INTRODUCTION

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Keywords: T cell receptor; lymphoproliferative disorders; HIV

Low-grade mucosa-associated lymphoid tissue (MALT) lymphoma develops exclusively in the background of chronic inflammation, either through an autoimmune process (e.g., Sjögren's syndrome, Hashimoto's thyroiditis) or chronic bacterial inflammation (e.g., Helicobacter pylori [HP]). MALT lymphomas contain large numbers of reactive tumor-infiltrating T lymphocytes (TITL). In the stomach, these lymphomas develop secondary to HP infection, and low-grade gastric MALT lymphoma may show regression after HP eradication (1). Considering that low-grade MALT lymphoma cells proliferate in vitro in response to activated TITL (2) and that the proliferation of low-grade MALT lymphoma cells is dependent on CD40 signaling and T-helper 2 (Th2) cytokines (3), TITL may play a critical role in supporting the progression of low-grade MALT lymphoma.

The T cell antigen receptor (TCR)-V and TCR-V genes rearrange during the early stages of T cell differentiation preceding TCR-V gene rearrangement. Regardless of whether a TCR-V is expressed, the TCR-V locus is more frequently rearranged than the TCR-V locus (4). A single analysis of the TCR-V locus is sufficient to determine the clonality of a T cell clonal population (5). In addition, TCR-V locus rearrangement is suitable for polymerase chain reaction (PCR) because the limited number of TCR-V gene repertoires makes it an easier system for which to optimize primers than the TCR-V gene, which has over 20 V regions. However, the simple genomic organization of the TCR-V locus and the presence of only one hypervariable N region in each joining segment are responsible for the small range of variability in the lengths of the different rearrangements. Owing to the limited junctional diversity of V gene rearrangements, the similar lengths of the amplified fragments could result in false clonal bands when analyzed by standard electrophoresis. Because the use of guanine- and cytosine-rich sequence (GC-clamp) primers and denaturing gradient gel electrophoresis (DGGE) separate DNA segments based on nucleotide sequence and not merely according to the length of the DNA, the resolution of DGGE and the accuracy of PCR are improved compared with those of methods previously reported (6, 7).

We used a DGGE procedure with a 40-nucleotide GC-clamp in the PCR and sequencing analysis to analyze the TCR-V gene repertoire of TITL in pulmonary lymphoproliferative disorders (low-grade MALT lymphomas and human immunodeficiency virus [HIV]-negative and HIV-related lymphocytic interstitial pneumonia [LIP]). Our data support the view that antigen-driven oligoclonal TITL expansion takes place in some pulmonary lymphoproliferative disorders (40% of low-grade pulmonary MALT lymphomas and all HIV-related and 30% of HIV-negative LIP).

	METHODS

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Patients

Materials from 30 patients with Epstein-Barr virus (EBV)-negative pulmonary lymphoproliferative disorders were collected from our own files for the years 1982 to 2001. Cases included 15 patients with low-grade pulmonary MALT lymphoma and 15 patients with LIPs (5 HIV related and 10 HIV negative) (Table 1). All HIV-related LIP cases had normal absolute CD4⁺ T cell numbers classified as group II or III according to the Centers for Disease Control (CDC) classification (8). In one case, case 5, mass shadows expanded very slowly, and subsequent percutaneous needle biopsy (PCNB) was done 8 yr after the open-chest biopsy procedure. Three patients with LIP (two HIV- related and one HIV-negative LIP) underwent transbronchial lung biopsies (TBLBs) 1 to 4 yr after open-chest biopsy.

Histopathologic Studies

The materials were fixed in formalin and embedded in paraffin wax. Sections were subjected to hematoxylin-eosin (H-E) and immunohistochemical stainings. Immunohistochemical studies were performed with the following markers: leukocyte-common antigen (LCA, Dakopatts, Globstrup, Denmark), CD20 (L26, Dakopatts), CD45RA (MB1 Euro-Diagnostica, Malmo, Sweden), CD43 (MT1, Euro-Diagnostica), CD45RO (UCHL1, Dakopatts), and anti-kappa and lambda light chains (Dakopatts). Microdissection was carried out by methods described elsewhere (9).

DNA Extraction and PCR

DNA was obtained from the microdissected materials and peripheral lymphocytes using TAKARA DEXPAT (Takara Corporation, Kyoto, Japan) and COLLECTAGENE (Takara Corporation, Kyoto, Japan).

The PCR method of Theodorou and coworkers was used (5). The V and J primers, described in Table 2, were mixed in a single reaction for the diagnosis of clonality. Each PCR experiment included a sample with DNA extracted from a pulmonary T cell lymphoma (as a positive control) whose V gene rearrangement was detected by Southern blotting. One PCR cycle consisted of annealing for 1 min at 56° C, extension for 1 min at 72° C, and denaturation for 1 min at 94° C. For amplification, the high-fidelity polymerase Pfu was used. After 40 cycles of PCR amplification, 30-µl aliquots of the amplified products were electrophoresed in a 10% denaturant/6% polyacrylamide-70% denaturant/12% polyacrylamide double-gradient gel (100% denaturant = 7 mol/L urea and 40% formamide) (10). The gel was developed at 90 V for 16 h in 1 × TAE buffer at a constant temperature of 60° C, stained with ethidium bromide, and photographed under ultraviolet transillumination.

Our procedure for PCR amplification of the EBV genome is given in detail in Oda and coworkers (11).

Sequence Analysis

Another portion of the PCR product was ligated to the pCR-BluntII-TOPO vector, and the ligation mixture was transfected into One Shot-competent cells using a Zero Blunt TOPO PCR Cloning Kit (Invitrogen Corporation, Carlsbad, CA). Ligated clones were chosen at random, and phage DNA was purified. Inserts in the pCR-BluntII-TOPO vector were sequenced by the Dye Primer method using a Taq Dye Primer Cycle Sequencing Core Kit (Applied Biosystems, Norwalk, CT). The TCR-V gene sequences were aligned to sequences in the EMBL/Gen Bank databases.

	RESULTS

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Histopathologic Findings

Fifteen cases of low-grade pulmonary MALT lymphoma were classified as extranodal marginal zone B cell lymphoma (low-grade B cell lymphoma of MALT type). In all cases, reactive follicles were sparsely present and surrounded by tumor cell infiltrations consisting of small- to medium-sized lymphoid cells. Every case demonstrated lymphoepithelial lesions, which are a characteristic feature of low-grade MALT lymphoma. All cases were classified as B cell lymphoma on the basis of the expression of at least two B cell markers. In patient 5, a subsequent PCNB specimen (8 yr later) demonstrated monomorphous lymphocytic infiltration suggestive of low-grade lymphoma (Table 1). These lymphoma cells were similar to those observed in the previous open-chest biopsy specimens.

All 15 LIP samples demonstrated dense and mixed interstitial infiltrates of lymphocytes, plasma cells, and histiocytes with prominent germinal centers. No cytologic atypia of the lymphoid infiltrates was seen. In cases 10, 11, and 12, subsequent TBLB specimens (1 to 4 yr later) demonstrated lymphocytic infiltration without cytologic atypia. Immunohistochemical staining of the specimens revealed polyclonal B cell populations with numerous T cells.

PCR and Sequencing Analysis of the TCR-V Gene Rearrangements of Pulmonary Lymphoproliferative Disorders

In no instance in which a case with pulmonary lymphoproliferative disorders was defined as EBV negative was any amplification of the EBV genome demonstrated by PCR analysis. Six of 15 (40%) pulmonary low-grade MALT lymphoma and eight of 15 LIP (five of five [100%] HIV-related and three [30%] of 10 HIV-negative LIP) cases showed oligoclonal bands of TCR-V genes on DGGE (Table 1 and Figures 1 and 2). Other cases demonstrated smear bands, representing polyclonal T cell infiltrates with the absence of a predominant clone. All peripheral blood samples from low-grade pulmonary MALT lymphoma and LIP cases also showed smear bands. In cases 5, 10, 11, and 12, some TCR-V rearrangements, identical to those found in the open-chest biopsy specimens, were found in the sequential biopsy specimens (1 to 8 yr later) (Figures 1 and 2).

View larger version (55K): [in this window] [in a new window]   Figure 1.   PCR analysis of TCR-V gene rearrangement in low-grade pulmonary MALT lymphoma. The numbers above the lanes correspond to the case numbers in Table 1. Note that multiple bands representing oligoclonal populations were observed in cases 1 through 6. In case 5, some TCR-V rearrangements, identical to those in the open-chest biopsy specimen, were found in subsequent PCNB specimens (8 yr later). A monoclonal single band is observed in the positive control. S, surgical biopsy specimen; P, percutaneous needle biopsy specimen; Pe, peripheral blood lymphocytes; Po, positive control; N, negative control.

View larger version (62K): [in this window] [in a new window]   Figure 2.   PCR analysis of TCR-V gene rearrangement in LIP. The numbers above the lanes correspond to the case numbers in Table 1. LIP cases (cases 7 through 14) also demonstrated multiple oligoclonal bands. For cases 10, 11, and 12, it was shown that some TCR-V gene rearrangements remained unchanged in sequential samples. S, surgical biopsy specimen; T, transbronchial biopsy specimen.

To confirm the specificity of our PCR reaction as well as the reliability of the DGGE electrophoresis, all PCR products were sequenced. The results of sequencing are presented as the incidence rates of the predominant sequences out of the total number of vector clones analyzed. All oligoclonal PCR products on DGGE demonstrated plural oligoclonal TCR-V sequences with frequencies ranging from 3 of 40 to 12 of 40 clones (Table 3). In other cases in which the TCR-V genes appeared as polyclonal smear bands on DGGE, the evaluation of 40 clones per specimen revealed all different TCR-V sequences (data not shown). Some identical rearrangements existed in both the open-chest and sequential biopsy specimens (Table 4).

	DISCUSSION

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This study was designed to address two main questions. First, is there a selective oligoclonal expansion of TITL within pulmonary lymphoproliferative lesions (low-grade pulmonary MALT lymphoma and LIP)? Second, if oligoclonal populations exist, do they differ in HIV-negative and HIV-related LIP?

Although the importance of TITL in low-grade MALT lymphomas appears clear, their role has remained unclear. In an attempt to characterize TITL of low-grade pulmonary MALT lymphomas, investigators have applied molecular techniques to detect T cells that are oligoclonal with respect to the highly polymorphic antigen binding third complementarity determining region (CDR3) of TCR, suggesting antigen-driven expansions at the site of low-grade MALT lymphoma lesions. Previous studies of TITL clonalities performed on low-grade MALT lymphomas have produced conflicting data regarding T cell clonalities (12). Although some studies have indicated the existence of a restricted T cell repertoire in MALT lymphomas, it remains unknown whether oligoclonal T cell populations exist in low-grade pulmonary MALT lymphomas.

Because it is necessary to use high-fidelity DNA polymerases in PCR in order to limit the introduction of amplification errors in the PCR products, we used the high-fidelity polymerase, Pfu. Unlike Taq DNA polymerase, Pfu possesses a 3' to 5' exonuclease proofreading activity that enables the polymerase-generated PCR fragments to have fewer errors than Taq-generated PCR inserts. The error rate for Pfu has been reported to be one mutation per 7.8 × 10⁵ bp per cycle (16).

The use of GC-clamp primers and DGGE improves the PCR method for the TCR-V gene, leading to an increase in clonal specificity. The GC-clamp allows for the partial denaturing of the PCR product as it electrophoreses through the denaturing gel, thus forcing the separation of the PCR products to be influenced by the unique sequence of the junction; the GC-clamp ensures the N sequence will be in the first melting domain of the V-J fragment (5). The PCR products are thus separated on the basis of melting rather than difference in length between N sequences at the V-J joining, resulting in a genetic imprint that is highly specific for every TCR-V rearranged allele. Using a modified DGGE procedure with a GC-clamp, we first analyzed TCR-V rearrangement in low-grade pulmonary MALT lymphomas. We detected oligoclonal T cell populations in six of 15 (40%) patients with low-grade pulmonary MALT lymphomas. Because no oligoclonal T cell populations could be detected in the peripheral blood, these clones undergo local expansion. It has been reported that there is an oligoclonal pattern with selectively expanded clones in MALT lymphoma TITL (12). Because we microdissected the MALT lymphoma areas carefully to avoid dilution by non-T cell DNAs and nonspecific reactive T cell DNAs, DNA extraction from a microdissected area of TITL in low-grade pulmonary MALT lymphomas improved the sensitivity of the TITL clonalities. More definitive molecular analyses will be required to understand the immunologic process of oligoclonal TITL expansion in pulmonary low-grade MALT lymphomas.

To determine whether oligoclonal T cell clones persist over time at the site of low-grade MALT lymphoma lesions, we had the opportunity to study PCNB specimens (case 5) obtained 8 yr after open-chest biopsy. Sequencing analysis of the PCR products showed some identical T cell populations stably detected between the open-chest biopsy and the PCNB specimens. These identical TITL clones suggest that some oligoclonal TITL are present continuously in the focal region of a low-grade pulmonary MALT lymphoma over a long time period and that these TITL may play a role in the pathogenesis of low-grade pulmonary MALT lymphomas.

T cells recognize antigens in two different forms: classical antigenic peptides that are presented to the specific T cells by major histocompatibility complex (MHC) molecules via their peptide-binding clefts, and superantigens that bind outside the MHC grooves. Both types can give rise to the use of restricted sets of TCR repertoires. In the first classical form, selected peptides can be presented by selected MHC molecules, which are preferentially bound by a selected TCR-V repertoire leading to an oligoclonal expansion (restricted CDR3) of the specific TITL clones. On the other hand, superantigens can stimulate T cells bearing a particular TCR-V repertoire giving rise to a polyclonal expansion (unrestricted CDR3). According to the results of the present study, it is unlikely that the oligoclonal TITL in low-grade pulmonary MALT lymphomas are a consequence of superantigen mechanisms; such a mechanism fails to provide an explanation for the restricted CDR3 (oligoclonal expansion) of the TITL. Based on the data obtained here, we suggest that reactive TITL may be stimulated by conventional antigens, resulting in oligoclonal expansion of the antigen-specific T cells. Conventional antigen-specific oligoclonal expansions may play a role in the pathogenesis of low-grade pulmonary MALT lymphomas. It has been reported that T cells in the intestinal mucosa are oligoclonal and are derived from the expansion of a relatively small number of T cell clones in which different TCR-Vs dominate in different individuals (17, 18). Although the origin of these oligoclonal T cell populations is not yet understood, the oligoclonal T cell clones observed in low-grade pulmonary MALT lymphoma may be associated with this phenomenon. Because all low-grade pulmonary MALT lymphoma cases except one (case 5) have remained asymptomatic without recurrence since surgical resection (1 to 19 yr), long-term follow-up is especially important for determining whether outcome variables will differ in oligoclonal TITL-positive and -negative patients with low-grade pulmonary MALT lymphomas.

After establishing these PCR techniques, we analyzed HIV-related and HIV-negative LIP. We detected oligoclonal T cell populations in five of five (100%) patients with HIV-related LIP and three of 10 (30%) patients with HIV-negative LIP. The frequency of oligoclonal TITL in HIV-related LIP was higher than that in HIV-negative LIP. To determine whether this finding is unique to HIV or whether other viral infections induce similar findings, we analyzed the TCR-V gene repertoire of TITL in five patients with EBV-positive LIPs. One of five patients with EBV-positive LIP showed oligoclonal TCR-V clones (data not shown). This result does not suggest an incidence of oligoclonal TITL higher than that observed in HIV-negative LIP. A high frequency of oligoclonal TITL may be unique to HIV-related LIP. More definitive molecular analyses are required to understand the immunologic process of HIV-related LIP. It was very interesting that all HIV-negative LIP cases showing oligoclonal TITL were complicated by collagen diseases (Sjögren's syndrome or rheumatoid arthritis). As some TITL in salivary glands from patients with Sjögren's syndrome and in synovial tissue from patients with rheumatoid arthritis are reported to expand oligoclonally, the existence of oligoclonal TITL populations in HIV-negative LIP may be associated with the pulmonary manifestation of collagen diseases (19).

TBLB specimens were obtained from three patients with LIP (two HIV-related and one HIV-negative LIP) during the interval (1 to 4 yr) after open-chest biopsy. By analyzing TBLB specimens, some oligoclonal TCR-V gene rearrangements identical to those found in the open-chest biopsies were identified in all 3 LIP cases. This finding lends further support to the concept that some oligoclonal T cell clones remain unchanged over the years, although a much longer follow-up interval is required before drawing such a conclusion. A longer follow-up of our patients could help to clarify the influence of TCR-V oligoclonality on the survival of patients with LIP.

Although it is presumed that HIV itself or other agents might promote oligoclonal TITL populations in HIV-related LIP, the pathogenetic mechanisms by which these oligoclonal TITL clones arise in HIV-related LIP remain unknown. Several reports suggest that HIV infection leads to a skewed T cell repertoire affecting both CD4⁺ and CD8⁺ T cells and that these alterations may contribute to the pathogenesis of acquired immunodeficiency syndrome (AIDS). The skewed TCR repertoire is suggested to be due to the expansion of HIV- specific CD8⁺ T cells, and a recent study involving several HIV-infected patients demonstrated the expansion of certain TCR-V families due to the clonal expansion of HIV-specific cytotoxic T lymphocyte (CTL) clones as shown by double staining with MHC tetramers and TCR-V family-specific antibodies (22, 23). Several considerations should be taken into account when interpretting the high incidence of oligoclonal TITL expansions observed in HIV-related LIP. Considering that the development of lymphocytic alveolitis in HIV patients may be an important host response against HIV, and that HIV-infected cells, including macrophages and T lymphocytes, with a high viral burden can be detected in the pulmonary microenvironment, an additional agent responsible for the oligoclonal TITL populations might be represented by the HIV virus itself. It has been reported that the V3 loop proviral nucleotides and the inferred amino acid sequences of mononuclear cells from bronchoalveolar lavage are more homogeneous than those from peripheral blood (24). This homogeneity might result in oligoclonal TITL expansions as a consequence of ongoing TITL reactions against lung-specific viral strains. It may be important to point out that the sequences of 59 V chains from five individuals with diffuse infiltrative lymphocyte syndromes share structural features suggesting antigenic clonal selection (25). Further examination of the expression of oligoclonal TITL in HIV-related LIP is necessary to clarify this possibility.

This study demonstrates that oligoclonal TITL expansions occur in some pulmonary lymphoproliferative disorders, and that the frequency of oligoclonal TITL expansions in HIV-related LIP is unusually higher compared with that in low-grade pulmonary MALT lymphomas or HIV-negative LIP.