polyclonal lymphocytosis of t-cells associated with human ......phocytosis, considered to be a...

(CANCER RESEARCH 48, 2585-2589, May I, I988|

Polyclonal Lymphocytosis of T-Cells Associated with Human T-Cell LeukemiaVirus I1

David N. Posnett,2 Helen A. McGrath, Rachelle A. Scott, N. Scott McNutt, Robert Folkl, Thomas D. Hickey, M. J.

Macera, and Paul SzaboDepartments of Medicine [D. N. P., H. A. M., R. A. S., R. F., P. S.Â¡and Pathology [N. S. M.. T. D. H.J, Cornell University Medical College, New York, New York10021, and Division of Genetics, Long Island College Hospital, State University of New York Health Science Center, Brooklyn, New York 11203 /M. J. M.J

ABSTRACT

A patient with antibodies to human I -coll leukemia virus type I andthe presence of integrated sequences of this virus in T-lymphocytes wasinvestigated. In contrast to previous reports, the T-cell lymphocytosiswas found to be polyclonal by analysis of human T-cell leukemia virustype I integration sites and T-cell antigen receptor rearrangements.Polyclonal T-cell infection by human T-cell leukemia virus type I mayrepresent an infrequently observed stage of leukemogenesis.

INTRODUCTION

HTLV-I3 is associated with a number of T-cell lymphoprolif-erative disorders, but in particular with adult T-cell leukemia(1,2). This virus has affinity for helper T-cells bearing the T4antigen, although it can infect other cells including T8 positiveT-cells (3), B-cells (4), and endothelial cells (5). In vitro studieshave demonstrated that the virus infects cultured T4 positiveT-cells in a polyclonal manner. Thereafter, a single clone ofinfected T-cells will acquire a growth advantage and the culturedT-cells will become monoclonal (6, 7). Human infection withHTLV-I might therefore be expected to produce an initialpolyclonal T-cell proliferation followed by a monoclonal malignant transformation. Such is the case in bovine leukemia (8),which is caused by a similar retrovirus (bovine leukemia virus).However, in humans, patients with ATL (2, 9), as well asasymptomatic patients with incidentally discovered T-cell lym

phocytosis, considered to be a preleukemic state of ATL (10),have monoclonal integration of the HTLV-I genome in thehost cell DNA. Even asymptomatic carriers from endemicsouthern Japan have a monoclonal pattern of HTLV-I genome

integration (6).This report describes a patient with a rash and with morpho

logically abnormal T-cells in the skin, blood, lymph nodes, andspleen. The T-cell lymphocytosis was associated with antibodiesto HTLV-I antigens. HTLT-I genome was integrated in apolyclonal manner and there was no monoclonal rearrangementof the T-cell antigen receptor 0-chain. Thus, this disorderrepresents a previously unrecognized polyclonal T-cell proliferation in association with HTLV-I.

MATERIALS AND METHODS

Immunofluorescence. Mononuclear cells were isolated from peripheral blood or teased spleen (obtained postmortem) by Ficoll-Hypaquecentrifugation and analyzed with monoclonal antibodies, by indirect

Received 7/7/87; revised 11/30/87, 1/26/88; accepted 2/8/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by USPHS Grants CA 40192, and CA35473 and an Investigator Award to D. N. P. from the Cancer Research Institute.

2Cornell Scholar in Biomedicai Science. To whom correspondence should beaddressed, at Department of Medicine, Cornell University Medical College, Rm.S811, 515 E. 71st St., New York, NY 10021.

3The abbreviations used are: HTLV-I, human T-cell leukemia virus type I;ATL, adult T-cell leukemia; MAb, monoclonal antibody; LGV, lymphogranulomavenereum.

immunofluorescence using flow cytofluorography as described elsewhere (11). Intracellular staining of bone marrow aspirate was performed as previously described (11).

The antibodies are a T3 MAb (IgG2.; United Biomedicai Inc., LakeSuccess, NY) which recognizes all T-cells, a T4 MAb (IgG2.; UnitedBiomedicai Inc.) which identifies helper T-cells, a T8 MAb (IgGi;United Biomedicai Inc.) which identifies suppressor and cytotoxic Icells, the Tac MAb, graciously provided by Dr. T. Waldmann, whichidentifies the interleukin 2 receptor Â«chain, a MAb to an invariabledeterminant of la (VG2.1), provided by Dr. Shu Man Fu, an antimon-ocyte MAb, 63DIII (12), provided by Dr. J. D. Capra, and MAb toimmunoglobulin heavy and light chains obtained from the AmericanType Culture Collection.

Southern Blots. Splenic tissue and abdominal lymph nodes wereobtained at autopsy and frozen at â€”80Â°Cfor future use. These tissues

were homogenized and DNA was extracted as described elsewhere (13).Southern blots were performed using the methods of Southern (14)with modifications (15). The probes used were X-23-3 (16), an HTLV-I probe containing the entire proviral sequence, and the Hin

POLYCLONAL T-LYMPHOCYTOSIS AND HTLV I

Table 1 Immunofluorescence studies

-- - -

Fig. 1. Photomicrograph of a skin biopsy showinand large lymphocytes with cleaved nuclei infiltrating

sy showing a Pautrier's microabscess

nfiltrating the skin, x 600.

Fig. 2. Electron micrograph of a lymphocyte with a convoluted nucleusinfiltrating the skin, x 3000.

metastatic squamous cell carcinoma of the sacral ulcer. The skin hadalmost total regression of the papular eruption. A routine section ofgrossly unremarkable abdominal skin contained areas of papillary dermal thickening, but almost no lymphoid infiltrate. Only a few residualclusters of lymphoid cells were found in the epidermis and papillarydermis. Serum was positive for HTLV-I antibodies in an enzyme linkedimmunosorbent assay graciously performed by Dr. C. Saxinger (NIH,Bethesda, MD), while antibodies to HTLV-II and to the human immunodeficiency virus were absent.

RESULTS

The morphologically abnormal lymphoid cells in the peripheral blood, which were found at a frequency of approximately5-10% of all mononuclear cells, were further characterized. Byindirect immunofluorescence 78% of the mononuclear cellswere T-cells and these had primarily a helper T-cell phenotype(Table 1; Ref. 19). When the T-cells were isolated by resettingwith neuraminidase-treated sheep erythrocytes, an abnormallylarge percentage expressed activation antigens such as the in-terleukin 2 receptor a chain (Tac) and membrane la. Tac-positive and Tac-negative T-cells were isolated by indirect ro-setting (19) and examined on a Wright stained smear. Theformer were enriched in large cells with cleaved nuclei, similarto those shown in Fig. 2 (data not shown). These data andresults presented elsewhere (19) suggested that the morphologically abnormal cell found in the blood corresponded to a T-cell with the following phenotype: T4+, T8â€”,T3+, Tac+, Ia+.

AntibodyT3T4T8TaclaÂ«

+XAnti-IgDAnti-IgMAnti-IgGAnti-IgA63DIIIMembraneimmunofluorescenceSplenicPBMNC"

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entire genome of this virus. The DNA was digested with therestriction enzyme Bamlll and analyzed by Southern blotting(Fig. 3A, RAC). Multiple fragments with sizes varying between20 and 2 kilobases contained sequences hybridizing specificallyto the HTLV-I probe. DNA from a normal individual contained

no such sequences (NL). In contrast to the findings with thepatient (RAC) T-cell DNA from 2 cases of ATL (HI and H2)contained only a small number of BamHl fragments hybridizingwith HTLV-I (Fig. 3Ã„).Two BamHl sites have been describedwithin the intact HTLV-I proviral genome, so that this enzymemay yield an internal fragment of 1 kilobase and two variableflanking fragments for each proviral integration site (2, 6, 9,10). For example, this was observed in patient H2, indicatingone integration site and monoclonality. In patient H l only one4.3-kilobase fragment was observed without the internal 1-kilobase fragment. This indicates absence of the internalBam\\\ sites and monoclonal integration. In patient RAC thelarge number of bands observed suggests polyclonality. As withpatient HI the internal /fami 11 fragment (1 kilobase) wasabsent. Like Konishi et al. (24), we have found considerablevariability of internal restriction enzyme sites in integratedHTLV-I sequences in a larger number of patients with ATL.4

This may be due to integration of defective proviral sequences(24).

While these data strongly suggest that HTLV-I infected T-cells in this patient are polyclonal, it is theoretically possiblethat a clone of T-cells contains numerous proviral copies in-

A BRAC NL Hl H2

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6,7-

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serted in the DNA at different sites. In the absence of internalBamHl sites each band observed would represent a differentintegration site. This possibility is not usually encountered withHTLV-I, which tends to integrate a very small number ofproviral gene copies per cell (2). However, to rule out thispossibility, we examined T-cell antigen receptor ÃŸchain generearrangement of the DNA extracted from an abdominal lymphnode, which also contained DNA hybridizing with the HTLV-I probe. DNA was digested with 2 restriction enzymes (Mudiliand EcoRl). DNA from the patient (RAC) contained no detectable ÃŸchain rearrangements with either restriction enzyme (Fig.4). These data clearly demonstrate the absence of detectableclonal proliferation of T-cells in the spleen and an abdominallymph node of this patient, in spite of the presence of integratedHTLV-I sequences within these same tissues.

DISCUSSION

The combined data presented herein strongly suggest thatthe patient's morphologically abnormal T-cells were infected

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Fig. 3. Southern blot of genomic DNA isolated from the patient's spleen

obtained at autopsy (RAC), of normal leukocyte genomic DNA (NL), and ofleukemic T-cells from 2 patients with ATL (HI and H2). Twenty-five microgramsof DNA were loaded on each lane. The DNA was digested with the restrictionenzyme BamHl. A full-length HTLV-I probe was used. Molecular size markerswere obtained from X phage DNA digested with Hindlll (A) or from knownrestriction fragments containing HTLV-I sequences (B).

4 M. J. Macera and P. Szabo, unpublished data.

Fig. 4. Southern blot of genomic DNA digested with Hindlll (lanes 1-5) orEcoRl (lanes 6-10). The probe was specific for the ÃŸchain of the T-cell antigenreceptor (17). Lanes S and 10 contain DNA obtained postmortem from anabdominal lymph node of the patient described herein, lanes 1 and 6 containDNA obtained from an Epstein-Barr virus infected B-lymphoblastoid cell line(representing germline for the T-cell antigen receptor genes), and lanes 2-5 and7-9 contain the same germline DNA mixed with increasing amounts of DNAfrom the T-cell leukemia, FF (26), which represents a monoclonal proliferationof T-cells with a specific ÃŸchain rearrangement detected with both restrictionenzymes. Germline restriction fragments (C>)of 7.5 and 3.3 kilobases (for 7/i'nd111)

and 11 and 4 kilobases (for EcoRl) were found, as described by others (25). Therearranged bands of FF DNA M) could still be distinguished at dilutions of 10-20%, thus defining the sensitivity of this assay.

2587

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with HTLV-I. This conclusion is based on the presence ofclinically suggestive findings such as the skin lesions, the morphology of the abnormal T-cells in various tissues, the correctimmunological phenotype of these T-cells (Tac+) (21 ), and thefindings of specific serum antibodies to this virus and integratedvirally encoded sequences in the patient's spleen and lymph

node tissue. Previous reports have uniformly described patternsof HTLV-I integration which suggest monoclonality of theinfected T-cells, even in patients with preleukemic states or inHTLV-I carriers (2, 6, 9, 10). Such reports contrast with invitro studies that demonstrate a stage of polyclonal infection ofT-cells prior to the development of a monoclonal proliferation(6, 7). In addition, bovine leukemia virus, a retrovirus similarto HTLV-I, causes a polyclonal T-lymphocytosis prior tomonoclonal leukemic transformation (8). This report describesa unique case of polyclonal T-cell proliferation in associationwith HTLV-I.

The spontaneous resolution of the papular rash, the lymph-adenopathy, and the T-lymphocytosis in this patient are inconsistent with a diagnosis of lymphoproliferative disorder. Alsothe alkaline phosphatase was not elevated and there was noevidence of hypercalcemia or lytic bone lesions to suggest adiagnosis of ATL (20). HTLV-I infection does not invariablyresult in T-cell leukemia since asymptomatic patients and carriers have been described. Similar cases of HTLV-I-infectedindividuals with morphologically abnormal T-cells, yet withoutapparent lymphoproliferative disorder, have been described bySarin et al. (27) and Matutes et al. (28), usually in the settingof a family study on relatives of ATL patients.

The patient's clinical history is remarkable for the severe

chronic infection due to Chlamydia trachomatis, the etiologicalagent of LGV (29). It is unlikely, however, that the abnormalT-cells were a direct consequence of this infection, since Ch.trachomatis is known to parasitize mucosa! columnar epithelialcells and cells of the monocyte-macrophage lineage (30), butnot T-cells. High serum IgG and IgA levels have been reportedin association with LGV (31 ). However, in the present case thehigh levels of IgG and IgA correlated with the ability of theabnormal T-cells to effect an isotype switch to these isotypes(19). Thus, the serum Ig levels were under control of thepatient's abnormal T-cells, thought to be infected with HTLV-

I. To what extent, if any, the sacral ulcÃ©rationand the squamouscell carcinoma contributed to the T-lymphocytosis will remainunresolved. However, the morphology of the T-cells, their Tac+phenotype, the presence of anti-HTLV-I antibodies, and thepresence of HTLV-I sequences in lymphoid tissues togetherrepresent strong evidence that HTLV-I was the cause of theobserved T-lymphocytosis.

In vitro studies, described in detail elsewhere ( 19) and summarized in Table 2, have characterized the T-cells isolated byPercoli density centrifugation either from the peripheral bloodof this patient or from the spleen obtained postmortem as"switch T-cells." The question could be raised of whether theswitch T-cell effect observed with these cells was due to in vitroHTLV-I infection of the hyper-IgM immunodeficient B-cells(22) and normal nllÃ¼gendeB-cells (19). However, this is unlikely since reports on the generation of HTLV-I-infected human B-cell lines do not suggest that such transformed cellsinvariably undergo the immunoglobulin switch (4, 32, 33). Infact, several of these B-cell lines produce IgM rather than IgGor IgA. In addition, we have examined HTLV-I-associated T-cell malignancies and HTLV-I-infected T-cell lines for switchT-cell effects in coculture experiments and have been unable toreproduce the findings obtained with the abnormal T-cells

(Trac) from the patient described herein. Possibly, transactiva-tion by HTLV-I (34) of a host T-cell gene resulted in switch T-cell activity in this particular patient. Since the very existenceof such switch T-cells has been difficult to prove, it is importantto identify further cases of human T-cells with the capacity toinduce the immunoglobulin isotype switch.

ACKNOWLEDGMENTS

We thank Drs. G. Siskind, W. Reeves, and R. Nachman for helpfulsuggestions.

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1988;48:2595-2589. Cancer Res David N. Posnett, Helen A. McGrath, Rachelle A. Scott, et al. T-Cell Leukemia Virus IPolyclonal Lymphocytosis of T-Cells Associated with Human

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