially in the first weeks after alloHCT, a high percentage of receptor expressing cells could be seen in patients without aGvHD compared to patients who developed aGvHD though. Therefore a relatively high expression of these receptors during lymphopenia did not seem to be associated with alloreactivity of the t cells. At the same time, an increased amount of receptor positive cells seemed to correlate positively with aGvHD. Yet, this was because patients with aGvHD showed overall higher rates of leukocytes and lymphocytes than the controls. The effect did not seem to be receptor specific so that receptors of the expression type 1 do not qualify for predicting aGvHD in clinical routine. Some receptors were constantely expressed on a medium level on helper t cells. A part of those showed higher expression levels when patients developed aGvHD grade 2-4 compared to patients with milder grade 1. The absolute amount of receptor positive cells was higher in severe aGvHD in all markers classified as expression type 2. Type 3 is characterized by an overall high but fluctuating homing receptor expression on t-cells. The receptors beta 7 integrin and CD 49 d alpha 4 were upregulated before patients developed severe aGvHD grade 2-4. The expression of the other type 3 receptors didn't seen to be influenced by later aGvHD. Further investigation in a larger cohort is needed to test the value of CLA, CCR4, CD 45 RA, beta 7 integrin and CD 49 d alpha 4 as predictive aGvHD biomarkers.

To study the pathogenesis and preventive strategies of acute and chronic graft-versus-host disease (aGVHD, cGVHD) after hematopoietic stem cell transplantation (HSCT), murine models of aGVHD and cGVHD were constructed. In addition, the role of CD4+CD25+ regulatory T cells (Tregs) in GVHD was also investigated in these models. My project consisted of three parts, including MHCI,II mismatched (part 1) and haploidentical BMT(part2) based aGVHD, and MHC matched, minor histocompatibility antigen (miHA)mismatched cGVHD(part 3). In the first model, aGVHD resulting from an MHC I, II mismatched aGVHD (B6(H-2b)→BALB/c(H-2d)) HSCT was studied, particularly with respect to the role that CD4+CD25+Tregs played. The results showed that CD4+CD25-T-cells induced more severe aGVHD than CD4+ T-cells, resulting in more extensive target organ lesions, especially in colon. The possible mechanism might be due to the enhanced proliferation and differentiation towards pathogenic Th1 cells. In the second model, haploidentical (B6(H-2b)→[C57BL/6×CBA/Ca]F1(H-2b×k)) HSCT was used to studied the role of CD4+or CD8+T-cells in aGVHD.Both high dose of donor CD4+-and CD8+-T-cells have the ability to induce lethal aGVHD in the hosts. However, the clinical and histological features of aGVHD induced by CD4+T-cells were significantly different from that induced by CD8+T-cells. Both donor CD4+-and CD8+-T-cells showed marked proliferation and differentiation towards CD4+IFN-γ+Th1and CD8+IFN-γ+cells, respectively. Polyclonalexpanded freshly isolatednTregs (exp-nTregs) showed obvious proliferation, increased apoptosis, and rapid loss of Foxp3 expression with impaired suppressive function. Exp-nTregs were further investigatedfor their preventive function in aGVHD in this haploidentical HSCT model. The results showed that exp-nTregs were capable of attenuating either CD4+-or CD8+-T-cell-induced aGVHD with significantly prolonged survival rate. In the third model, cGVHD was investigatedin DBA/2 (H-2d)→BALB/c (H-2d) HSCT, where the biologic readout was proteinuria and skin fibrosis. The results showed that donor CD4+T-and B220+B-cells were the main effectors in the pathogenesis of cGVHD. Notably, a more active germinal center (GC) reaction existed in the cGVHD cohorts compared with the control syngeneic cohort. Furthermore, Tfh and GC B-cells were shown to have originated from donor CD4+T-and B220+B-cells, respectively. Importantly, Tfh and GC B cells were mutually stimulatory and inter-dependent. In conclusion, three murine models were used to investigate aGVHD and cGVHD. The results showed that Tregs played a significant suppressive role in aGVHD complicating haploidentical HSCT. Furthermore, a hyperactive germinal center reaction might be the main cause of cGVHD. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

BACKGROUND: Graft versus host disease (GVHD) is a significant cause of morbidity and mortality following stem cell transplantation. Donor T cells are thought to be the main mediators of this disease, although we have recently identified that host T cells are present and active during acute GVHD suggesting contributions from both donor and host T cells. Whether both donor and host T cells can survive GVHD and coexist harmoniously after disease resolves is unknown. OBJECTIVE: The goals of this thesis are two-fold: (i) to study T cell chimerism in post-GVHD skin and (ii) to understand what effect, if any, treatment has on T cell chimerism in skin. METHODS: Acute GVHD and post-GVHD skin samples were obtained from male patients that had been transplanted with female donor cells. Chimerism was assessed using fluorescence in situ hybridization for the X and Y chromosomes concurrently with immunofluorescence staining for CD3, a T cell marker. Regulatory T cells were stained by immunofluorescence for CD3, CD4 and Foxp3. Medical record data was collected for all patients. RESULTS: We found that the percent of host T cells decreased significantly after resolution of acute skin GVHD compared to during active acute skin GVHD in skin samples obtained from five male patients that had been transplanted with female donor cells. The T cell composition in these patients in post-GVHD skin was primarily donor. We identified chimerism shifted toward donor T cells in patients treated with systemic steroids and this correlated with an increased number of donor T cells infiltrating into skin rather than a decrease in the number of host T cells in skin. With regard to frequency of Tregs, there was no significant difference between the group that had been treated with systemic steroids prior to biopsy and the group that had not. CONCLUSIONS: We discovered that donor chimerism predominates in post-GVHD skin and in active skin GVHD of patients who received systemic steroids, suggesting a role of donor cells in acute GVHD resolution. We were not able to identify a higher frequency of regulatory T cells in the treatment group. It is possible that the Treg recruited to skin by steroid treatment is Foxp3 negative, and therefore missed by our staining approach. The use of another marker is required for future studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble serum stimulation-2 (sST2) are elevated during human and murine aGVHD and are correlated to a type 1 T cell response. Membrane-bound ST2 (ST2) on donor T cells has been shown to be protective against aGVHD. ST2 signals through the adapter protein myeloid differentiation primary response 88 (MyD88). The role of MyD88 signaling in donor T cells during aGVHD remains unknown. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of interleukin 1 receptor (IL-1R) and toll-like receptor 4 (TLR4) signaling, both of which also signal through MyD88, in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells, leading to a decreased type 1 response without affecting T cell proliferation, apoptosis, or migration. In our aGVHD models, loss of intrinsic MyD88 signaling is not responsible for the observed protection. However, transplanting donor MyD88-/- T conventional cells (Tcons) with wild type (WT) or MyD88-/- T regulatory cells (Tregs) ameliorated aGVHD severity and lowered aGVHD mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine ten days post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, and Jak2, among others. Decreased sST2 was confirmed at the protein level with less secretion of sST2 and more expression of ST2 compared to WT T cells. Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs mirrored observations when using donor MyD88-/- Tcons. This suggests that Treg suppression from lack of MyD88 signaling in Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways. The results of our study confirm that ST2 represents an aGVHD therapeutic target that spares Treg function.

Hematopoietic stem cell transplant was developed as a curative therapy to treat onco-hematological diseases and recently indications for this therapy have expanded to include solid tumors, hemoglobinopathies and other genetic diseases and disorders. Two major types of hematopoietic stem cell transplant have been developed. Autologous transplants aim to deliver a massive dose of radiation and/or chemotherapy that is capable of ablating the hematopoietic stem cells in the bone marrow. The patient is then "rescued" from this lethal dose of treatment by an infusion of their own hematopoietic stem cells. Allogeneic transplants are designed to either functionally replace a cell class, or an enzyme or biological function absent in the patient, or to consolidate a remission in a onco-hematological disease via the graft-versus-tumor effect . Two of the largest causes of non-relapse mortality from an allogeneic hematopoietic stem cell transplant are acute and chronic graft-versus-host disease, in which immune cells derived from the graft recognize normal host tissue as foreign and attack these tissues. A host of biomarkers for acute graft versus host disease have been identified, but there is almost none for chronic graft versus host disease. Herein, a methodology to discover and validate a biomarker(s) for the most common organ system affected by chronic graft versus host disease is proposed.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references.

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Dec 22