Lisbet Sviland

In situ dissection of the graft-versus-host activities of cytotoxic T cells specific for minor histocompatibility antigens.

Minor histocompatibility antigens (mHags) are immunogenic peptides from polymorphic cellular proteins that induce strong T-cell responses after human leukocyte antigen (HLA)-matched, mHag-mismatched stem-cell transplantation. mHags with broad or limited tissue expression are target antigens for graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactivities. Separation of these activities is crucial for adoptive immunotherapy of leukemia without GvH disease. Therefore, using a skin-explant assay we investigated the in situ activities of cytotoxic T lymphocytes (CTLs) specific for the ubiquitously expressed mHag H-Y and for the hematopoietic-restricted mHags HA-1 and HA-2. H-Y-specific CTLs, visualized by tetrameric HLA–mHag peptide complexes, infiltrated male skin sections within 24 hours, induced severe GvH reactions of grade III–IV and produced high levels of IFN-γ. In contrast, CTLs specific for the hematopoietic system–specific mHags HA-1 and HA-2 induced no or low GvH reactions above background and produced little or no interferon-γ, unless the skin sections were preincubated with HA-1/HA-2 synthetic peptides. These results provide the first in situ dissection of GvH effects by mHag-specific CTLs and show that ubiquitously expressed mHags are the prime targets of GvH disease.

MHC class II and ICAM-1 expression and lymphocyte subsets in transbronchial biopsies from lung transplant recipients.

The expression of MHC class II antigens and ICAM-1 and the composition of lymphocyte infiltrates have been studied in frozen sections of transbronchial biopsies from lung transplant recipients. First, biopsies obtained from patients who showed acute rejection, OB, and normal features were compared. Second, we compared first-year biopsies from patients developing OB and patients with a good clinical outcome. HLA-DR was widely expressed on epithelia and vascular endothelium. Increased vascular HLA-DP expression was found in OB biopsies. In OB patients there was a significantly increased frequency of bronchial HLA-DP and vascular HLA-DQ expression. Expression of ICAM-1 by bronchial and bronchiolar basal cells, a phenomenon not reported previously in humans, was seen in a small number of biopsies. CD8 predominant lymphocytic infiltrates were present in all groups and were increased in OB biopsies and OB patients. Increased numbers of CD4-positive cells were found in rejection and OB when compared with normal biopsies. These findings support an immunological basis for the development of OB.

Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis.

The clinical and histological criteria used to diagnose lymphadenitis caused by Mycobacterium tuberculosis complex organisms have poor specificity. Acid-fast staining and culture has low sensitivity and specificity. We report a novel method for diagnosis of tuberculosis that uses immunohistochemistry to detect the secreted mycobacterial antigen MPT64 on formalin-fixed tissue biopsies. This antigen has not been detected in non-tuberculous mycobacteria. Polymerase chain reaction (PCR) for amplification of IS6110 from DNA obtained from the biopsies was used as a gold standard. Fifty-five cases of granulomatous lymphadenitis with histologically suspected tuberculosis obtained from Norway and Tanzania were evaluated. Four known tuberculosis cases were used as positive controls, and 16 biopsies (12 foreign body granulomas and four other non-granulomatous cases) as negative controls. With immunohistochemistry, 64% (35/55) and with PCR, 60% (33/55) of granulomatous lymphadenitis cases were positive. Using PCR as the gold standard, the classical tuberculosis histology had sensitivity, specificity, positive and negative predictive values of 92, 37, 60, and 81%, respectively, and immunohistochemistry had sensitivity, specificity, positive and negative predictive values of 90, 83, 86, and 88%, respectively. The observed agreement between PCR and immunohistochemistry was 87% (κ=0.73). Immunohistochemistry with anti-MPT64 antiserum is a rapid, sensitive, and specific method for establishing an etiological diagnosis of tuberculosis in histologic specimens. Immunohistochemistry has the advantages over PCR of being robust and cheap, and it can easily be used in a routine laboratory.

Class Ii Antigen Expression By Keratinocytes And Enterocytes—an Early Feature Of Graft-versus-host-disease

HLA-DR expression by keratinocytes and enterocytes was studied in 23 patients undergoing BMT (12 autologous; 11 allogeneic). Two monoclonal antibodies were used to detect the HLA-DR antigen. Only in two patients before transplant and in one following autologous BMT was HLA-DR expressed on keratinocytes. Of 11 allogeneic recipients, 7 developed clinical GVHD, and HLA-DR-positive keratinocytes were seen in 6 of these. HLA-DR was expressed by enterocytes in 5 patients with GVHD and 4 of these also showed HLA-DR expression by keratinocytes. HLA-DR expression by keratinocytes correlated well with clinical GVHD. Expression of this antigen by enterocytes was associated with characteristic histological appearances of GVHD, even in the absence of intestinal symptoms. A combination of traditional and immunocytochemical techniques offers a sensitive and accurate method of confirming GVHD before it becomes florid.

Predicting graft-versus-host disease in HLA-identical bone marrow transplants : A comparison of T-cell frequency analysis and a human skin explant model

BACKGROUND Graft-versus-host disease (GVHD) occurring after HLA-identical sibling bone marrow transplantation (BMT) is considered to be mainly caused by minor histocompatibility antigen (mHag) disparities between the recipient and donor. In our laboratory, a human skin explant model has been successfully used to predict acute GVHD in HLA-identical sibling BMT. More recently, the frequency analysis of host-reactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp, respectively) has been shown to have potential application for predicting GVHD. In the present study, HTLp and CTLp frequency analysis and the skin explant model were directly compared for their ability to predict acute GVHD in HLA-identical sibling BMT. METHODS Host-reactive HTLp and CTLp frequencies were determined using a combined limiting dilution assay. A human skin explant model was used to detect graft-versus-host reactions in vitro. The results from the skin explant model (graft-versus-host reaction grades I-IV) and T cell frequency analysis (>/< 1:100,000) were correlated with posttransplant GVHD outcome, respectively. RESULTS The skin explant model correctly predicted GVHD outcome in 77% of cases (P=0.03). HTLp frequencies were very low in all patient/donor pairs tested. None of them exceeded 1:100,000, although 9/18 recipients developed GVHD (> or =clinical grade II) after transplant. In all patients tested, the relationship between either high (>1:100,000) or low (<1:100,000) CTLp frequency and occurrence of GVHD appeared to be random (P=1.0). CONCLUSIONS HTLp and CTLp frequency analysis did not predict the occurrence of acute GVHD after HLA-identical sibling BMT. The human skin explant model, however, remained an accurate indicator of acute GVHD and probably detects mHag disparities.

Organizing pneumonia following pulmonary transplantation and the development of obliterative bronchiolitis

Twelve patients receiving lung transplants between 1988 and 1992 who developed clinical and histological features of obliterative bronchiolitis (OB) were compared with a group of 13 patients with good stable lung function (FEV1 more than 80% of predicted). Histological features of 180 biopsies were studied from the first postoperative year in order to assess whether any were associated with the development of OB. Clinically and histologically defined pulmonary rejection occurring after the first month was more frequent in OB patients (P=0.03). Organizing pneumonia that was associated with acute rejection but not with nonviral infection was also seen more frequently in OB patients (P=0.003). When all available lung transplant recipients surviving beyond 18 months were included in analyses, organizing pneumonia in the first year was associated with an increased relative risk of developing OB of 2.26 (95% CL 1.19–4.29), and the occurrence of coexistent organizing pneumonia and pulmonary rejection gave a relative risk for OB of 6.33 (95% CL 1.61–24.94). An increased incidence of histologically defined organizing pneumonia in OB patients has not been described previously. Furthermore the coexistence of organizing pneumonia with pulmonary rejection in the first year posttransplantation is a strong predictive factor for the development of OB.

Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64

BackgroundThe aim of this study was to evaluate the diagnostic potential of immunohistochemistry using an antibody to the secreted mycobacterial antigen MPT64, in abdominal and lymph node tuberculosis.MethodsWe used formalin-fixed histologically diagnosed abdominal tuberculosis (n = 33) and cervical tuberculous lymphadenitis (n = 120) biopsies. These were investigated using a combination of Ziehl-Neelsen method, culture, immunohistochemistry with an antibody to MPT64, a specific antigen for Mycobacterium tuberculosis complex organisms. Abdominal and cervical lymph node biopsies from non-mycobacterial diseases (n = 50) were similarly tested as negative controls. Immunohistochemistry with commercially available anti-BCG and nested PCR for IS6110 were done for comparison. Nested PCR was positive in 86.3% cases and the results of all the tests were compared using nested PCR as the gold standard.ResultsIn lymph node biopsies, immunohistochemistry with anti-MPT64 was positive in 96 (80%) cases and 4 (12.5%) controls and with anti-BCG 92 (76.6%), and 9 (28%) respectively. The results for cases and controls in abdominal biopsies were 25 (75.7%) and 2 (11.1%) for anti-MPT64 and 25 (75.7%) and 4 (22%) for anti-BCG. The overall sensitivity, specificity, positive and negative predictive values of immunohistochemistry with anti-MPT64 was 92%, 97%, 98%, and 85%, respectively while the corresponding values for anti-BCG were 88%, 85%, 92%, and 78%.ConclusionImmunohistochemistry using anti-MPT64 is a simple and sensitive technique for establishing an early and specific diagnosis of M. tuberculosis infection and one that can easily be incorporated into routine histopathology laboratories.

Heat shock protein 70: correlation of expression with degree of graft-versus-host response and clinical graft-versus-host disease.

Background. The heat shock proteins are increasingly becoming associated with immunopathologic phenomena, being induced in response to inflammation. They are highly immunogenic and are postulated as playing a role in both innate and adaptive immunity. Their proposed role in peptide binding and antigen presentation could suggest a potential role in the alloreactive process that leads to graft-versus-host disease (GVHD) after bone marrow transplantation. Methods. In this study we examined the expression of the widely studied heat shock protein 70 (hsp70) in an in vitro–generated graft-versus-host reaction in human skin, using streptavidin biotin immunohistochemistry and laser scanning confocal microscopy. Results. Hsp70 expression was correlated with high graft-versus-host responses (P <0.001) and was confirmed using laser scanning confocal microscopy. Increased expression of hsp70 was further defined due to increases in the inducible form of hsp70. Expression of inducible hsp70 was predictive of both clinical acute GVHD (P =0.001) and incidence of chronic GVHD (P <0.001). Conclusions. This investigation has demonstrated for the first time the expression of hsp70 in a human model of GVHD, suggesting involvement in the pathogenesis of the disease and providing the basis for further investigation. Increased expression of inducible hsp70 in the model could provide a biologic marker for the prediction of clinical acute and chronic GVHD.

Histological features of skin and rectal biopsy specimens after autologous and allogeneic bone marrow transplantation.

The histological appearances of skin and rectal biopsy specimens were studied in 31 bone marrow transplant recipients (13 autologous, 18 allogeneic) before transplant, at 28 days, at six months, and as soon as graft versus host disease (GVHD) was clinically suspected. Grades I and II skin changes were commonly seen in patients before transplant and in the autologous group after transplant, as well as in most of the allogeneic recipients with suspected GVHD. Epidermal lymphocytic infiltration was seen only in allogeneic recipients, with clinical GVHD following transplant, but this was not a consistent finding and no other histological features were seen which would distinguish early GVHD from changes caused by cytotoxic agents. Rectal biopsy specimens, however, were normal in patients before transplant and in autologous recipients at 28 days; single cell necrosis of crypt cells was seen only in six of 13 allogeneic recipients studied after transplant with clinical skin GVHD but no gastrointestinal symptoms. Skin changes greater than I and II are required for the histological diagnosis of GVHD. Rectal changes are more specific and may be present despite a lack of intestinal symptoms.

A human skin explant model for predicting graft-versus-host disease following bone marrow transplantation.

Graft-versus-host disease (GVHD) is the most serious complication following bone marrow transplantation, with an incidence of 40-60%. The disease can be fatal in 50% of cases, even in patients receiving marrow from an HLA identical sibling. Several assays have been developed to try to predict the development of GVHD, including mixed lymphocyte culture reaction, cytotoxic T lymphocyte precursor, and helper T lymphocyte precursor frequency assays. This review describes an in vitro skin explant model which has been used since 1988 for both predicting acute GVHD in HLA identical sibling bone marrow transplantation and studying the pathophysiology of the disease. The model involves sensitising donor lymphocytes in vitro in a primary mixed lymphocyte reaction and then evaluating the secondary response on patient skin biopsies by grading the graft-versus-host reactivity (grades I-IV) histopathologically. From analysis of collective data the model is a clear predictor of GVHD and superior to the other assays widely used, with a correlation of 82% with clinical outcome. The skin explant model allows the investigator to study the pathogenesis of GVHD. The cytokines TNF alpha and IFN gamma are shown to be important mediators of cellular damage in graft-versus-host reactions. Recent work has also involved using the model to study the alloreactivity of cord blood. The model is currently being assessed in several bone marrow transplantation centres in Europe on different patient groups including those who receive marrow from haploidentical and matched unrelated donors.