[Show abstract][Hide abstract] ABSTRACT: Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.
Modern Pathology 05/2012; 25(9):1246-57. DOI:10.1038/modpathol.2012.74 · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main objective of this 1.5-year prospective study was to evaluate the value of T-SPOT®.TB as compared to the tuberculin skin test (TST) for the first-line assessment of uveitis.
A total of 108 consecutive non-immunocompromised patients with acute or chronic uveitis, including 92/108 (85.2%) with previous BCG vaccination, underwent a general examination, a TST, and a T-SPOT.TB test (Oxford Immunotec; Oxford, UK), blood and serological tests, and chest imaging. Concordance between tests was assessed using kappa coefficients. The performance of binary classification tests was evaluated with sensitivity, specificity, and likelihood ratios.
The results of the TST and the T-SPOT.TB test differed significantly (55.5% versus 29.6% positivity, P < 0.001), with a low concordance between the two tests (κ = 0.362, P = 0.001). The sensitivity of the TST was 100% (diagnosis of suspected tuberculous uveitis included a positive TST), but its specificity was only 53.3%. The sensitivity and the specificity of the T-SPOT.TB test were 94.4% and 83.3%, respectively. The positive and negative likelihood ratios of the T-SPOT.TB test were 5.67 and 0.07, respectively.
In uveitis patients with frequent previous BCG vaccination, the T-SPOT.TB test is more specific than the TST and therefore allows more accurate selection of patients requiring extensive investigations to rule out TB diagnosis.
Annals of Medicine 10/2010; 42(7):546-54. DOI:10.3109/07853890.2010.518155 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In chronic hepatitis C (CHC), HCV-specific T-cell responses are often dysfunctionnal. In vitro data point out that regulatory T cells (Treg) are able to suppress HCV-specific lymphocyte proliferation and cytokine secretion but their implication in this pathology is still debated.
Three complementary approaches were performed to investigate phenotype, frequency or localization of intra-hepatic Treg in treatment naïve CHC patients. Double immunohistochemical analysis was performed in 20 formalin-fixed biopsies with CD8/FoxP3 and CD4/FoxP3 antibodies. Cellular markers and cytokines were investigated by quantitative RT-PCR in 27 additional frozen biopsies. Eight other fresh liver biopsies were selected for complementary analysis of immunophenotyping and frequency of intra-hepatic Treg.
Immunohistochemical analyses showed the presence of intra-hepatic CD4(+)FoxP3(+)T cells while CD8(+)FoxP3(+)T cells were very scarce. CD4(+)FoxP3(+)T cells were located in necro-inflammatory areas in contact with CD8(+)T cells, suggesting that Treg-mediated inhibition of CD8(+)T cell proliferation may occur by cell-cell contact. RT-PCR analyses showed strong correlations between CD8, FoxP3, and IL-10 with emergence of four distinct gene clusters, CD8-FoxP3, CD8-IL-10, TGF-beta-IL-10, and TNF-alpha-TGF-beta. No correlation was found between serum viral load and any immune markers. Interestingly, the FoxP3(+)/CD8(+) cells ratio significantly decreased in severe fibrosis (F>3) due to the dramatic decline of FoxP3 cells.
This study provides new insights into the histological localization of Treg within HCV-infected liver, with a special accumulation of CD4(+)FoxP3(+)Treg cells in necro-inflammatory areas, in contact with CD8(+)T cells. Our results suggest a link between Treg, CD8, and IL-10 which altogether could balance immune responses against the virus to avoid immunopathogenesis.
Journal of Hepatology 07/2010; 53(1):25-35. DOI:10.1016/j.jhep.2010.02.024 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After HLA-identical hematopoietic stem cell transplantation, minor histocompatibility (mH) antigen alloreactivity plays a dominant role in the development of graft-versus-host disease (GVHD) and graft versus leukemia (GVL).
We have analyzed the mH alloreactivity (enzyme-linked immunospot [ELISpot] for interferon-gamma[IFN-gamma] assay) from 24 donor/recipient pairs over a period of 2 years of follow-up and correlated such alloreactivity with the development of GVHD or absence of relapse. Circulating specific T cells anti-mH with multimer HLA-peptides were also studied.
We show by ELISpot IFN-gamma assay that alloreactivity during the first 3 months from donor versus recipient or donor versus mismatched identified mH antigens is associated with acute GVHD and GVL effect. In addition, we demonstrate that the donor-versus-recipient reactivity observed after the third month is highly associated with chronic GVHD and GVL (p = 0.0007). Finally, we show by multimer HLA-peptide assay that mH epitope-specific T cells present after 3 months are statistically related to the GVL effect.
Our results provide a robust method to monitor mH antigen graft-versus-host reaction and suggest that current identified mH have predictive value on GVHD and GVL.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the T-cell receptor (TCR) repertoire by flow cytometry proved to be relevant for investigating T-cell diversity and detecting reactive cells in blood samples. We used this approach to characterize non-malignant T-lymphocytes in lymph nodes and give insights into their origin. The TCR repertoire of CD4+ and CD8+ T-cells from 81 lymph nodes was analyzed with a four-color flow cytometer using a wide panel of 25 anti-Vbeta monoclonal antibodies. Flow cytometry proved to be a useful and informative technique. We demonstrated a diversified TCR-Vbeta repertoire, and only low level expansions, in 53% of the samples. They involved nearly all Vbeta families, were more frequent in the CD8+ subset of older patients, but were not related to pathology. No evidence could be demonstrated in favor of stimulation by common antigens. Interestingly, the TCR-Vbeta repertoire proved to be very similar in lymph nodes and blood samples. Our results argue that in the cases studied, lymph node enlargement is mainly due to an increased homing of circulating T-cells. They also provide reference values for expression of 25 TCR-Vbeta in lymph nodes, which could serve as a basis for further applications in diagnosis of T-cell lymphoproliferative disorders.
Cytometry Part A 09/2009; 75(9):743-51. DOI:10.1002/cyto.a.20767 · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood.
Blood and intrahepatic CD56(+) NK cell subsets (expressing NKG2A, CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies.
A significant reduction in NK cell frequency and a quantitative imbalance between CD56(bright) and CD56(dim) subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56(+)CD3(-) cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56(+)CD3(-) cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3(-)CD56(bright)NKG2A(+) and CD3(-)CD56(dim)NKG2A(+) cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients.
HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3(-)CD56(dim)NKG2A(+) and CD3(-)CD56(bright)NKG2A(+) cells, may be of interest for disease monitoring.
Journal of Hepatology 07/2009; 51(3):458-67. DOI:10.1016/j.jhep.2009.05.030 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary C3 deficiency, a rare autosomal inherited disease (OMIM 120700), was identified in a 2-year-old male suffering from recurrent pyogenic infections from early infancy with undetectable total complement hemolytic activity (CH50) and C3 values. The nonconsanguineous parents and the two patients' two siblings had 50% normal serum C3 concentration. The molecular abnormality associated a paternal allele coding C3 with the missense mutation p.Ser(550)Pro and an apparently null maternal allele, with production of a defective protein that could no longer be secreted. Vaccination of the child did not induce a long-term Ab response. Accordingly, switched memory IgD(-)CD27(+) B cells were barely detected, amounting to only 2.3% of peripheral blood CD19(+) cells. Cells were significantly defective in stimulating alloreactive responses. The in vitro development of immature dendritic cells and their maturation capacity were greatly impaired, with decreased CD1a expression and IL-12p70 secretion ability. These cells were unable to induce autologous B cell proliferation and Ig secretion in the presence of CD40L and C3. Finally, the regulatory T cell development ability of CD4(+) T cells after CD3 and CD46 activation in the presence of IL-2 was significantly impaired. Thus, the association of important functional defects of dendritic cells, acquisition of B cell memory, and regulatory T cells with human C3 deficiency strongly supports a major role for C3 in bridging innate and adaptive immunity in humans.
The Journal of Immunology 11/2008; 181(7):5158-66. DOI:10.4049/jimmunol.181.7.5158 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD8(+) T cells represent a sizable component of the liver inflammatory infiltrate in chronic hepatitis C and are thought to contribute to immune-mediated tissue injury. Because chronic stimulation may promote the expression by CD8(+) T cells of distinct human leukocyte antigen class I-specific natural killer cell receptors (NKRs) susceptible to both inhibiting effector functions and promoting cell survival, we examined the distribution and characteristics of CD8(+) T cells with such receptors in chronic hepatitis C patients. NKR CD8(+) T cells were detectable in liver and peripheral blood from hepatitis C virus (HCV)-infected patients but were not major subsets. However, the frequency of NKG2A(+) CD8(+) in the liver and in a lesser extent in the peripheral blood was positively correlated to histological activity in HCV-infected patients. No such correlation was found with KIR(+) T cells in liver in HCV-infected patients and with the both NKR CD8(+) T cells in hepatitis B virus (HBV) infected patients. Circulating CD8(+) T cells expressing KIRs exhibited phenotypic features of memory T cells with exacerbated expression of the senescence marker CD57 in patients. NKG2A(+)CD8(+) T cells were committed T cells that appeared less differentiated than KIR(+)CD8(+) T cells. In HCV-infected patients, their content in perforin was low and similar to that observed in NKG2A(-)CD8(+) T cells; this scenario was not observed in healthy subjects and HBV-infected patients. Both NKG2A and KIRs could inhibit the response of HCV-specific CD8(+) T cells ex vivo. CONCLUSION: These results support the concept that an accumulation in the liver parenchyma of NKR(+)CD8(+) T cells that have functional alterations could be responsible for liver lesions. They provide novel insights into the complexity of liver-infiltrating CD8(+) T cells in chronic hepatitis C and reveal that distinct subsets of antigen-experienced CD8(+) T cells are differentially sensitive to the pervasive influence of HCV.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of liver cell injury during chronic hepatitis C (CHC) is poorly understood. The cellular immune response is thought to play a key role in both inhibition of viral replication and liver pathology. However, little is currently known about which lymphocyte populations and which immune effectors contribute to or control liver damage. We investigated a panel of 15 phenotypic and functional markers of intrahepatic T-lymphocyte subsets irrespective of their antigen specificity in 48 hepatitis C virus (HCV)-infected patients and 8 healthy control subjects. Lymphocyte characteristics were evaluated from liver biopsy specimens both at gene expression level by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and by immunochemistry, in relation with the degree of liver injury and with intrahepatic HCV-RNA levels. As compared with controls, we found major changes in T-lymphocyte subsets in HCV-infected patients, with a significant decrease of T-cell antigen receptor (TCR) delta and CD56 gene expression, associated with a concomitant increase of TCRalpha and CD8beta that were correlated with cytotoxic factors, proinflammatory chemokines, and chemokine receptors including peforin, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), RANTES, and CXCR3. The gene expression of CD8beta, a specific marker for conventional TCRalpha+CD8+ lymphocytes, was correlated by multivariate analysis with both alanine aminotransferase (ALT) serum levels and histologic activity index. Furthermore, CD8 staining was observed by immunochemistry in the areas of lobular and piecemeal necrosis. In contrast, no lymphocyte marker was correlated with viral load, measured both in serum and in liver. In conclusion, these results strongly suggest key roles for CD8+ T cells as effectors of liver damage during chronic HCV infection and for their inability to control viral replication.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of liver cell injury during chronic hepatitis C (CHC) is poorly understood. The cellular immune response is thought to play a key role in both inhibition of viral replication and liver pathology. However, little is currently known about which lymphocyte populations and which immune effectors contribute to or control liver damage. We investigated a panel of 15 phenotypic and functional markers of intrahepatic T-lymphocyte subsets irrespective of their antigen specificity in 48 hepatitis C virus (HCV)-infected patients and 8 healthy control subjects. Lymphocyte characteristics were evaluated from liver biopsy specimens both at gene expression level by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and by immunochemistry, in relation with the degree of liver injury and with intrahepatic HCV-RNA levels. As compared with controls, we found major changes in T-lymphocyte subsets in HCV-infected patients, with a significant decrease of T-cell antigen receptor (TCR) δ and CD56 gene expression, associated with a concomitant increase of TCRα and CD8β that were correlated with cytotoxic factors, proinflammatory chemokines, and chemokine receptors including peforin, interferon gamma (IFN-γ), tumor necrosis factor α (TNF-α), RANTES, and CXCR3. The gene expression of CD8β, a specific marker for conventional TCRα+CD8+ lymphocytes, was correlated by multivariate analysis with both alanine aminotransferase (ALT) serum levels and histologic activity index. Furthermore, CD8 staining was observed by immunochemistry in the areas of lobular and piecemeal necrosis. In contrast, no lymphocyte marker was correlated with viral load, measured both in serum and in liver. In conclusion, these results strongly suggest key roles for CD8+ T cells as effectors of liver damage during chronic HCV infection and for their inability to control viral replication. (Hepatology 2003;38:829–841).
[Show abstract][Hide abstract] ABSTRACT: During viral chronic hepatitis C (CHC), the intra-hepatic lymphocyte infiltrate is mainly composed of T lymphocytes expressing alphabeta T-cell receptors (TCR). Since little is known about the TCRalphabeta diversity of intra-hepatic T lymphocytes (IHL), we evaluated the IHL repertoire from CHC patients (n=8) as compared to healthy subjects (n=4), total peripheral blood mononuclear cells, and purified peripheral and intra-hepatic CD8(+) cells (n=2).
The diversity of TCRalphabeta receptors was evaluated by determining the size and the sequence of the TCRbeta chain complementarity determining region 3 (CDR3). The number of total T lymphocytes in liver was estimated by real-time quantitative reverse transcription-polymerase chain reaction of TCRalpha and CD3 epsilon transcripts.
Our results show that transcripts encoding all TCR V beta (BV) families and all TCR J beta (BJ) segments were present in healthy and CHC livers. No biased TCR repertoire, in terms of preferential BV or BJ gene use or restricted CDR3 sequence, was observed in infected livers. When corrected for equivalent numbers of T lymphocytes, BJ segments utilization and CDR3 length diversity were similar in IHL and PBMC, indicating that the TCRbeta chain diversity is comparable in both cases. In addition, TCR diversity was similar in both peripheral and intra-hepatic CD8+ T cells.
This study shows limited expansions of intra-hepatic T lymphocytes in CHC patients. The increase of T lymphocytes in infected livers correlates with diversification of TCR, arguing for the establishment of a multi-specific immune response.
Journal of Hepatology 06/2003; 38(5):651-9. DOI:10.1016/S0168-8278(03)00089-8 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In agreement with good practices for therapeutic use of human cells, quality control has to be performed to valid the process of extracorporeal photopheresis (ECP) with the Vilbert-Lourmat system. Since no protocol exists, we evaluated a technique based on the measurement of the inhibition of mitogen (PHA, Con-A, OKT3)-induced proliferation, in 164 procedures from 16 patients. Whatever the pathology, we observed a high proliferation rate in most samples, and we obtained over 90% ECP-induced inhibition in as many as 94% of the cases. Since this approach proved to be relevant regarding our objective, a protocol for the ECP process validation is proposed.
[Show abstract][Hide abstract] ABSTRACT: Intrahepatic lymphocytes are believed to be involved in the immunopathogenesis of hepatitis C virus (HCV) infection and the evolution of HCV-induced hepatitis. In the present study, we examined the three main intrahepatic lymphocyte subsets, namely CD3+CD56- conventional T lymphocytes, CD3+CD56+ natural T (NT) lymphocytes and CD3-CD56+ natural killer (NK) lymphocytes in HCV-infected patients. The proportion of each lymphocyte subset was evaluated both in liver biopsies and in samples of peripheral blood lymphocytes (PBL) by flow cytometry in 21 patients with histologically proven chronic hepatitis C. Simultaneously, alanine aminotransferase (ALT) levels, viral load and histological lesions were assessed. Neither NT nor NK populations correlated with any biochemical, viral or histological parameters. Furthermore, Valpha24+ NT lymphocytes showed no preferential enrichment in the liver of HCV-infected patients. Regarding conventional T lymphocytes, a highly significant linear correlation was found between intrahepatic CD3+CD56- T lymphocytes and the Knodell score, a numerical score for assessing histological activity and fibrosis (r = 0.715, P < 0.0001) and more specifically with the periportal necrosis parameter, which is the main lesion of chronic hepatitis C. In addition, analysis of the peripheral compartment revealed a high correlation between values of CD3+CD56- lymphocytes and both Knodell score (r = 0.624, P = 0.003) and serum ALT levels and again with periportal necrosis. The strong correlation between the proportion of peripheral CD3+CD56- conventional T lymphocytes and the severity of hepatic lesions leads us to propose that evaluation of this accessible peripheral population could be used as an indicator test for the severity of histological lesions in chronic hepatitis C. Abbreviations:
[Show abstract][Hide abstract] ABSTRACT: We present a case of documented acute hepatitis C that occurred in a health care worker who sustained a needlestick injury while caring for an individual who was infected with both hepatitis C virus (HCV) and human immunodeficiency virus (HIV). According to the findings of third-generation serological assays performed during a follow-up of >1 year, the health care worker, who was treated with interferon-alpha (during weeks 2-6) and ribavirin (during weeks 5-9), did not develop antibodies against HCV, in spite of documentation of an HCV-specific T cell response.
[Show abstract][Hide abstract] ABSTRACT: Angioneurotic edema (AE) is a rare but severe disease. Hereditary AE is the more well-known form. The acquired form is exceptional: the symptoms are the same but there are some biologic and treatment differences. We investigated the clinical and biochemical features in nine patients with acquired angioneurotic edema (AAE).
Four of the patients with type I AAE presented an accelerated metabolism of C1Inh, associated with a hematology disease. Their C4, C1q and C1Inh plasma levels were low. Four patients had type II AAE associated with an autoantibody to C1Inh. Their C1Inh plasma levels were normal or low but the functional levels were low in all four. One patient had AAE induced by oral contraceptives. The C1Inh plasma level was normal but the functional level was very low; there were no autoantibodies. Symptoms resolved when oral contraceptives were withdrawn and the C1Inh level returned to normal.
Treatment of AAE is a difficult matter. For type I AAE, it consists in treating the associated disease. For type II AAE, the treatment goal is to lower the autoantibody level. Management of these diseases requires close collaboration between clinicians and biologists.
La Presse Médicale 05/2000; 29(12):640-4. · 1.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Innate defence mechanisms play a role in acquired immune defence, which has been underestimated for a long time. Lectins, polysaccharide binding molecules, represent a very important component of antimicrobial defence; these proteins preceded antibodies in the course of evolution, and are involved in agglutination and endocytosis of many antigens. Collectins are complex proteins, combining the functions of the lectin part with the functions of a collagen-like part. Receptors for this collagen represent a link with cell-mediated immune defence; the association with certain proteases also allows one of these collectins to participate in activation of complement. Complement represents a significant component of innate immunity following trigger by activators and by participation of proteolysis fragments, such as major C3 fragments, in opsonisation and endocytosis-phagocytosis of antigens. Complement plays a significant role in the antigen processing phase of acquired immunity.
Revue Française d Allergologie et d Immunologie Clinique 01/1999; 39(4):255-262. DOI:10.1016/S0335-7457(99)80050-1 · 0.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The syndrome of acquired angioneurotic edema (AAE) is characterized by the adult onset of angioedema, the lack of evidence for inheritance of the disorder, and the frequent association of the C1-inhibitor (C1-INH) deficiency with lymphoproliferative or other malignant diseases. Recently, a new type of AAE (type II AAE) has been described. The two major biologic differences of this new syndrome compared with all other previously reported AAE cases (type I AAE) are the presence in patients' sera of both anti-C1-INH autoantibodies, often monoclonal, and a circulating low molecular weight (95 kd) C1-INH protein. From the clinical point of view, the absence of underlying lymphoproliferative disease is the hallmark of type II AAE compared with type I AAE. However, the distinction between type I and type II AAE may not be so clear-cut. We report three patients with monoclonal gammopathies and AAE for whom the initial diagnosis was type I AAE. The demonstration by ELISA of the C1-INH binding ability of their monoclonal immunoglobulins in addition to the presence of 95 kd C1-INH protein enables us to change the diagnosis to type II AAE. From the therapeutic point of view, it is crucial to detect the anti-C1-INH antibody and to analyze the C1-INH size to distinguish type I and type II AAE, especially if patients have a monoclonal gammopathy, to give the appropriate treatment (attenuated androgens vs immunosuppressive regimen, respectively) to prevent a fatal outcome.
Journal of Allergy and Clinical Immunology 05/1996; 97(4):998-1008. DOI:10.1016/S0091-6749(96)80076-5 · 11.48 Impact Factor