Complement component C3 mediates Th1/Th17 polarization in human T-cell activation and cutaneous GVHD.

The complement system has been shown to regulate T-cell activation and alloimmune responses in GVHD. Mice deficient in the central component of complement system C3 have significantly lower GVHD-related mortality/morbidity, and C3 modulates Th1/Th17 polarization in mouse GVHD. To investigate whether anticomplement therapy has any impact on human T-cell activation, a drug candidate Compstatin was used to inhibit C3 activation in this study. We found the frequency of IFN-γ (Th1)-, IL-4 (Th2)-, IL-17 (Th17)-, IL-2- and TNF-α-producing cells were significantly reduced among activated CD4(+) cells in the presence of Compstatin. Compstatin treatment decreased the proliferation of both CD4(+) and CD8(+) T cells upon TCR stimulation. However, Compstatin does not affect the production of IL-2 and TNF-α in activated CD8(+) T cells, and the differentiation of CD8(+) T cells into distinct memory and effector subsets remained intact. Furthermore, we examined complement deposition in skin and lip biopsy samples of patients diagnosed with cutaneous GVHD. C3 deposition was detected in the squamous epithelium and dermis, blood vessels and damaged sweat glands, and was associated with gland damage and regeneration. We conclude that C3 mediates Th1/Th17 polarization in human T-cell activation and skin GVHD in patients.

Effects on tumor development and metastatic dissemination by the NKG2D lymphocyte receptor expressed on cancer cells.

The stimulatory NKG2D lymphocyte receptor together with its tumor-associated ligands enable the immune system to recognize and destroy cancer cells. However, with dynamic changes unfolding, cancers exploit NKG2D and its ligands for immune evasion and suppression. Recent findings have added yet another functional dimension, wherein cancer cells themselves co-opt NKG2D for their own benefit to complement the presence of its ligands for self-stimulation of parameters of tumorigenesis. Those findings are here extended to in vivo tumorigenicity testing by employing orthotopic xenotransplant breast cancer models in mice. Using human cancer lines with ectopic NKG2D expression and RNA interference (RNAi)-mediated protein depletion among other controls, we show that NKG2D self-stimulation has tumor-promoting capacity. NKG2D signals had no notable effects on cancer cell proliferation and survival but acted at the level of angiogenesis, thus promoting tumor growth, tumor cell intravasation and dissemination. NKG2D-mediated effects on tumor initiation may represent another factor in the observed overall enhancement of tumor development. Altogether, these results may have an impact on immunotherapy approaches, which currently do not account for such NKG2D effects in cancer patients and thus could be misdirected as underlying assumptions are incomplete.

Ex vivo expansion of canine dendritic cells from CD34+ bone marrow progenitor cells.

BACKGROUND: The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. METHODS: CD34+-selected cells and CD34+-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-alpha 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. RESULTS: Two main cell populations were identified, DR++(bright)/CD14- and DR+(dim)/CD14+. Ex vivo expanded CD34+-selected cells showed increased allostimulatory activity compared to both cultured CD34+-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34+-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34+-selected bone marrow to enrich for DR++/CD14- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR+/CD14+ cells (P=0.02). CONCLUSIONS: Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34+-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.

Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study.

We evaluated 18,014 patients who underwent allogeneic bone marrow transplantation (BMT) at 235 centers worldwide to examine the incidence of and risk factors for posttransplant lymphoproliferative disorders (PTLD). PTLD developed in 78 recipients, with 64 cases occurring less than 1 year after transplantation. The cumulative incidence of PTLD was 1.0% +/- 0.3% at 10 years. Incidence was highest 1 to 5 months posttransplant (120 cases/10,000 patients/yr) followed by a steep decline to less than 5/10,000/yr among >/=1-year survivors. In multivariate analyses, risk of early-onset PTLD (<1 year) was strongly associated (P <.0001) with unrelated or human leukocyte antigen (HLA) mismatched related donor (relative risk [RR] = 4.1), T-cell depletion of donor marrow (RR = 12.7), and use of antithymocyte globulin (RR = 6.4) or anti-CD3 monoclonal antibody (RR = 43.2) for prophylaxis or treatment of acute graft-versus-host disease (GVHD). There was a weaker association with the occurrence of acute GVHD grades II to IV (RR = 1.9, P =.02) and with conditioning regimens that included radiation (RR = 2.9, P =.02). Methods of T-cell depletion that selectively targeted T cells or T plus natural killer (NK) cells were associated with markedly higher risks of PTLD than methods that removed both T and B cells, such as the CAMPATH-1 monoclonal antibody or elutriation (P =.009). The only risk factor identified for late-onset PTLD was extensive chronic GVHD (RR = 4.0, P =.01). Rates of PTLD among patients with 2 or >/=3 major risk factors were 8.0% +/- 2.9% and 22% +/- 17.9%, respectively. We conclude that factors associated with altered immunity and T-cell regulatory mechanisms are predictors of both early- and late-onset PTLD.

Iron overload in bone marrow transplant recipients.

We examined the degree of hepatic iron overload in patients receiving marrow transplant for hematologic malignancy and evaluated a new method of morphometric analysis of marrow iron content as a means of estimating hepatic iron stores in these patients. The iron content of marrow and liver specimens from 10 consecutive patients who died between 50 and 100 days after transplant was determined by spectrophotometry. Their mean age was 34.9 years (range 10-59). The mean time to death from disease onset was 2.2 years (0.5-8.7). Patients had received 30.2 +/- 17.4 units of red cells during the transplant period and 47.6 +/- 25.9 red cell units from diagnosis to death. The median hepatic iron concentration (HIC) was 4307 microg/g dry weight (range 1832-13120; normal 530-900) and the median hepatic iron index (HIC (micromol/g dry weight/age (years)) was 3.85 (0.76-8.14). The median biochemical marrow iron content was 1999 microg/g dry weight (range 932-3942). Morphometric analysis of the marrow iron content was performed on digital photomicrographs of a single Prussian blue-stained section of marrow. Strong correlations were demonstrated between morphometric marrow iron content and (1) biochemical marrow iron content (r = 0.8, P= 0.006) and (2) biochemical hepatic iron index (r = 0.82, P = 0.004). We conclude that marrow transplant recipients have a high liver iron content at 50-100 days post transplant with the hepatic iron index in the hereditary hemochromatosis range. Computerized morphometric marrow iron determination is a readily available means of estimating hepatic iron stores in these patients.

The effect of prophylactic fluconazole on the clinical spectrum of fungal diseases in bone marrow transplant recipients with special attention to hepatic candidiasis. An autopsy study of 355 patients.

We reviewed 355 autopsies performed between 1990 and 1994 at a major marrow transplant center to determine whether fluconazole prophylaxis prevented visceral fungal infection. Fluconazole prophylaxis was defined by a minimum of 5 prophylactic doses. Fungal infection (any site) was found in 40% of patients transplanted and autopsied at the center. Overall, the proportion of autopsies with any fungal infection was not different for those patients receiving no fluconazole prophylaxis versus those with prophylactic fluconazole. With fluconazole prophylaxis, candidal infections were less frequent, decreasing from 27% to 8%, while Aspergillus infections were more frequent, increasing from 18% to 29%. No increase in deaths related to non-albicans Candida infections was seen. Of the 329 patients with livers examined, hepatic infection caused by Candida species was significantly less common in patients who had received fluconazole. Fungal liver infection was found in 31 patients (9%), 16% of those who were not treated with fluconazole and 3% of those who were treated with fluconazole. Since patients with candidal infections died earlier after marrow transplant than patients with mold infections, we speculate that a longer length of survival may dispose toward acquisition of mold infections. Fluconazole prophylaxis in this cohort of marrow transplant patients undergoing autopsy resulted in a significant reduction in infection caused by Candida species and an increase in mold infections.

Multidimensional flow cytometry of marrow can differentiate leukemic from normal lymphoblasts and myeloblasts after chemotherapy and bone marrow transplantation.

Serial bone marrow aspirates from patients previously given a diagnosis of acute lymphoblastic leukemia (ALL) who had undergone chemotherapy, bone marrow transplantation (BMT), or both were analyzed by multidimensional flow cytometry (MDF) to detect residual disease (lower limit of detection 0.3%). Correlation between the results of morphologic examination and MDF showed concordant results on 100 of 118 specimens. The MDF-positive, morphologic examination-negative specimens were positive by cytogenetic examination or were obtained from patients in whom the ALL eventually relapsed. Similar correlations between MDF and the results of cytogenetic examination were obtained. Leukemic cells were detected in 29 of 62 patients before BMT and 12 of 52 after BMT Normal regenerating lymphoblasts were identified and quantified by MDF in patients without detectable leukemic lymphoblasts. Patients with leukemic lymphoblasts found by MDF in specimens obtained immediately before BMT were 3.28 times more likely to experience relapse after BMT compared with MDF-negative patients, even when leukemic lymphoblasts were undetectable by histopathologic examination, cytogenetic examination, or both. All patients who had undergone BMT with leukemic lymphoblasts found by MDF, with or without morphologic or cytogenetic confirmation, experienced relapse according to conventional criteria within 42 days of the MDF analysis. The detection of residual disease before overt relapse may provide information for early intervention, while definitive recognition of normal recovering blasts may prevent unnecessary treatment.

Acute pancreatitis in marrow transplant patients: prevalence at autopsy and risk factor analysis.

Pancreatitis has been described as an infrequent complication of marrow transplantation. This study investigated the prevalence of pancreatitis at autopsy in marrow transplant patients and determined risk factors for its development. We reviewed consecutive autopsy reports from 1991 to 1993. Medical records and laboratory reports were reviewed for analysis of clinical variables. Autopsy findings and clinical variables were correlated with the autopsy diagnosis of pancreatitis. Pancreatitis was found in 51 of 184 (28%) patients at autopsy. Of those with pancreatitis, 35% had abdominal pain, 10% had measurements of serum pancreatic enzymes, and 20% had abdominal imaging studies in the week prior to death. By univariable analysis, risk factors associated with development of pancreatitis included clinical grades 3 and 4 GVHD, GVHD at autopsy, liver GVHD at autopsy, major infection at autopsy, and increasing days of survival. By multivariable analysis, independent risk factors for its development included any GVHD at autopsy, increasing length of survival after transplantation, and major infection at autopsy. We conclude that pancreatitis is a common but often subclinical complication of marrow transplantation. Its development may be associated with a high prevalence of biliary sludge and prolonged treatment of GVHD with cyclosporine and prednisone.

Does graft-versus-host disease attack epithelial stem cells?

Graft-versus-host disease (GvHD) is the opposite of graft rejection in that a transplant containing donor lymphocytes attacks the host's skin, liver and gut. This disease can usefully also attack host tumor cells. There is a peculiar distribution of normal tissue targets in epithelial stem cell sites, suggesting that GvHD may be the abnormal counterpart of a physiological growth control system. Efforts to understand how the graft-versus-tumor effect could be therapeutically separated from GvHD require further understanding of the mechanisms involved in GvHD.

Fungal liver infection in marrow transplant recipients: prevalence at autopsy, predisposing factors, and clinical features.

To determine the prevalence of fungal liver infection at autopsy in marrow transplant recipients, we reviewed autopsy results for the period 1980-1989. Cases were compared to randomly chosen autopsied controls without fungal infection. Fungal liver infection was found in 67 (9%) of 731 patients. Fungal cultures of liver lesions were positive for 34 of 67 patients, most of whom had been culture-positive for the same fungal species (largely Candida) during life. Multivariate analysis revealed that independent predictors of fungal liver infection were deep fungal infection after transplantation (RR, 35), colonization or superficial infection after transplantation (RR, 13), and severe liver dysfunction caused by veno-occlusive disease of the liver and/or graft-versus-host disease (RR, 7). Clinical and laboratory findings during the last month of life revealed no differences between cases and controls. Liver imaging studies performed during the last 15 days of life had a sensitivity of only 18% for detecting fungal liver lesions.

CD8+ activated T lymphocytes produce an in vitro skin graft-versus-host reaction in an organotypic skin culture model.

We adapted organotypic skin cultures to the dog as a model for skin graft-versus-host reaction (GVHR) to explore the relative roles of T cells and cytokines. To produce GVHR, activated lymphocytes from bulk mixed leukocyte cultures (bMLC) from 2 dog leukocyte antigen-unrelated dogs were injected into organotypic skin cultures. Additionally, effects of separated CD4+ and CD8+ activated lymphocytes as well as cytokine-containing (TNF alpha and IFN gamma) supernatants from bMLC were studied. Noninjected cultures as well as cultures injected with autologous (cultured and uncultured) lymphocytes and allogeneic uncultured lymphocytes served as controls. The unseparated bMLC-activated cell populations induced histopathological changes similar to in vivo skin GVHR along with very prominent class II antigen expression on keratinocytes. Separated CD8+ cells were directly involved in tissue damage by producing necrosis of epidermis at the site of injection, with less class II antigen expression on keratinocytes, and predominantly distributed intraepidermally. CD4+ cells, located mostly in the dermal regions, induced prominent class II antigen expression on keratinocytes, but no histological changes of GVHR. High levels of TNF alpha and IFN gamma were found in the supernatant of allogeneic bMLC cultures, although when the supernatant was injected into the organotypic skin cultures, keratinocytes failed to express surface class II antigen and histologically did not show changes of skin GVHR. This study demonstrated that organotypic skin cultures can serve as a model for studying the etiology of GVHR, and indicated direct involvement of CD8+ cells in tissue damage.

Parafollicular bulges, but not hair bulb keratinocytes, are attacked in graft-versus-host disease of human skin.

Graft-versus-host disease (GVHD) has been shown to preferentially attack epithelial stem cell regions in the rete ridge of epidermis, the parafollicular bulge stem cells of the hair follicle, the limbus of the eye, the basal cells of filiform papilla of the tongue and the stem cell regions of gut crypts. The parafollicular bulge, but not the papillary bulb tip, has recently been shown to be the site of follicular stem cells. The hair bulb at the tip of the papilla contains progeny of the parafollicular bulge stem cells. These progeny in turn are a proliferative population which gives rise to the hair. We asked the question: Does GVHD preferentially attack the younger, less differentiated population of cells in the parafollicular bulge but spare the also cycling, later generation of cells in the papillary bulb? We had 22 evaluable cases from our prior study of 38 biopsies of GVHD patients: 19 showed preferential bulge involvement, 2 showed both involved, 1 showed neither involved and none showed bulb positive and bulge negative. The data support a strong preference for GVHD to attack the less differentiated of two cell populations both of which are cycling, thereby supporting the hypothesis that preferred targets are related to the stem cell populations in epithelial and not just all cycling or proliferating populations.

Gamma-irradiation of pretransplant blood transfusions from unrelated donors prevents sensitization to minor histocompatibility antigens on dog leukocyte antigen-identical canine marrow grafts.

Pretransplant blood transfusions from a dog leukocyte antigen (DLA)-identical canine littermate marrow donor will sensitize the recipient to non-DLA-linked polymorphic minor histocompatibility antigens, which uniformly results in graft rejection. We observed previously that 2000 cGy gamma-irradiation of marrow donor blood transfusions prevented this sensitization and subsequent marrow graft rejection. The purpose of the present study was to determine whether treatment of unrelated blood transfusions with gamma-irradiation would also prevent sensitization. Conceivably, sensitization to minor histocompatibility antigens might be more efficient or potent and thus more difficult to prevent when those antigens are seen in the context of disparity for DLA antigens. Furthermore, this model, in which sensitization to DLA-identical littermate marrow is caused by unrelated blood transfusions, is directly relevant to the clinical circumstances of human marrow transplantation. We assessed sensitization caused by unrelated blood transfusions by monitoring graft outcome in recipients transplanted with DLA-identical littermate marrow after conditioning with 920 cGy total body irradiation. Two thousand cGy gamma-irradiation of unrelated blood transfusions significantly reduced the incidence of transfusion-induced sensitization of recipients. There was successful marrow engraftment in 15 of 16 (94%, P < 0.003) of these animals in contrast to the previous study in which only 7 of 16 (44%) animals engrafted after they were transfused with unmodified blood on the same schedule. These results suggest that blood transfusions for use in humans, especially for patients with aplastic anemia, should be gamma-irradiated in order to reduce the incidence of marrow graft rejection caused by sensitization to minor histocompatibility antigens.

Positive identification of enterocytes by keratin antibody staining of sloughed intestinal tissue in severe GVHD.

Nine marrow allograft recipients who developed GVHD of the gastrointestinal tract accompanied by the passage of ropey necrotic material per rectum were studied. The material, resembling necrotic intestinal mucosa, was evaluated for the presence of epithelial cells using monoclonal antibodies for keratin and macrophages. Two keratins (AE1/AE3 and 34 beta E12) were detected within cells in all cases while macrophages were found in all but one case. In three cases, some cells were positive for both antibodies suggesting the presence of phagocytosed keratin in macrophage cytoplasm. Search for organisms with Gram and methenamine silver stains showed bacteria in six cases and fungus in one. Some cases of severe GVHD will be associated with the passage of ropey tan material grossly resembling sloughed mucosal tissue, but microscopically consisting largely of fibrin clots. The presence of free intestinal epithelial cells in this material is confirmed by the present study.