Kathrin Rieger

Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation

HIV entry into CD4(+) cells requires interaction with a cellular receptor, generally either CCR5 or CXCR4. We have previously reported the case of an HIV-infected patient in whom viral replication remained absent despite discontinuation of antiretroviral therapy after transplantation with CCR5Δ32/Δ32 stem cells. However, it was expected that the long-lived viral reservoir would lead to HIV rebound and disease progression during the process of immune reconstitution. In the present study, we demonstrate successful reconstitution of CD4(+) T cells at the systemic level as well as in the gut mucosal immune system after CCR5Δ32/Δ32 stem cell transplantation, while the patient remains without any sign of HIV infection. This was observed although recovered CD4(+) T cells contain a high proportion of activated memory CD4(+) T cells, ie, the preferential targets of HIV, and are susceptible to productive infection with CXCR4-tropic HIV. Furthermore, during the process of immune reconstitution, we found evidence for the replacement of long-lived host tissue cells with donor-derived cells, indicating that the size of the viral reservoir has been reduced over time. In conclusion, our results strongly suggest that cure of HIV has been achieved in this patient.

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation.

Human cytomegalovirus (CMV) is a major cause of death after transplantation. The frequency of pp65-specific T cells was examined in 38 HLA-A2+ stem cell recipients during the first year after transplantation. Patients were divided into four groups based on donor/recipient serostatus: d+/r+ (n=17), d+/r− (n=7), d−/r+ (n=9) and d−/r− (n=5). Peripheral blood mononuclear cells were stimulated with the CMVpp65 peptide NLVPMVATV, and the specific T-cell frequency was assessed by interferon gamma (IFN-γ) ELISPOT assay. Responding T cells were characterized by flow cytometry revealing a terminal differentiated effector phenotype. Surveillance of CMV infection was carried out by real-time polymerase chain reaction (n=26) or immunofluorescence (n=12). Infection was present in 7/9 d−/r+ high-risk patients, and CMV disease occurred exclusively in this group with delayed or absent virus-specific T-cell recovery. In contrast, 16/24 intermediate-risk patients showed CMV-specific T cells. Our data suggest that CMV infection and disease rates are elevated in high-risk patients with delayed CMV-specific T-cell immune reconstitution and lower in those with early recovery of T-cell immunity. We recommend preferring CMV seropositive donors for CMV seropositive recipients, as this should lead to durable CMV-specific T-cell responses soon after transplantation with consecutive protection from CMV disease.

Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan

Radioimmunotherapy (RIT) of relapsed lymphoma is gaining increasing importance. Especially the commercially available anti-CD20 antibody 90Y-ibritumomab tiuxetan is currently under investigation in various trials including dose escalation and autologous hematopoietic progenitor cell support. It is not clear, however, whether the implementation of this radiolabeled antibody into another treatment option for relapsed or poor risk lymphoma patients—allogeneic hematopoietic cell transplantation—interferes with or delays successful engraftment. This study reports encouraging results with 2 relapsed lymphoma patients (1 transformed marginal zone lymphoma and 1 mantle cell lymphoma) who underwent allogeneic hematopoietic cell transplantation from HLA-matched donors. The conditioning regimen consisted of Rituximab 250 mg m−2 on days −21 and −14, 0.4 mCi kg−1 body weight 90Y-ibritumomab tiuxetan on day −14 and fludarabine (30 mg m−2) plus cyclophosphamide (500 mg m−2) on days −7 to −3. The data demonstrate that engraftment is fast and reliable with leukocytes >1 × 109 L−1 on day 12 and platelets >50 × 109 L−1 on day 10. Thus, the incorporation of radioimmunotherapy into allogeneic transplant protocols combines established modalities with proven anti-lymphoma activity and, hence, offers an attractive new therapeutic option for relapsed lymphoma patients.

Rabbit antithymocyte globulin (Thymoglobulin®) impairs the thymic output of both conventional and regulatory CD4+ T cells after allogeneic hematopoietic stem cell transplantation in adult patients

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA+CD31+) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4+CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4+ T cells. The sustained depletion of conventional and regulatory CD4+T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

Serotherapy with thymoglobulin and alemtuzumab differentially influences frequency and function of natural killer cells after allogeneic stem cell transplantation.

Although thymoglobulin and alemtuzumab are frequently used in hematopoietic stem cell transplantation (HSCT), little is known of their effects on NK cells, which mediate important functions in post-transplantation immunology. In the present study, we determined NK cell death in vitro using propidium iodide and Annexin V. The NK cell activity in 34 patients at day +30 after allogeneic HSCT was assessed using the CD107a assay. Alemtuzumab and thymoglobulin were similarly very potent in inducing NK cell death in vitro. Even in low concentrations (<1 μg/ml) the antibodies induced apoptosis and necrosis in a relevant percentage of NK cells (>30%). However, the number of tumor reactive (CD107a+) NK cells was 13.16 per μl and 1.15 per μl (mean) in patients receiving T-cell depletion with 6 mg/kg thymoglobulin and in patients receiving 100 mg alemtuzumab, respectively (P=0.02). Although thymoglobulin and alemtuzumab are equally NK cell toxic in vitro, the recovery of NK cell frequency and anti-tumor reactivity is reduced in recipients of alemtuzumab. Our findings can be explained by a longer half-life of alemtuzumab as compared to active thymoglobulin under therapeutic conditions. Prolonged immunosuppression with increased risk of infections and tumor relapse are a potential threat to patients undergoing HCST and receiving alemtuzumab as T-cell depletion.

Remission Induction Using Alemtuzumab can Permit Chemotherapy-refractory Chronic Lymphocytic Leukemia (CLL) Patients to Undergo Allogeneic Stem Cell Transplantation

The outcome of allogeneic stem cell transplantation depends upon the disease status before transplantation. Patients with refractory disease are at high risk for relapse. To improve the curative potential of the transplant procedure, we treated 3 chemotherapy-refractory CLL patients with alemtuzumab before allogeneic stem cell transplantation. Prior to therapy, all patients suffered from B-symptoms, and had massive adenopathy, splenomegaly, thrombocytopenia, and anemia; two patients had hepatomegaly. Alemtuzumab greatly reduced tumor mass in blood and bone marrow, B-symptoms resolved, and organomegaly improved. Two patients became blood product independent. All patients proceeded to transplantation after conditioning with TBI 2 Gy (n = 1) or Treosulfan (n = 2) in combination with Fludarabine either from an HLA-matched sibling (n = 2) or from an HLA-matched unrelated donor (n = 1). All patients engrafted, and are alive and well. Two patients reached complete remission (CR); one patient attained stable partial remission (PR). These heavily pre-treated refractory patients gained substantial clinical benefit from alemtuzumab, and received successful allografts.

Rabbit antithymocyte globulin induces rapid expansion of effector memory CD8 T cells without accelerating acute graft versus host disease

Rabbit antithymocyte globulin (Thymoglobulin®) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin® therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapid expansion, whereas the fraction of naive CD8 T cells decreased. Importantly, this shift towards effector memory CD8 T cells did not accelerate the incidence of GvHD.

Confocal endomicroscopy in diagnosis of intestinal chronic graft‐versus‐host disease

Graft‐versus‐host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High‐resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E‐stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD.

Epithelial barrier dysfunction as permissive pathomechanism in human intestinal graft-versus-host disease

Acute graft-versus-host disease (A-GVHD) is characterized by a strong inflammatory reaction of the graft against the recipient, mainly in the skin, the intestine and the liver. The gastrointestinal manifestation of the disease is characterized by nausea, vomiting, abdominal cramps and diarrhea with extensive fluid loss. Histologically, diagnosis of A-GVHD in colonic biopsies is characterized by crypt abscesses and epithelial apoptosis [1]. Pathophysiologically, A-GVHD is described as a three-stage model with activation of antigen presenting cells in the host, donor T-cell activation and target tissue destruction [2]. Within this model barrier dysfunction with translocation of microbial products and luminal antigens is thought to be a major perpetuating factor and a major cause of the “cytokine storm” through immune activation [3]. However, epithelial barrier function in AGVHD may not only be relevant for the pathophysiology of immune activation but also for the pathomechanism of diarrhea in this disease. Therefore, we investigated the epithelial barrier function in human biopsies ex vivo in Ussing-type chambers with subsequent molecular characterization of potential pathomechanisms. In this study, 22 patients with diarrhea and clinical suspicion of intestinal GVHD after bone marrow transplantation underwent sigmoidoscopy at the CharitéUniversitätsmedizin Berlin. Infectious causes for symptoms were previously ruled out including stool analysis for Clostridium difficile toxin and whole blood CMV-PCR and immunohistochemistry of biopsies. Diagnosis of A-GVHD was made from histological biopsies in 16 patients (AGVHD; grade I: n= 6, grade II: n= 8, grade III: n= 1, grade IV: n= 1). The remaining six patients without histopathological diagnosis of GVHD served as a further control group (no GVHD). A group of 15 patients which underwent endoscopy within this period of time for tumor exclusion without signs of pathology served also as controls (control). All patients gave their written consent for taking biopsies for scientific purpose and the study was approved by the local ethics committee of the Charité Berlin (229-32). A miniaturized Ussing-type chamber was used for impedance spectroscopy. This technique can differentiate the epithelial and subepithelial portion of the transmural electrical resistance [4]. Paracellular permeability was determined by H-Mannitol flux. For macromolecular flux the transcellular passage of horse radish peroxidase (HRP; 44 kDa; Sigma-Aldrich) was analyzed in six consecutive patients with A-GVHD grade I/II. Tight junction protein expression was analyzed by Western blotting. For cryosections, tissues were fixed with 2% paraformaldehyde. Antibodies were rabbit anti-claudin-2, rabbit anti-occludin, mouse anti-claudin-5 (Life Technologies GmbH, Darmstadt, Germany) and mouse anti-ZO-1 (BD Biosciences, San Jose, CA). Secondary antibodies were Alexa Fluor 594 goat anti-mouse or rabbit IgG and 488 goat anti-rabbit or mouse IgG (Molecular Probes, Eugene, OR). Nuclei were counterstained with DAPI. Images were obtained with a confocal laser scanning microscope (Zeiss LSM 510 Meta, Jena, Germany). Results are given as means ± SEM. These authors contributed equally: Kathrin Rieger, Jörg-Dieter Schulzke