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Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 94 No. 7 (October 1), 1999: pp. 2374-2382 Clonal Expansion of -T Lymphocytes With Inverted J1 Bias in Familial Hemophagocytic Lymphohistiocytosis By Mitsuyuki Nagano, Nobuhiro Kimura, Eiichi Ishii, Nobuyuki Yoshida, Tetsuya Yoshida, Masahiro Sako, Shigeyoshi Hibi, Shinsaku Imashuku, Sumio Miyazaki, Toshiro Hara, and Shuki Mizutani From the First Department of Internal Medicine, Fukuoka University School of Medicine, Fukuoka, Japan; the Division of Pediatrics, Hamanomachi Hospital, Fukuoka, Japan; the Department of Pediatrics, Saga Medical School, Saga, Japan; the Division of Pediatrics, Osaka City General Hospital, Osaka, Japan; the Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan; the Department of Virology, National Children's Hospital Medical Research Center, Tokyo, Japan; and the Department of Pediatrics, Kyushu University, Fukuoka, Japan.     ABSTRACT TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Familial hemophagocytic lymphohistiocytosis (FHL) is a rare but fatal disease in infancy. There are no previous reports on the clonality of T cells in FHL patients. We analyzed here the clonality of -T cells in 5 FHL patients using an inverse reverse transcriptase-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region gene (TCR V), a joining region gene of the  chain (J)-PCR, a single-strand conformation polymorphism (SSCP), and sequence analysis. A high frequency (15%) of V and V families was observed in 3 of 5 and 4 of 4 patients examined, respectively. In 19 V repertoires, including all highly frequent V, the J-PCR analysis showed restricted usage of the J family, indicating a marked bias to J1 subsets (the mean rate of J1:J2 was 87:13 in 65% of the -T cells) in widespread -T cells (in all patients but 1). In all patients, the clonality of specific V-J fragment expanded was confirmed by SSCP and sequence analysis. These results suggest that the existence of clonal expansion and restricted J1 usage of T cells in FHL is genetically associated with the pathogenesis and the immunodysfunction of the disease. These results help to explain some of the abnormal functional behaviors of T cells in FHL and raise new questions regarding the mechanisms responsible for the restricted clonal diversity. © 1999 by The American Society of Hematology.     INTRODUCTION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES FAMILIAL HEMOPHAGOCYTIC lymphohistiocytosis (FHL) is a rare genetic disorder of the mononuclear phagocyte system characterized by fever, hepatosplenomegaly associated with pancytopenia, hypertriglyceridemia, and hypofibrogenemia.1,2 Despite intensive chemotherapy, most patients with FHL relapse and die of progressive disease.3,4 Only bone marrow transplantation (BMT) has achieved a long remission in some cases.5,6 FHL has been considered a disorder secondary to T-cell dysfunction.7,8 Uncontrolled T-cell activation by an abnormal immune response results in a large amount of inflammatory cytokines that promote macrophage infiltration and the formation of a cytokine network.2,9 Increased numbers of activated T cells or atypical lymphoid cells are observed in the circulation of affected individuals.4,10 High levels of interferon- (IFN-), soluble interleukin-2 (IL-2) receptor produced by activated T cells, and IL-6 and tumor necrosis factor (TNF) secreted mainly by activated macrophages have been detected in FHL patients.9,11,12 Immunosuppressive drugs, such as cyclosporine A and steroids, are sometimes effective for maintenance therapy in FHL patients.13 The clonal dissemination of T cells has recently been reported in patients with hemophagocytic syndrome. Especially in the majority of patients with Epstein-Barr virus (EBV)-associated hemophagocytic syndrome, infected cells have been shown to proliferate monoclonally.14,15 However, there is no evidence of the clonal origin of T cells in FHL patients. In the present study, we analyzed the clonality of T cells increased in the circulation of 5 FHL patients using an inverse reverse transcriptase-polymerase chain reaction (RT-PCR) for T-cell receptor variable region gene (TCR V), a joining region gene of the  chain (J)-PCR, followed by a single-strand conformation polymorphism (SSCP) and sequence analysis. Our findings suggest that the existence of clonal expansion and restricted J1 usage of T cells in FHL is genetically associated with the pathogenesis and immunodysfunction of the disease.     MATERIALS AND METHODS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Patients.   Samples from a total of 5 patients were available for the study. In the absence of a specific marker for the disease, the diagnoses of all 5 patients were based on the criteria of FHL by the Histiocyte Society and as described elsewhere.1,16,17 Clinical findings, including cytopenia, hypofibrinogenemia, and hypertriglyceridemia, were observed in all patients. They showed the increased histiocytes with hemophagocytosis in peripheral blood, bone marrow, and/or cerebrospinal fluid. Hepatosplenomegaly was also observed in all patients at onset. All of the patients had a family history of affected siblings. No apparent infectious agents, including cytomegalovirus and EBV, were detected in the peripheral blood of all the patients. The natural killer (NK) cell activity and lymphocyte subpopulation at onset in the 5 patients are shown in Table 1. Low NK activity (1% to 9%) was observed in 3 of the 4 patients examined; the NK activity in patient no. 2 was 19% at onset, but it decreased to 1% 1 month after the treatment. An increased number of activated T cells was observed in 4 of the 5 patients examined. Although most of the patients with FHL described in previous reports showed low NK activity or a high number of activated T cells,17,18 these findings were not necessary for the definite diagnosis of FHL.19 Chemotherapy, including prednisolone, vincristine, etoposide, cyclophosphamide, methotrexate, 6-mercaptopurine, cytosine arabinoside, or anthracyclines, was administered to these patients. Allogeneic hematopoietic stem cell transplantation (HSCT) was applied for 3 patients. Only the 2 patients who received cord blood stem cell transplantation (CBSCT) have survived, with several relapses; 2 patients died of progressive disease despite chemotherapy, and 1 achieved remission with chemotherapy but died of disease progression after allogeneic BMT.                                View this table: [in this window] [in a new window]   Table 1. NK Cell Activity and Lymphocyte Subpopulation of 5 FHL Patients at Onset V and V analysis of T cells in FHL patients.   Blood samples of peripheral blood (4 patients) or bone marrow (1 patient) from the FHL patients were obtained at onset and showed an increased number of atypical lymphoid cells or histiocytes with hemophagocytosis. Mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation, and total cellular RNA was extracted according to a previously described method.20,21 The T-cell receptor  and  chain variable region (TCR V and V) repertoires were analyzed by the newly devised RT-PCR method.22 Briefly, double-stranded cDNA was synthesized, followed by circularization and the inverse PCR using 2 constant region primers that are in opposite orientations.23 Oligo(dT)-primed double-stranded cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus-derived reverse transcriptase, RNase H, Escherichia coli DNA polymerase I, and E coli DNA ligase, followed by incubation with T4 DNA polymerase for blunt-end formation. The blunt-ended cDNA was circularized with T4 DNA ligase in a volume of 10 µL. The ligated material (5 µL) was used as template for the PCR. The PCR constant (C) primers used were as follows: C forward primer, C inverse primer, C forward primer, and C inverse primer. After 35 cycles of PCR (denaturation at 95°C for 0.5 minutes, annealing at 62°C for 0.5 minutes, and extension at 72°C for 1 minute), the Klenow fragment of E coli DNA polymerase I was added to ensure full-length DNA synthesis. The sequences of C and C primers used are shown in Table 2. The common nucleotide sequences in 2 constant regions of human TCR  chain genes were used.                                View this table: [in this window] [in a new window]   Table 2. Primers Used for PCR Assays Two hundred nanograms of each V fragment (from V1 to V20) or V fragment (from V1 to V18, V21, and V24) was dotted on the filters. Each V-specific fragment was prepared from the series of HBVT/HBVP or HAVT/HAVP plasmids originated from thymus or peripheral T cells.24-26 Fifteen microliters of amplified PCR products was labeled by -32P-dCTP and hybridized to the filter, including V segments. Using densitometry, a semiquantitative assessment of V gene usage was made from the amount of hybridized products. In vitro stimulation by superantigen.   Staphylococcusaureus enterotoxin (SEB, SEA, SEC2, SEC3, and SEE) and toxic shock syndrome toxin 1 (TSST 1) were used as bacterial superantigens to stimulate T cells in a V-specific fashion. Control peripheral T cells were isolated from the blood of 2 healthy donors and analyzed before or after stimulation with bacterial superantigen (1 µg/mL; Toxin Technology, Madison, WI). A solution of the protein (1 µg/mL) was incubated on the well surface for 8 hours at 4°C. Nonadherent protein was removed by extensive washing. This plastic-adherent superantigens were used to stimulate the peripheral blood T cells (5 × l05/mL). Three days later, live cells were collected and RNA was prepared for the analysis of TCR V repertoires. As shown in Fig 1, although the in vitro stimulation with a superantigen appears to have almost no effect on the V repertoire (data not shown), the results obtained with the new PCR method suggested that all of the toxins used preferentially stimulated T cells expressing particular Vs, consistent with the results of previous studies.27 View larger version (36K): [in this window] [in a new window]   Fig 1. V repertoire stimulated with superantigens in healthy controls. () Healthy donors as the control; () stimulated with a superantigen. Peripheral T cells were isolated from the blood of 2 donors and analyzed before or after in vitro stimulation with a bacterial superantigen (1 µg/mL) for 3 days. All of the toxins preferentially stimulated T cells expressing particular Vs.27 Clonal analysis of V family.   A clonal analysis was then performed using PCR with 13 sets of J primers (J-PCR) and an SSCP and sequence analysis.28,29 The sequences of V and J primers used are also shown in Table 2.30 Besides the FHL specimens, as control subjects, 3 infants (a 3-month-old healthy infant, a 2-month-old patient with hepatitis, and a 2-year-old patient with diarrhea) were investigated. cDNA was amplified using primers specific to each V family and J primer. The amplified products (V-J) were examined with the same volume of loading solution (0.05% xylene cyanol, 20 mmol/L EDTA, and 95% formamide), incubated at 96°C for 5 minutes, and rapidly chilled on ice. Samples were electrophoresed at a constant voltage of 100 V for 3 hours on a 0.5× MDE gel (AT Biochem, Malvern, PA). The gel was stained with SYBR green (1 µg/mL; FMC Bio Products, Rockland, ME), and the bands were detected and photographed on a UV translluminator.31 Finally, sequence analysis was performed. The V-J amplification fragments were purified by electrophoresis in low melting point agarose. Fragments were ligated into pT7Blue T-Vector (Novagen, Madison, WI). After transformation of Epicurian Coli XL II-Blue (Stratagene, La Jolla, CA), 6 individual colonies were selected for sequencing by ABI PRISM 377 DNA sequencer (Perkin-Elmer, Forster City, CA). Data were analyzed by PC-Gene (IntelliGenetics, Mountain View, CA).     RESULTS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES TCR V and V repertoire in FHL patients.   The frequency of the TCR V repertoire in peripheral -T cells of 4 FHL patients (patients no. 1 through 4) and in bone marrow -T cells of patient no. 5 is shown in Figs 2 and 3. Although there was no preferential usage of a V family member in patients no. 1 and 3, high expressions (>15%) of V5a (39%) and 5b (32%) in patient no. 2, of V6 (17.5%) and 13 (24%) in patient no. 4, and of V6 (61%) in patient no. 5 were observed. The TCR V repertoire was also analyzed in the 4 FHL patients except patient no. 1. High frequencies (>15%) of V3 (20%) and 9 (25%) were detected in patient no. 2, of V3 (47%) in patient no. 3, and of V9 in patient no. 4 (28%) and patient no. 5 (15.4%). There was no correlation between V and V family usage in the FHL patients. View larger version (150K): [in this window] [in a new window]   Fig 2. Dot blotting of TCR V and V repertoires of 5 patients with FHL. Fifteen microliters of products that was amplified by an inverse RT-PCR using the set of constant region primers was labeled by -32P-dCTP and hybridized to the V and V filter prepared with 200 ng of each variable gene segment, respectively. View larger version (32K): [in this window] [in a new window]   Fig 3. Frequency of TCR V and V family in FHL patients. With densitometry, a semiquantitative assessment of V gene usage was made from the amount of hybridized products. The dotted line shows 15% of -T cells. TCR V gene segments were regarded as highly expressed when their relative frequency was more than 15%. A highly expressed V family member was observed in 3 of the 5 patients: V5a and 5b of patient no. 2 and V6 of both patients no. 4 and 5. A high frequency of a V family member was detected in all 5 patients: V3 and 9 in patient no. 2, V3 in patient no. 3, and V9 in patients no. 4 and 5. J usage in V of FHL patients.   A J-PCR was then performed on the highly expressed V subset(s) (>15%) for each patient using each V specific primer and a set of J primers (Figs 4 and 5). In patient no. 2, V5a and 5b used J1.5 (24%) or J1.6 (56%) and J1.2 (75%), respectively. In patient no. 4, V6 exclusively used J1.1 (95%), whereas V13 used several genes of only the J1 subgroup (99%). V6 in patient no. 5 exclusively used J1.1 (86%). These findings show that the highly frequent V families observed in FHL patients use only the J1 subgroup. View larger version (28K): [in this window] [in a new window]   Fig 4. Southern blot analysis of PCR-amplified V-J segments in FHL patients. The amplified products were respectively hybridized with each V fragment probe. *Underlining shows a V gene with high frequency (15%). Preferential J usage in highly frequent V families is observed. View larger version (68K): [in this window] [in a new window]   Fig 5. Frequency of J usage in V repertoire in FHL patients. Autoradiograms were scanned by computerized densitometry, providing an absolute value for each autoradiographic spot. Each V-J spot is expressed as a percentage of the sum of all of the respective V-J signals detected on the autoradiogram. Some V family members expressed within the normal range were also investigated in each patient. In the TCR V genes with a high frequency (15%), only 1 member of the J1 gene subgroup was highly expressed in 3 patients. Even among the other V family members expressed within the normal range, 1 of J1 with overexpression and J prediposed bias were observed: V13 in patient no. 1, V3 and 4 in patient no. 3, V1 and 15 in patient no. 4, and V13 in patient no. 5. The transplantation of peripheral mononuclear cells in patient no. 1 to scid mice caused an FHL-like physiological disorder. CD4CD8 -T cells were infiltrated in mice organs and the clonal expansion of V13-J1.2 was recognized, which was described elsewhere.32 Therefore, the clonality of V13 (5.6%) in peripheral -T cells was studied in patient no. 1 and the accumulation of the same J1.2 (88%) was observed (Figs 4 and 5). Several V subsets with a frequency rate less than 15% were investigated in each patient as well at random. These V family members used a variety of J gene families, suggesting polyclonality: V3 (12%) in patient no. 1, V4 (4%) in patient no. 2, V2 (4.9%) and 8 (4.8%) in patient no. 3, V4 (9.6%) in patient no. 4, and V3 (11.6%) in patient no. 5. Polyclonal V13 was recognized in patients no. 2 (4%), 3 (9.5%), and 5 (3%) (Figs 4 and 5). Even in part of those V subsets, a bias of J1 gene usage was observed. Both V1 and V15 in patient no. 4 exclusively used J1.1 (73%) and J1.4 (99%), suggesting clonality in each of these V family members. The J usage of V13 and 6 in 3 control individuals is shown in Fig 6. Compared with FHL patients, both V6 and V13 generally used a variety of 13 J genes with a bias for J2; the average J1:J2 ratios as a whole were 42:58 for V6 and 33:67 for V13. There was no J gene usage greater than 20%. View larger version (42K): [in this window] [in a new window]   Fig 6. J usage of V13 or V6 in control infants. Both V6 and V13 generally used a variety of 13 J genes with a bias for J2; the average J1:J2 ratios as a whole were 42:58 for V6 and 33:67 for V13. There was no J gene usage greater than 20%. SSCP analysis.   Part of the PCR products amplified by each V and J primer were examined by the SSCP analysis to investigate the clonality of V-J families. On the SSCP gel (Fig 7), there were no distinct bands detectable in the amplified products of each V family of the normal control or in expanded T cells (V4-J1.4) by the in vitro stimulation with TSST-1 superantigen (Fig 7). By contrast, a distinct band(s) was detected in amplified products of several highly frequent V family members in the FHL patients; V5a-J1.6 in patient no. 2 (lane 4) and V6-J1.1 in both patients no. 4 and 5 (lanes 6 and 7). Even in low expressed V repertoires, a clear band(s) suggesting clonality was observed: V13-J1.2 in patient no. 1 (lane 1) and V1 and 15 in patient no. 4 (not shown). These data suggest that some restricted -T-cell clone(s) dominantly proliferates in the peripheral blood or bone marrow of FHL patients. View larger version (93K): [in this window] [in a new window]   Fig 7. Clonality by SSCP analysis. Healthy peripheral blood mononucler cells (PBMC) as a control were investigated. Lane 2 (V5a-C) and lane 10 (V13-C); lane 8, V4-J1.4 of healthy PBMC stimulated with TSST1; lane 1, V13-J1.2 in patient no. 1; lanes 3, 4, and 5, V5a-C, V5a-J1.6, and V5b-J1.2 in patient no. 2, respectively; lanes 6 and 12, V6-J1.1 and V13-C in patient no. 4, respectively; lane 7, V6-J1.1 of bone marrow in patient no. 5; lanes 9 and 11, V4-J1.4 and V13-C in patient no. 3, respectively. Smears are shown in lanes 2, 5, 8, 10, 11, and 12. Distinct bands are observed in lanes 3, 4, 6, 7, 9, and 1, suggesting clonal expansion. Sequence analysis of TCR -chain transcript.   To confirm that the SSCP analysis of the predominant V fragment amplified from each patient represented the clonal expansion, the fragments were subcloned into plasmids and sequenced. Six individual colonies were analyzed in each fragment. As summarized in Table 3, the expressed V gene in patient no. 1 was V13.6, and residues comprising the NDN junction were TSAL. A similar analysis was performed on the dominant V gene for the remaining 4 patients. The sequence identified in 83% and 50% of the recombinant plasmids were identical, which indicates clonally expanded cells.                                View this table: [in this window] [in a new window]   Table 3. Structural Features of TCR -Chain Transcripts Expressed by FHL Clone(s) There are submembers in some V families. It was indicated that V5a in patient no. 2 is V5.1, that V4 in patient no. 3 is V4.1, and that V6 in patients no. 4 and 5 is V6.4 and V6.5, respectively. In all patients, the junctional sequences expressed by each clone were unique. Table 4 shows the summarized data. In all 5 FHL patients, the clonality of -T cells was demonstrated. The high frequency of 5 V subsets was found in 3 FHL patients. Three of these five V subsets clonally expanded using 1 of 6 J1 genes. A total of 19 V subsets, including 14 within the normal range, were investigated. Of the 14 V with a frequency rate less than 15%, 4 V were also suggested or confirmed to be a clone(s). Fifteen of the 19 V subsets showed a bias for J1 subsets (>70%). In patient no. 2, 79.3% of the peripheral -T cells use J1 over J2 (89.7:10.3). The marked bias for J1 usage (the mean rate of J1:J2 was 87:13) was to be confirmed by densitometry in all but 1 patient (patient no. 1).                                View this table: [in this window] [in a new window]   Table 4. Clonal Expansion and J Bias of -T Cells in 5 FHL Patients     DISCUSSION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES The genetic basis of FHL, a rare inherited disorder characterized by multivisceral infiltration by lymphocytes and histiocytes, is still unknown. Although it is now accepted that FHL is a disorder of T-cell dysfuction, clonality of T cells in FHL patients has not been reported. In the present study, we obtained evidence of clonal expansion of -T cells with J1 bias in all 5 FHL patients studied. Immunodysfunction has been speculated in widespread pan--T cells. Because activated T cells and various cytokines derived from T cells are increased in the circulation of FHL individuals, it can now be speculated that those clonally expanded T cells may produce high levels of inflammatory cytokines followed by the activation of macrophages with hemophagocytosis. Concentrating on the TCR  chain, a paucity of clonotypic T-cell expansion has been demonstrated in the peripheral blood of healthy individuals by using the PCR method and a subsequent SSCP analysis.31 A number of recent reports have established that oligoclonality and/or clonal expansion is a common feature of the CD8+ T-cell population. Posnett et al33 documented that clonal TCR T-cell populations are frequent in normal subjects more than 65 of age, and they showed that these populations are usually clones of CD8+CD28 cells. In addition, Morley et al34 reported that oligoclonal CD8+ T cells are preferentially expanded in the CD57+ subset and that the oligoclonal expansion is a characteristic feature of the normal immune system. In the present study, low NK activity and an increased number of activated T cells (CD3+/HLA-DR+ cells) were observed in 4 of 4 and 4 of 5 FHL patients examined, respectively. In patient no. 1, with an increased number of activated T cells, the clonal T lymphocyte with V13.6-J1.2 was CD4CD8 (double negative [DN]), and these T cells caused an FHL-like physiological disorder in transplanted scid mice.32 In a previous analysis of normal donors, the expressions of V2, 8, 11, and 13 were reported to be markedly increased in DN -T cells.35 Therefore, it is unlikely that clonally expanded T-cell subsets (V5a, 6, and 6 in patients no. 2, 4, and 5, respectively) are DN. As described above, it appears that expanded T-cell subsets in each FHL patient are not limited to only one common T-cell subclass. It would be worthwhile to determine whether the clonal T cells expand by their own abnormality or by the reaction to some antigens or superantigens. Based on the preferential usage of different V and J genes in each of our patients, we propose that FHL is not a superantigen-mediated disease. In FHL patients who exhibit an excess of HLA DR+CD3+ T cells, it would be worthwhile to check for an abnormal repertoire in separated HLA DR+ and HLA DR T cells. This study would help to recognize whether possible oligoclonality reflects selected T-cell activation or a truly abnormal repertoire. In the present study, the highly frequent V family members observed in FHL patients used only the J1 subgroup. In addition, a markedly inverted bias to J1 usage (the mean J1:J2 ratio of 87:13) was observed in many -T cells (65%), suggesting an association with the genetical pathogenesis in FHL. All helper and cytotoxic T cells reported so far have been shown to rearrange and express TCR  and  genes. The use of either of the 2 C regions does not correlate with either the class of T cells or the class of major histocompatibility complex (MHC) molecules that are recognized, although the C2 gene is used more often in the T-cell population as a whole.36 An analysis of the J gene in TCR chain message (human T cells) showed that J1 or J2 genes are expressed in a VDJC fragment including C1 or C2, respectively.30 The J2 family was used more commonly than J1 in healthy individuals, indicating the preferential use of J2 over J1 (72%:28%).37 Our results obtained from 3 controls also indicated a similar trend of the J bias. Rosenberg et al37 indicated that this J bias must result from events associated with the rearrangement itself, before subsequent selection pressures are applied to the repertoire. Therefore, the marked inverted bias in only the J1 usage of FHL may be associated with the genetic pathogenesis and/or with thymic-positive selection. It should be clarified as to whether the clonal T cells and the J1 bias in widespread T cells of FHL patients are responsible for the mechanism of T-cell dysfunction. In severe combined immunodeficiency (SCID) patients, the unusual functional behavior of maternal T cells has been explained as the result of profoundly reduced T-cell receptor (TCR) diversity.38 Those investigators demonstrated the lack of one or several TCRBV segments in SCID patients.38,39 Sottini et al40 documented that TCRBV transcripts were characterized by extremely restricted V-D-J junctional diversity in an SCID patient. The functional alteration of these cells appears to be ascribable to an insufficient TCR diversity. The abnormal inverted J1/J2 usage rate might mean the limitation of the size of the TCR member. Subtle changes in the T-cell functions that may be due to the J bias can genetically influence the regulation of the T-cell network in FHL, resulting in hyperactivation and widespread multiorgan infiltration by lymphocytes and histiocytes. FHL is generally considered a nonneoplastic disorder, but its treatment requires chemotherapy and subsequent HSCT. Stephan et al8 recently recommended immuno-suppressive agents (steroids, antithymocyte globulins, and cyclosporine A) as alternative primary and maintenance therapy. Their effective results also support the key role of T cells in the disease. During the clinical course of our FHL patients, polyclonal V13 T-cell lymphoproliferative disease developed in patient no. 1 with a relapse after an allogeneic BMT, and the clonal change (V5.1-J1.6 to V4) of expanded T cells in patient no. 2 occurred; both patients died. An association with EBV was suggested in both of these relapse cases.41 Patients no. 3 and 4 received CBSCT and are now in remission. In FHL patients with low NK activity and unbalanced regulation of T cells by widespread J bias who are undergoing immunosuppressive therapy, virus infection might induce more aggressive disease. Henter et al1 suggested that various viral infections may elicit a bout of FHL disorders in genetically predisposed individuals. Finally, FHL is often indistinguishable from other types of hemophagocytic syndromes42 in neonates and infants. Clonal T cells may also be present in patients with infection-associated HL. A comparison of the V repertoire and J usage in such patients would be of interest. An unbalanced regulation of T cells by widespread J bias in -T cells may contribute to the pathogenesis of FHL. The association of the J bias of clonal T-cell subpopulations and the genetic pathogenesis of FHL remain topics of ongoing study.     FOOTNOTES Submitted August 3, 1998; accepted May 27, 1999. Supported in part by Grants-in-Aid for General Scientific Research from the Ministry of Education, Science, and Culture. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. Address reprint requests to Nobuhiro Kimura, MD, The First Department of Internal Medicine, Fukuoka University School of Medicine, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-01, Japan.     REFERENCES TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES 1. Henter J, Elinder G, Ost A: Diagnostic guidelines for hemophagocytic lymphohistiocytosis. Semin Oncol 18:29, 1991[Medline] [Order article via Infotrieve] 2. Loy TS, Diaz-Arias AA, Perry MC: Familial erythrophagocytic lymphohistiocytosis. Semin Oncol 18:34, 1991[Medline] [Order article via Infotrieve] 3. Fischer A, Virelizier JL, Arenzana-Seisdedos F, Perez N, Nezelof C, Griscelli C: Treatment of four patients with erythrophagocytic lymphohistiocytosis by a combination of epipodophyllotoxin, steroids, intrathecal methotrexate, and cranial irradiation. Pediatrics 76:263, 1985[Abstract/Free Full Text] 4. Janka GE: Familial hemophagocytic lymphohistiocytosis: Diagnostic problems and differential diagnosis. Pediatr Hematol Oncol 6:219, 1989[Medline] [Order article via Infotrieve] 5. Blanche S, Caniglia M, Girault D, Landman J, Griscelli C, Fischer A: Treatment of hemophagocytic lymphohistiocytosis with chemotherapy and bone marrow transplantation: A single center study of 22 cases. Blood 78:51, 1991[Abstract/Free Full Text] 6. 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