METHODS Clonalities of Î±Î² T cell receptor bearing the complementarity-determining region 3 in portal tracts isolated from liver biopsy sections of 30 patients with a laser capture microdissection technique were studied by size spectratyping. CDR3 profiles of liver-infiltrating lymphocytes (LIL) were also compared with those circulating in the blood. The representative results of Î±Î² TCR by CDR3 were also obtained from liver tissues and peripheral blood lymphocytes (PBL) of 21 chronically HCV-infected patients without MC and from PBL of 30 healthy subjects.
RESULTS LIL were highly restricted with evidence of TCR Î±Î² clonotypic expansions in 23 of 30 (77%) and in 15 of 30 (50%) MC and non-MC patients, respectively. The blood compartment contained TCR Î±Î² expanded clones in 19 (63%) MC and in 12 (57%) non-MC patients. Though the TCR repertoire profiles between the two compartments were generally distinct, in almost 30% of the patients identical T cell subsets were revealed in both liver and blood. The occurrence of LIL clonalities was detected irrespective of the degree of liver damage or circulating viral load, whereas it positively correlated with higher levels of intrahepatic HCV RNA. The removal of HCV RNA was associated with a progressive decline of baseline T cell clonalities.
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CONCLUSION These data suggest that HCV antigenic pressure is likely responsible for the T cell repertoire skewing either in patients with or without MC.
Key words: Hepatitis C virus, Mixed Cryoglobulinemia, T Cell Repertoire, Laser Capture Microdissection
Persistence of a virus is evidence of its ability to escape host's immune surveillance through the exploitation of several strategies, including integration into the host genome, silencing of viral gene expression, inhibition of antigen processing and presentation,[3-4] synthesis of proteins homologous to known immune regulatory molecules, mutations that either inhibit viral responsiveness to antiviral cytokines or preclude recognition by neutralizing antibodies or else modify residues that are critical for recognition by the major histocompatibility complex or T cell receptor (TCR).
Hepatitis C virus (HCV) infection is characterized by a striking tendency to become chronic in a high proportion of patients. A 60 to 80% range of persistent infection has been documented by detection of HCV RNA. Infection with HCV results in liver disease of variable severity, which is thought to arise from an immune mediated reaction. This is because the virus is not cytopathic, and histopathology of HCV-infected liver frequently shows a large infiltration of inflammatory cells containing virus-specific HLA class I-restricted cytotoxic T lymphocytes (CTL). Some have argued that HCV-specific CTL are preferentially sequestered in the liver, where they can cause persistent hepatic damage without complete virus eradication.
Histopathology of HCV-infected liver is characterized by varying degrees of portal tract and parenchymal infiltration of T lymphocytes. The immune response is initiated following interaction between the TCR on T lymphocytes and antigenic peptides associated with major histocompatibility molecules on the antigen-presenting cells. TCR consists of four polypeptides (Î±, Î², Î³, Î´) that form two types of heterodimers (Î±Î² and Î³Î´). Each polypeptide has variable (V), joining (J) and constant (C) regions and Î² and Î³ chains also have diversity (D) regions. TCR diversity is generated by the rearrangement of V, D, J , and C regions. The random insertion of non-germline-encoded nucleotides at the junctions of these rearranged segments provides additional diversity and is the main site of antigen recognition.
Restrictions of T cell immune response as evidenced by oligoclonal T-cell expansions are preferentially found in the memory T cell population of healthy older individuals and are possibly the result of the increasing number of antigens encountered during life.
The size and diversity of HCV-specific T-cell populations in the liver and their relevance for the outcome of HCV infection are still unknown. Whether unsuccessful immune response is targeted against fewer antigens or consists of smaller effector T cell populations, is one of the major questions being currently investigated. HCV mutations involving a TCR contact residue significantly diminish T cell recognition and fail to effectively prime naÃ¯ve T cells.
Studies in chimpanzees and humans infected with HCV have shown that broadly reactive HCV-specific T cell responses, established early after infection, are promptly associated with clearance of the virus. On the contrary, diversity of clonal TCR usage is considered a factor in the development of escape mutations and correlates with a poor response to Î±-interferon therapy.
HCV infection can be detected in mixed cryoglobulinemia (MC), a chronic immune complex-mediated disease with underlying skewing of B-cell repertoire.[22-23] Morphologically, MC is characterized by bone marrow and liver multifocal infiltrates of monoclonal B cells.[21, 23] The liver, indeed, is considered an "ectopic" lymphoid organ, in that B cells bearing antigen-specific receptors are stimulated to proliferate and differentiate. Intrahepatic B cell clonotypes contribute to the formation of intraportal lymphoid nodules. B cell repertoire in MC is rather restricted and the occurrence of B cell clonal expansions profoundly influences the clinical expression of HCV-infection.[23, 26] B-cell clonal expansions, which mainly involve rheumatoid factor (RF)-secreting cells, represent the cogent molecular mechanism underlying production of cryoprecipitating immune complexes.
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To assess whether a limited TCR diversity exists in MC patients with HCV infection, a comprehensive analysis of TCR Î±Î² repertoire of LIL and peripheral blood lymphocytes (PBL) has been performed. Their changes following antiviral therapy with pegylated interferon-Î± (Peg-IFN-Î±) and ribavirin (RBV) have also been investigated.
MATERIALS AND METHODS
Fifty-one patients were enrolled in this study. Thirty with MC and 21 without. All patients were recruited from the cohort of patients receiving Peg-IFN-Î± and RBV combination therapy at our Institution after providing their written informed consent. MC diagnosis was based on the following criteria: a) detection of serum cryoglobulins as described previously; b) clinical symptoms comprising the triad purpura-weakness-arthralgia. All patients were anti-HCV and HCV RNA positive. Other potential causes of hepatitis (i.e., alcoholic liver disease, toxic or metabolic etiology, chronic hepatitis B, autoimmune hepatitis) were ruled out. None of the patients had been previously treated. Liver biopsy specimens were obtained percutaneously from all patients. Histological activity and fibrosis grading were defined according to Ishak et al. Eighteen and 15 patients completed 12 months of Peg-IFN-Î± plus RBV combination treatment in the MC and non-MC groups respectively. Patients were treated with Peg-IFN-Î± 2b at a dose of 1.5 Î¼g/Kg or Peg-IFN-Î± 2a at a dose of 180 Î¼g once a week and RBV 800-1200 mg/day for 48 weeks. Patients were reassessed 6 months after discontinuation of treatment to determine whether they had achieved a sustained virologic response. Complete response of cryoglobulinemic patients was defined as previously established, namely reduction of the cryocrit levels to less than 75% of the initial value, associated with disappearance of clinical symptoms and signs.
Isolation of Peripheral Blood Lymphocytes (PBL)
PBL from patients and control subjects were isolated by Ficoll/Hypaque (Pharmacia, Freiburg, Germany) density gradient centrifugation.
Measurement of Intrahepatic HCV RNA
Percutaneous liver biopsies were performed as part of the diagnostic evaluation. A tissue portion (~ 2mg) was used for HCV RNA measurement, whereas the remaining part was put in RNase and DNase-free microtube and immediately frozen in liquid nitrogen until sectioning, as described elsewhere.
HCV RNA in tissue was measured by signal amplification with a branched DNA probe assay (Quantiplex, Chiron, Emeryville, CA, USA). Cold guanidine-HCl homogenizing solution (8M guanidium thyocyanate, 50 mM Tris-HCl pH 7.5, 25 mM EDTA, 8% (v/v) 2-ME containing 3 M sodium acetate) was added to the frozen tissue and homogenized with pellet pester mixer. Sarkosyl (10%) was added and gently mixed. Tubes were then centrifuged to sediment particulates. Supernatants were removed and added to the tubes containing poly A (10 mg/mL). Ethanol was then added and thoroughly mixed. Tubes were placed at -20Â°C overnight and centrifuged for 20 min at 4Â°C. Supernatants were aspirated, and the pellets were dried down with a speedvac rotatory vacuum device (Eppendorf-Netheler-Hinz, Hamburg, Germany). After solubilization in nuclease-free H2O, HCV RNA was measured. Duplicate samples were added to the wells in which lysis, hybridization, capture and signal amplification occurred. A standard curve was constructed with reference sera obtained from Acrometrix OptiQuant HCV RNA Panel (Egret Court Benicia, CA, USA). Results were expressed as pg HCV RNA per gram of bioptic tissue [conversion factor 0.52 obtained by dividing HCV RNA molecular mass by the number of viral copies/mL (3.13 x 106 g/mol/6.023 x 1023 copies /mol) x 105].
Isolation of Portal Tracts from Liver Biopsy Sections
Portal tract structures were isolated by Laser Capture Microdissection (LCM) technique, as decribed elsewhere. Briefly, frozen liver tissue specimens were cut as a series of 5 Î¼m sections mounted on slides coated with a thermoplastic membrane (Leica Microsystems, Wetzlar, Germany). Portal tracts were selectively dissected by focal melting of the membrane with an ultraviolet laser beam by Leica SVS LMD System (Leica Microsystems). Dissected microsamples were dropped into cap tube under microscope inspection. Microsamples were lysed in 50 Î¼L lysis buffer and tubes were centrifuged. Then, pellets were washed with 70% ethanol and air dried. DNA was isolated via QIAshedder columns and DNeasy Kit from Qiagen (Hilden, Germany). Portal tracts from liver biopsy sections of patients with non-alcoholic steatosis and with near normal liver obtained during cholecystectomy were employed as controls.
Analysis of TCR Repertoire by PCR and Capillary Electrophoresis
DNA-based PCR approaches were established for detection of TCR gene rearrangements. The PCR approach for Î±Î² TCR was designed as a multiplex PCR for the detection of VÎ²-JÎ² and incomplete DÎ²-JÎ² rearrangements. With 23 family-specific VÎ² primers designated to mainly recognize functional VÎ² gene segments, 2 specific DÎ² primers and 13 specific JÎ² primers theoretically most complete VÎ²-JÎ² and all incomplete DÎ²-JÎ² gene rearrangements can be detected.
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Each 25 Î¼L PCR reaction contained 200 ng DNA, 12.5 Î¼L 2x Taq PCR Master mix (Qiagen) and 0.5 Î¼mol/L of each primer. PCR used one cycle at 94Â°C for 3 min followed by 40 cycles at 95Â°C for 60 seconds, 60Â°C for 30 seconds, and 72Â°C for 30 seconds with a final 10 min extension at 72Â°C and 4Â°C hold. The PCR product was diluted 1:10 in water and 1 Î¼L was mixed with 12 Î¼L of deionized formamide and 0.5 Î¼L of Gene Scan 400 HD Rox size Standard (Applied Biosystems, Faster City, CA, USA). The mixture was injected onto ABI PRISM 310 (5 second injection, 15 Kv, GSSTR POP 4 (1mL) D module, 60Â°C, 24 min run-time). Data were analyzed using Genescan (Applied Biosystems) which records the fluorescence intensities in each peak. Run variations of the run-off are the results of different CDR3 lengths, reflecting an imprecise V-D-J joining process. The graphs representig CDR3 size patterns were standardized at 100% for the highest peak and data used to generate these graphs could be used to determine the intensity of each peak expressed as relative fluorescence units and to evaluate the background. Dominant (oligoclonal) peaks were arbitrarily defined as a predominant length of CDR3 with a peak area of fluorescence corresponding to at least 40% of the sum of fluorescence intensities of all the peaks in a given family.
To eliminate risk of false-positive results due to "background" amplification of similar rearrangements in polyclonal cell populations, heteroduplex analysis was used. Homo and heteroduplexes resulting from denaturation at 94Â°C and renaturation at lower temperature were separated in non-denaturing polyacrylamide gels. PCR products of clonally rearranged TCR genes give rises to homoduplexes, whereas in case of clonally rearranged TCR genes, they give rise to heteroduplexes. TCR Î±Î² primer sequences depicted in Table 1 were labeled with 5' FAM (6-carboxyfluorescein) and 5' HEX (6-hexachlorofluorescein). Amplifiable DNA was confirmed in all samples by Î²-globin primers generating a 268 bp product (5'-HEX-CAACTTCATCCACGTTCACC-3' and 5'-GAAGAGCCAAGGAGAGGTAC-3').
Characteristics of patients are shown in Table 2. In the cryoglobulinemic group, 28 had type II MC, being IgMk the monoclonal component in all. Type III MC was demonstrated in the remaining 2 patients. All patients were shown to harbor productive HCV infection by repeated confirmation of HCV viremia. The number of females was higher in the MC group. Liver disease was histologically characterized in all. Obvious cirrhosis was demonstrated in 3 patients of both groups. A moderate prevalence of genotype 2 was noticed in MC patients. Liver enzymes were elevated in all. MC patients had measurable cryocrit (3.5Â±1.8%).
DNA-based PCR approach for TCR Î±Î² gene rearrangements were designed as multiplex PCR for detection of complete VÎ²-JÎ² and incomplete DÎ²-JÎ² rearrangements. TCR Î² gene rearrangements were carried out in the liver tissues and in PBL collected in step with liver biopsy. The normal pattern which consisted of 7 to 11 peaks in a Gaussian pattern with a 3-nucleotide interval, was observed in all but one healthy subjects (97%), in 7 (23%) and 6 (29%) liver tissues and in 11 (37%) and 9 (43%) PBL of MC and non-MC patients, respectively. The abnormal patterns, which contained a single or a few dominant peaks, were observed in multiple variable segments. Quantitation of DNA fragments was defined by measurement of laser-light-induced fluorescence. The fluorescence intensity of the amplicons was expressed as an area under the curve (AUC) in relative fluorescence units. Clonal deviation of CDR3 profile was taken when the AUC percentage exceeded the value of reference panel by 3.0 standard deviation.
Discrepancy between liver and PBL was found in terms of frequency of TCR Î² gene rearrangements and expansions. These findings comprised T cell clonal expansions in 23 (77%) and 15 (71%) liver tissues and in 19 (63%) and 12 (57%) PBL in MC and non-MC patients respectively.
Spectratyping profiles showed that identical expanded T cell populations were recognized in the two biological compartments in 8 (27%) and 6 (28.5%) MC and non-MC patients. Conversely, in 10 (33.3%) MC and in 5 (23.8%) non-MC patients, T cell clonalities in the liver were demonstrated to be different from those found in PBL. Analyses of circulating T cell clonal expansions showed that they were unchanged during the clinical survey.
In 5 (17%) MC and 3 (14.%) non-MC patients T cell ß gene rearrangements were demonstrated just in the liver, but not in the peripheral blood. In one (5%) non-MC patient, circulating T cell clonotypes occurred without evidence of intrahepatic TCR gene rearrangements. VB5, VB7, VB8, VB14, VB16 families were among the most frequently represented in the liver and in the periphery of both groups of patients.
Demonstration of TCR gene rearrangements is strictly related to the detectability threshold which is, indeed, intrinsic to the current assays. The sensitivity levels of PCR assays in our hands reached the detection of one clonally-rearranged cell in 100 non-rearranged control cells tested by diluting Jurkat cells in normal samples. However, inability to detect TCR gene rearrangements includes many other reasons, such as atypical rearrangements, or rearrangements of non-functional gene leading to incapability of PCR primers to anneal appropriately. Another factor responsible for attenuating efficiency of PCR assays is the dilution of antigen-specific lymphocytes among liver-resident T cell population. It can be assessed that primers currently used amplify CDR3 of T cells, but the products of a small clonal population may be obscured by polyclonal T cells in the sample. This drawback is particularly critical when considering total DNA extracted from the entire bioptic sample, which contain a mixture of normal and abnormal nucleic acids that likely interfere with the molecular analyses of cell of interest.
To overcome this critical point TCR ß gene rearrangements were assayed on DNA extracted from portal tracts containing lymphoid aggregates obtained from liver biopsy sections. LCM was used to precisely separate inflammatory cells from surrounding contaminating cells as depicted in Fig.1.
The relationship between occurrence of expanded T cell clonalities and epidemiological, virological and laboratory parameters in MC and non-MC patients is summarized in Table 3. Mean age and duration of HCV infection were not dissimilar between the groups. Comparable values of ALT activity and liver histological features were noticed. Interestingly, in spite of comparable average levels of circulating viral load, a significant higher concentration of intrahepatic HCV RNA was demonstrated in patients with TCR gene rearrangements than in those without in both groups of patients.
Changes of TCR ß gene rearrangements were investigated in 18 and 21 MC and non-MC patients respectively, after 12 months of efficient Peg-IFNÎ± and ribavirin combination. Patients were reassessed in terms of circulating T cell expansions 6 months after discontinuation of therapy. A sustained response occurred in 10 (56%) and in 12 (57%) of MC and non-MC patients, respectively. This resulted in the clearance of HCV RNA from the circle, a dramatic improvement of cryoglobulin-related signs and symptoms with disappearance or remarkable reduction of cryocrit values. Follow-up after the end of therapy showed that responsive patients had no clinical event related to liver disease or vasculitis. It was demonstrated, indeed, that T cell clonal expansions were down-regulated. The reduction of repertoire perturbation corresponded to a down-regulation of baseline clonal expansions after successful antiviral treatment. Spectratype analysis of PBL revealed that 5 of 12 MC and 6 of 12 patients who were dominated by single or few clones during the first 6 months of therapy changed their CDR3 length profile with polyclonal subset obtained at 6 months after therapy. In the remaining 7 and 6 MC and non-MC non-responding patients, viral replication was never efficiently suppressed and the TCR repertoire remained largely perturbated even during treatment. Individual clonal expansions remained remarkably stable for the extended period of time (Fig. 2).
As shown by spectratyping profiles of TCR repertoire, MC patients with chronic HCV infection are characterized by clonally-expanded T cells either in the liver or in the peripheral blood. Almost 80% of examined livers and more than 60% of circulating blood samples showed rearrangements of the TCR Î±Î² gene. Similar features were found in HCV-infected patients without MC, in that T cell clonal expansions occurred in more than 70% of liver tissues and in almost 60% of peripheral lymphocytes.
Spectratype profiles of LIL and PBL from each patient have been demonstrated to be distinct in 43.5% (10/23) and in 33.3% (5/15) of MC and non-MC cases showing TCR gene rearrangements. This reflects a unique pattern of T cells within each compartment. Comparison of T cell repertoire between liver and peripheral blood is of obvious importance if one assumes that T cell clonalities more frequently found in the liver than in the blood are likely to be relevant as regards HCV infection, in that HCV specific T cells would be expected to home to the site of infection. On the contrary, T cell clonal expansions were more frequently detected in the blood than in the liver and possibly represent memory T cells induced by previously encountered pathogens. LIL include HCV-specific T cells, probably as a minor population. It seems reasonable to hypothesize that activated T cells become trapped in the liver irrespective of their specificity. Furthermore, chronic inflammation in local tissues is capable of inducing additional T cell activation through production of several cytokines, resulting in the expansion of activated T cells and switching of TCR repertoire. This, indeed, may represent a mechanism that can enrich liver tissue of expanded T cell clones, thus diversifying intrahepatic T cell repertoire. Otherwise, since the adult liver is devoid of constitutive lymphoid components, any intrahepatic T cell must have migrated into the liver. It can be argued that, during an immune response to hepatotropic microorganisms, clonal expansions of antigen-specific lymphocytes occur in lymph nodes which drain the sites of infection. Then, activated lymphocytes enter the blood stream and home to the liver. This implies that the pool of intrahepatic lymphocytes is maintained through the constant influx from extrahepatic sites. In this context, chemokines have been shown to orchestrate migration to and preferential sequestration in the liver of B and T cells. Intrahepatic T cell sequestration likely reflects deregulation of T cell traffic as the consequence of locally high production of chemokines. In this context, the formation of lymphoid follicle-like structures in the portal tracts of HCV-related MC patients is considered the morphologic counterpart of ectopic lymphoid tissue which includes naÃ¯ve B cells in the central zone surrounded by mature B and T cells. These are structures that contribute to antigen presentation in situ, and clonal expansion of antigen-specific cells.
In the present series, 21.7% (5/23) MC and 20% (3/15) non-MC patients showed expanded T cell clones in the liver, but not in the circulation. This, indeed, may represent a biological condition potentially able to recruit a large number of inflammatory cells in the liver, suggesting that additional factors participate in the recruitment process. Similar mechanisms have been emphasized in animal model of HBV chronic infection. Whether T-regulatory CD4+CD25+ cells are defective in MC patients and influence the production of chemokines by stromal cells, is a matter which deserves further investigation. Studies aimed at defining the role of in situ production of factors involved in intrahepatic accumulation of inflammatory cells are warranted.
In 34.8% (8/23) and 40% (6/15) MC and non-MC patients respectively, clonal expansions of T cells in the peripheral blood closely reflected the intrahepatic population. Spectratype profiles between LIL and PBL appeared virtually identical, suggesting that T cell populations can freely circulate from one compartment to the other.
In a single instance, T cell clonalities were demonstrated in the peripheral blood, but not in the hepatic compartment. This possibly reflects an inability of the liver to entrap expanded T cells or else it can be the result of sampling error, in that T cell clonotypes occur in selected areas of liver sections, as previously demonstrated for expanded B cell clones.
T cell clonalities reflect down-regulation of the immune response in chronic HCV infection. It has been suggested that accumulated T cell clonal expansions represent a late differentiation stage of antigen-specific effector cells, whose functional impairment cannot be improved by antiviral therapy (30, 41).[30, 41] However, viral clearance following IFN-Î± administration also suggests that this cytokine changes the quality of the cellular immune response. Interestingly, the higher concentration of liver HCV RNA in patients with intrahepatic T cell clonal expansions supports the contention that HCV is directly involved in the pathogenesis of initial expansion and maintenance of T cell populations. Our data emphasize previous observations regarding clonally-expanded B cell population in HCV-related MC patients. Overload of HCV-encoded proteins has been proposed to play a direct role in stimulating and maintaining intrahepatic B cell clones. CDR3 spectratyping determined T-cell repertoire regardless of antigen specificity, although clonal expansions of HCV-specific T cells could potentially be hidden by other T cell populations present in the liver. A formal differentiation between virus-specific and non-specific T cell populations must be defined at the site of infection and inflammation to assess the weight of the cellular components of the immune system. Nevertheless, the fact that T cell clonal expansions decrease dramatically only when HCV replication is efficiently suppressed, whereas insignificant changes in TCR repertoire perturbation occur when HCV antigens remain expressed, strongly suggests that the latter are HCV-related.
Antigen-driven selection of T and B cell inflammatory populations has a direct impact on clinical and therapeutic outcome of MC. In these patients, interaction between T and B cells may be of great relevance, in that intrahepatic production of rheumatoid factor molecules results in their reaction with human class I HLA molecules involved as part of antigen-binding pocket, thus influencing peptide recognition by cell-mediated immune response.[43-44]
It has been reported that clonally-expanded intrahepatic T cells in HCV-infected patients without MC induce an immune regulatory environmental defect which predisposes to progressive inflammatory liver damage, thus establishing the basis for correlating clonal expansions of T cell populations to liver pathology.[45-46] Based on the these results, commitment of both arms of the immune system may be considered a feature of patients with HCV-related MC. Restriction of both cellular and humoral immune responses must be considered in the context of a limited host response to a pathogen capable of undergoing long-term spontaneous mutations that heavily contribute to the viral burden.
Although further studies are needed to clarify the functional basis underlying termination of chronic persisting virus infection and regression of T and B cell clones, emerging evidence indicates that some dominant clones may persist in MC patients in spite of HCV clearance, suggesting that inappropriate survival signals may be active irrespective of antigen stimulation.
This study was supported in part by:
Italian Ministry of University and Scientific and Technologic Research, National Project "Chronic liver damage induced by hepatitis C virus" (DS);
AIFA - Agenzia Italiana del Farmaco, funds for independent studies, 2007, contract no. FARM7SJX (DS);
Funds from University of Bari (FD, DS)
"Associazione Italiana per la Ricerca sul Cancro" (AIRC), Milan, Italy (FD).