The identification of clonal rearrangements of T cell receptor (TCR) genes is central to the diagnosis of T cell lymphomas. However, in angioimmunoblastic lymphadenopathy (AILD), first described as a nonneoplastic proliferation associated with immunodeficiency, the heterogeneity of TCR and IgH gene rearrangements suggest that some cases may harbor multiple lymphoid clones. In this study we have isolated DNA from archival paraffin biopsy material from 22 cases of AILD identified on the basis of classical histological and immunohistochemical features with the aim of establishing the occurrence of clones and oligoclones, the frequency of TCR and immunoglobulin heavy chain (IgH) variable (v) gene use, and the relationship of these findings to the presence of Epstein-Barr virus. DNA extracted from the biopsies was amplified using the polymerase chain reaction (PCR) and sequenced to detect functional and nonfunctional gene rearrangements. Epstein-Barr virusencoded short RNA species (EBERs) were detected using in situ hybridization combined with immunochemistry to identify the phenotype of the EpsteinBarr virus-infected cells. Fifty-seven clonal products were found in 20/22 patients: TCR␥ clonal products were identified in 16/22, TCR␤ clonal products in 16/22 and IgH clonal products in 6/22 cases. Oligoclonal PCR products were seen for TCR in 3/22 and for IgH in 3/22 cases. In one biopsy PCR products from all reactions were polyclonal. Sequence analysis revealed functional TCR␥, TCR␤, and IgH sequences in 6/12, 9/11, and 8/8 cases, respectively. Functional TCR and/or IgH oligoclones were detected in 6/20 (30%) cases. In addition, nonfunctional TCR and IgH sequences were found in 11 cases. EBERs were identified in 18/20 cases varying from occasional to 25 to 30% nuclei staining and were associated with both T and B cells, although the majority were of indetermi- Early reports of AILD stressed the nonneoplastic nature of this disease, 1,2 noting that neither the cytology nor the pattern of infiltration seen in biopsies or autopsies was that of a neoplasm. The disease was thought to be due to abnormal immune regulation, the majority of patients dying of infections, often with opportunistic organisms.2 An association with lymphoma was, however, noted by Lukes and Tindle, 3 who reported the development of immunoblastic lymphoma in 3 of their 32 patients. Subsequently, there has been controversy over the separation of AILD from AILD with immunoblastic lymphoma (IBL) based on histological features such as atypical clear cells, which has contributed to the variation in the reported incidence of lymphoma in AILD from 0 to 35%. 4 -6 In addition, both groups show clonality or lack of clonality. 4,7 Frizzera et al 8 has proposed that three AILDrelated disorders can be recognized as AILD, AILD-like dysplasia, and AILD-like peripheral T cell lymphoma (PTCL) based on a combination of morphology, phenotype, clonal-

T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.

Aims-Immunohistological and molecular characterisation ofa case ofperipheral T-cell lymphoma (PTCL) of angioimmunoblastic T-cell lymphoma (AILD) type. Methods-Frozen and paraffin wax sections of the diagnostic lymph node were stained with a panel of T-and B-cell lineage monoclonal antibodies. DNA was isolated from the paraffin wax embedded biopsy material for T-cell receptor (TCR) and immunoglobulin (Ig) PCR amplification, and resultant PCR products were cloned and sequenced. Results-Immunohistological analysis of the presenting lymph node was consistent with an extensive infiltrate ofpleomorphic CD3+CD8+ lymphocytes. Most (>80%) of these infiltrating CD3+ cells were also positive for the TCR VB5.1 gene family product, and were shown to be oligoclonal by TCRB PCR amplification and sequencing. Three oligoclones of B cells were also demonstrable by PCR amplification with Ig heavy chain primers and sequencing, a finding at variance with the diagnosis of AILD.Conclusions-These data demonstrate the complexity and heterogeneity of PTCL which require extensive histological examination and molecular characterisadon.

This paper describes the immunohistology and molecular genetics of 18 cases of T-cell-rich B-cell lymphoma (TCRBL). In all cases, the large B cells stained strongly for CD20, with more variable expression of CD79a, and were negative for CD30 and CD15. The majority of T cells were predominantly positive for TIA-1 and negative for CD57; a large population of histiocytes was present in all cases. Epstein-Barr virus (EBV)-coded RNA (EBER) was found in B blasts from four cases and in one case was present among the background lymphoid cells. IgH PCR products were generated in 16/18 cases and revealed clonal, oligoclonal and polyclonal PCR products in 12, two and two cases, respectively. In addition, TCRG clonal gene rearrangements were identified in two cases. TCRB gene rearrangements were polyclonal. Sequence analysis of seven cases with clonal/oligoclonal IgH gene rearrangements revealed functional sequences with predominant V(H)3 gene usage associated with various D genes and J(H)4 or J(H)6 gene segments. Four cases displayed varying degrees of replacement and silent mutations (1.8-21%), with one case exhibiting intraclonal heterogeneity; the distribution of mutations was indicative of antigen selection in three cases. The remaining three cases, including two cases with functional oligoclonal IgH rearrangements, harboured unmutated V region genes. The EBV-positive cases were associated with clonal, oligoclonal and polyclonal PCR products and with mutated and germline clonal sequences. These data indicate that TCRBL may be a heterogeneous entity associated with clonal and oligoclonal B cells derived from both germinal centre and naïve B cells.

In this paper we document a case of peripheral T cell lymphoma (PTCL) that exhibited variable T cell histology at presentation and follow-up. Southern blot analysis for T cell receptor (TCR) and immunoglobulin (Ig) receptor gene rearrangements failed to reveal clonal T or B cell populations. TCR gamma (TCRG) and beta (TCRB) chain gene polymerase chain reaction (PCR) amplification of DNA isolated from biopsies was also consistent with polyclonal T cell populations, however Ig PCR revealed clonal Ig rearrangements in follow-up biopsies but not in the presentation biopsy. There was no histological evidence for a neoplastic B cell population in these biopsies although occasional EB virus positive blasts were present. The significance of a cryptic B cell clone is unknown but suggests a relationship with the proliferating polyclonal T cells in this case of PTCL. These data reflect the complexity of PTCL with implications for treatment and patient management.