Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-{gamma} rearrangement analysis. To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC. To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded. In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR. These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INF{gamma} and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients' CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls. In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma.

Molecular and Functional Analysis of Peripheral Lymphocytes in Churg Strauss Syndrome Reveals Several Monoclonal Expansions of CD8+Cells with a Th1/Proinflammatory Profile

BOITA, MONICA;CIRCOSTA, Paola;GUIDA, Giuseppe;HEFFLER, Enrico Marco;ROLLA, Giovanni;CIGNETTI, Alessandro
2012

Abstract

Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-{gamma} rearrangement analysis. To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC. To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded. In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR. These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INF{gamma} and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients' CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls. In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma.
American Society of Hematology (ASH) Annual Meeting
Atlanta
8-11/12/2012
120
21
Abstract 1051
Abstract 1051
Boita M; Circosta P; Guida G; Elia AR; Heffler E; Geuna M; Tarella C; Rolla G; Cignetti A
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/141014
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