Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia

RECENTLY WE described that T cells from most patients with hairy cell leukemia (HCL) exhibit a marked skewed repertoire of the T-cell receptor (TCR) genes.1 In individual patients, different oligoclonal patterns were observed after polymerase chain reaction (PCR) amplification of the junctional regions of the rearranged TCRG and TCRB genes.1 These abnormalities can in part explain the abnormal cellular host defense and extreme sensitivity to opportunistic infections of these patients.2-8 We wondered whether the TCR repertoire would normalize with treatment of the disease. Most patients respond with partial remissions upon interferon-α (IFN-α) therapy with normalization of blood parameters8-10 and gradual restoration of T-cell immunity.11-13 Although newer therapy regimens with 2-deoxycoformycin and 2-chlorodeoxyadenosine are followed by more complete remissions than with IFN-α,14-16 both cytostatic drugs are characterized by a longstanding T-cell cytopenia with persistently decreased CD4⁺ T-cell counts.15,17-19 Therefore, to evaluate the role of treatment on the T-cell repertoire in patients with HCL, we studied a cohort of patients solely treated by IFN-α. Here we show that indeed many T-cell abnormalities disappeared with time. Analysis of the TCRBV repertoire at the protein level with TCRBV-specific monoclonal antibodies (MoAbs) matched the results obtained by reverse transcriptase (RT)-PCR analysis of TCRBV transcripts before and during IFN-α therapy. Moreover, these MoAbs made it possible to study specific subsets of TCRBV family-expressing T cells by multicolor immunofluorescence stainings. Finally, to confirm that the phenotypical improvement also reflected immunological improvement, we measured T-cell reactivity before and during recovery, focusing on helper, proliferative, and cytotoxic alloreactive T-cell precursor frequencies against major histocompatibility complex (MHC)-unrelated individuals.20 21 

Peripheral blood (PB) cells, collected during the last decade, from seven patients with HCL treated by IFN-α and who had undergone splenectomy were studied. The age of the patients during the present analysis varied from 29 to 61 years, with a median of 41 years (see Table 1). The diagnosis was confirmed by histology of the spleen and bone marrow (BM), cytomorphology, and immunophenotyping (reactivity with MoAbs against CD11c, CD19, CD25, CD103, and expression of monotypic Igs). Patient characteristics are summarized in Table 1. The cell samples tested were obtained before initiation and treatment with IFN-α. The majority of patients was treated with initial doses of 1.5 to 3 million international units (MIU) IFN-α2b, three times a week, followed by a further reduction of the dose when at 1 year of therapy a hematologic remission of the HCL was obtained. In most patients the IFN-α therapy was continued at ultra-low doses (about 1 MIU/wk22). Patient G (61 years old and known with HCL for 12 years) was analyzed while receiving his second course of IFN-α. Blood was collected at regular intervals during follow-up. Mononuclear cells were isolated by Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) (1.077 g/mL) density gradient centrifugation. In most cases the cells were cryopreserved in liquid nitrogen in 10% dimethyl sulfoxide until further study.

Total RNA was isolated by the guanidium isothiocyanate method as described23 or with Trizol (GIBCO-BRL, Gaithersburg, MD), according to the manufacturer's procedure. cDNA was prepared from samples of 2 μg RNA each (derived from at least 2 × 10⁶ T cells) using Moloney murine leukemia virus BRL RT (GIBCO-BRL) for 60 minutes at 37°C as described.23,24 One fiftieth of each cDNA reaction was individually amplified using a TCRBV family-specific primer and a Cβ primer. All primers have been described previously.1,25 26 Each 50-μL PCR reaction contained cDNA in 10 mmol/L Tris HCl, pH 8.4, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 20 μg/mL bovine serum albumin, 20 pmol of a TCRBV family-specific primer and the C primer, 50 μmol/L dNTPs, 20 Pm [α-³²P]dCTP (3,000 Ci/mmol/L; Amersham, Arlington Heights, IL), and 1.25 U Taq DNA polymerase. The amplification was started with a denaturation step of 4 minutes at 94°C, followed by 25 to 30 cycles, each cycle consisting of 1 minute at 94°C, 55°C, and 72°C. The number of V-Cβ amplification cycles required for the individual cDNA samples was first determined by amplifying the C region of the TCRβ cDNA for 20 to 30 cycles and visualization on an ethidium bromide–stained agarose gel. The cycle number at which the constant-region amplicons were clearly visible was chosen for amplifying the TCRBV repertoire. Clonality within each family was determined by denaturing polyacrylamide gel electrophoresis (dPAGE) and single-strand conformation polymorphism (SSCP) analysis. After electrophoresis, amplified DNAs were visualized by autoradiography.

In addition to visual analysis, the dPAGE and SSCP gels were exposed to a Storage Phosphor Screen, secured with a Phosphor Imager 455 SI and analyzed by ImageQuant NT software (all from Molecular Dynamics, Sunnyvale, CA). A selection of 1,500 images was scored by three different persons, of whom two were not aware of the clinical data. The scores for the three were concordant in greater than 90% of all images; the inter-observer variability focusing on the absence or presence of oligoclonal patterns varied between 1% and 10% per patient and per TCRBV family. The images usually visualized with a virtual Y-axis were also expressed using a Y-axis representing the relative use of total expression. To this end the counts for each pixel in a graph were added, the integral surface per graph was calculated, and the amount of radioactivity related to the total amount of all families in a single experiment.

Out of an extensive (n = 50) panel of TCRBV-specific MoAbs of the TCRBV Workshop, the following were used: BV2: E2.2E7.2 and MPB2/D; BV3: CH92, 8F10, and 5E4; BV5.1: IMMU157; BV5.2/5.3: 4H11; BV6.1: CRI304.3; BV6.7: OT145; BV7.1: 3G5; BV8.1/8.2: 56C5.2; BV9.1: FIN9; BV11.1/11.2: C21; BV12.2: VER2.32; BV13.1/13.2: BAM13; BV13.6: JU74.3; BV14; CAS1.1.3; BV16: TAMAYA1.2; BV17: C1 and E17.5F3; BV18: BA62.6; BV20: ELL1.4; BV21.3: IG125; BV22(22.1): IMMU546; BV23: AF23.27,28 These antibodies were kindly provided by Immunotech (Marseille, France), T Cell Diagnostics (Cambridge, MA), and T Cell Sciences (Cambridge, MA). Reactivity of the TCRBV MoAbs was assessed by fluorescein isothiocyanate–labeled goat–anti-mouse antibodies. After blocking with normal mouse serum, cells were further stained with phycoerythrin (PE)-conjugated MoAbs and PE-Cyanine 5 or PerCP-conjugated MoAbs of the following clusters: CD3 (Leu-4), CD4 (Leu-3a), CD8 (Leu-2a), anti–TCR-α/β (WT31 or BMA031), anti–TCR-γ/δ (11F2) from Becton Dickinson (Mountain View, CA). Fluorescence was assessed by flow cytometry (FACScan; Becton Dickinson) using CellQuest software (Becton Dickinson). Appropriate controls were included to rule out aspecific fluorescence. Only viable cells were analyzed using LDS.29 

From one patient with 13% TCRBV2⁺ cells, the CD8⁺BV2⁺ T-cell subset was sorted using a FACStar (Becton Dickinson). After amplification by RT-PCR, the PCR product was cloned into the pCR3.1 vector with a TA cloning system (Invitrogen, Leek, The Netherlands) and sequenced with the T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden) using the vector primers.

Cytotoxic and helper T-lymphocyte precursor frequencies (CTLp and HTLp) were analyzed as described.20,21 In short, responder T cells from the patient at various time points of IFN-α treatment were twofold diluted across the wells of two 96-well plates from 40,000 cells per well until a concentration of 625 cells per well was reached. To determine whether contaminating HCL cells in the responder cell populations could possibly suppress the proliferation of these cells, responder cells from active disease were tested with and without depletion of HCL cells. In addition, as a control, the same HCL cells recovered during these depletion steps were added to responder T-cell suspensions obtained after several years of IFN-α treatment. All of these cocultures were done with frozen PB mononuclear cells, and always simultaneously realized in a single experiment. Irradiated stimulator lymphocytes obtained from healthy donors, which differed at four or more HLA class I and II antigens from the responder cells, were added at 50,000 cells per well and cultured for 3 days. For the HTLp analysis, 80 μL of supernatant was obtained and interleukin-2 (IL-2) release was measured using an IL-2–dependent murine CTLL-2 cell line.21 Subsequently, IL-2 was added (120 U/mL) and the cultures were continued until day 10. IL-2 plus phytohemagglutinin (PHA)-stimulated target cells were labeled with europium (Eu) chelated to diethylenetriaminopentaacetate (DTPA) and added at a concentration of 5,000 cells per well to each responder cell concentration. After 4 hours of incubation, released Eu was measured in the supernatant using a time-resolved fluorometer, and expressed in counts per second. A well was scored positive if the counts exceeded the mean ± 3 × SD of the wells with stimulator cells and corresponding target cells only. CTLp frequencies were calculated for that responder cell concentration resulting in 37% of the tested wells remaining negative. As controls, identically treated autologous target cells were used.

Proliferative T-lymphocyte precursor frequencies (PTLp) were identically set up as described above. However, instead of IL-2 release, ³H-thymidine incorporation was measured at day 6 of culture.

All T-precursor frequencies were expressed per 10⁶ T cells.

In four of seven patients IFN-α induced a hematologic response with normalization of hemoglobin levels, white blood cell and thrombocyte counts, disappearance of circulating hairy cells, and reappearance of monocytes. In the remaining patients, the blood counts normalized but a few circulating HCL cells were still detectable (see Table 1).

The seven patients were studied before the start of IFN-α and during therapy for a period of 315 to 1,646 days. Some of the results in six patients obtained before the start of IFN-α have been described before.1 RT-PCR analysis with a panel of 24 primers of junctional regions of TCRB transcripts from 22 well-established TCRBV families analyzed on dPAGE gels disclosed oligoclonal and polyclonal (ie, regular ladder patterns) configurations for the individual TCRBV families in all seven patients. Markedly abnormal patterns were found with clonal bands in many different BV families. In addition, several gaps in the TCRB repertoire were present with BV families missing or showing abnormal weak signals compared with normal age-matched controls. For each family the junctional region size distribution patterns were recorded using a Phosphor Imager, allowing comparison of the samples during treatment (Fig 1). SSCP gel analysis of the TCRBV repertoire confirmed the clonal excess patterns seen on the dPAGE gels. Follow-up during IFN-α therapy disclosed a remarkable improvement of the skewed TCRBV repertoire in all patients, which became more clear only after 2 or more years of therapy. To illustrate the slow rate of this response, a detailed overview of seven abnormal BV families in one patient followed for 3.3 years is given in Fig 1. Not only did oligoclonal populations disappear, but also a reappearance of polyclonal patterns was observed. The latter is illustrated for several BV families in two patients in Fig 2, which clearly shows the restoration of some of the affected BV families, both quantitatively and qualitatively. The combined results of the RT-PCR analyses of 22 TCRBV gene families in the seven patients at the start of IFN-α therapy and after long-term follow-up are summarized in Fig 3.

To match the findings at the RNA level with TCRBV protein expression, we determined in four patients with active disease the percentages of CD3⁺ T cells in the blood that expressed particular TCRBV families. For this purpose we used a panel of 25 MoAbs covering about 65% of the TCRBV repertoire in healthy individuals.27 28The results were compared with a panel of five normal donors, as shown in Fig 4. In all four patients large T-lymphocyte expansions (percentual and numerical) expressing a single BV family were seen. In two of four patients tested, more than 22% and 25% of the CD3⁺ T lymphocytes expressed a single TCRBV family. Detailed flow cytometric analysis showed that in most cases the expanded TCRBV families concerned the CD8⁺ T lymphocytes, although “clonal” CD4⁺ populations were also seen (Table 2). We calculated the absolute numbers of circulating TCRBV⁺ cells to illustrate the presence of large expansions of single BV⁺ expressing T cells (Table 3). For example, patient A with 40% of the CD8⁺ cells being BV3⁺ harbored a total of 617 × 10⁶BV3⁺ cells/μL in the blood during active disease. An excess of BV-family expression was almost always concordant with clonal populations found by RT-PCR.

To analyze whether the excessive expression of some BV families represented true clonal expansions of individual members, we sorted the CD8⁺BV2⁺ cells from patient G. cDNA obtained from the sorted cells was amplified with TCRBV2 and TCRBC primers. The TCRBV RT-PCR products were cloned and 30 colonies were subjected to dPAGE and SSCP analysis. These analyses showed that 28 of the 30 fragments comigrated under various electrophoresis conditions, indicating their clonal origin. Sequencing of 1 of the 28 fragments showed a functionally rearranged TCRB gene consisting of the TCRBV2S1D2S1J2S7C2 gene segments with a junctional region amino acid sequence of (BV2S1)CSA_IPLNGTD_EQY(BJ2S7).

In two patients the TCRBV domain expression before and during IFN-α therapy was measured. Results are shown in Table 2. Patient A was analyzed after 3 years and showed a complete hematologic response with normalization of the leukocyte differential (disappearance of circulating hairy cells, reappearance of monocytes). The very high percentage of BV3⁺ cells decreased from 24.6% to 7.5% (normal, 3.3% ± 1.2%). By RT-PCR a faint TCRBV band was still visible 3 years after the start of IFN-α (Fig 3). Also, the increased percentages of BV17⁺ (10%; normal, 5.4% ± 1.3%) and BV18⁺ (6.8%; normal, 1.2% ± 0.6%) normalized during IFN-α. This normalization was also seen for the absolute BV17⁺ and BV18⁺ cell counts (Table3). In contrast, patient B, who was assessed after 1.3 years as far as these experiments are concerned, showed only minor improvements. The leukocyte differential still yielded 1% hairy cells, 27% granulocytes, and only 1% monocytes. In this patient both the increased percentage of BV2⁺ (12.8%; normal, 6.9% ± 0.5%) and BV20⁺ (7.2%; normal, 2.5% ± 1.0%) remained elevated, and the RT-PCR analysis also showed persistent clonality.

To analyze whether the improvement of the TCRBV repertoire correlated with a functional immunologic improvement, we assessed the alloreactivity to HLA-nonidentical donors before and during IFN-α therapy. The results from the analysis of helper (HTLp), proliferative (PTLp), and cytotoxic (CTLp) T-cell precursor frequencies before and during IFN-α are shown in Table 4. In all three cases HTLp, PTLp, and CTLp frequencies were markedly abnormal at start of IFN-α. Patients A and B were considered good responders upon IFN-α treatment and showed in parallel a good restoration of the immunologic T-cell functions. Patient G, without a complete hematologic remission and with a persistently abnormal TCRBV repertoire during 1.3 years of treatment, continued to have impaired T-cell responses.

HCL cells present in the cultures might have suppressed the functional reactivity of the responder cells collected during active disease. Therefore, control experiments were performed and equal numbers of HCL cells were added to the cultures performed with responder T cells obtained during remission of the disease. The outcome was not different from the results shown in Table 4 (data not shown).

The mechanism of acquired T-cell immune dysfunction in the absence of T-cell lymphocytopenia observed in patients with hematologic malignancies is poorly understood. Untreated patients with HCL have a T-cell dysfunction, amongst others characterized by severe opportunistic infections.2,7 Furthermore, defective K, natural killer, and lymphokine-activated killer (LAK) cell functions30,31 are found, as well as a remarkable decrease in the percentage and number of circulating (CD4⁺CD45R0⁺) memory T-helper cells.32 

Recently we showed that the T-cell immune deficiency in HCL is associated with a marked restriction in the TCRBV repertoire with oligoclonal T-lymphocyte subsets and large gaps in the use of BV families.1 Patients with active disease and not yet treated, who were monitored several times for more than 1 year, showed a further progression of these abnormalities.1 

Clonal T-cell populations have also been found in other B-cell malignancies, eg, in chronic lymphocytic leukemia33,34 and multiple myeloma.33,35 Such oligoclonal T cells may play a role in antitumor surveillance. Remarkably, in the studies by Wen et al,33 only patients with early disease (stage 0 chronic lymphocytic leukemia and smoldering myeloma) harbored circulating clonal T cells. Moss et al35 found, by immunopheno- typing, oligoclonal CD4⁺ and CD8⁺T cells in the blood of patients with benign paraproteinemia and multiple myeloma. The highest percentage of clonal expansions was seen in a patient with presumably benign IgM paraproteinemia. Farace et al34 succeeded in the expansion of a BV19⁺ T-cell clone that specifically recognized autologous CLL cells. We analyzed patients with B-cell malignancies other than HCL¹ and confirmed the occurrence of oligoclonality in some patients with chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or prolymphocytic leukemia, but the TCRBV abnormalities in these cases were much less pronounced than in HCL.

Here we show for the first time that a skewed TCRBV repertoire can normalize upon treatment of the associated malignancy. IFN-α induced a remarkable restoration of the T-cell abnormalities, which took much more time (at least 2 to 3 years) than was needed for hematopoietic recovery. This is not unexpected given the long period necessary for a full recovery of the T-cell repertoire such as is seen after BM transplantation.36,37 We observed not only a gradual disappearance of the clonal expansions, but also a polyclonal reappearance of those BV families that were very low or even absent at the start of IFN-α therapy. Although many of the oligoclonal T-lymphocyte populations could not be detected after several years, in some patients with a hematologic remission persistent T-cell clones could be recognized by RT-PCR. This might be comparable to the recent observation by Callan et al,38 who showed large clonal expansions in patients with infectious mononucleosis; analysis 6 months after recovery did not yield an expansion in any of the eight patients. However, the investigators could not exclude the persistence of small clones, because they used the immunophenotyping technique instead of RT-PCR analysis.38 Similar to acute Epstein-Barr virus infections, the clonal expansions found in HCL may reflect T-cell responses to an HCL-specific antigen which obviously needs further determination.

We correlated the restrictions in the TCRBV repertoire assessed at the genotypic and immunophenotypic levels with functional tests. We measured a restoration of the T-cell function by a combined assay in which HTLp, PTLp, and CTLp frequencies were assessed.

In conclusion, patients with HCL harbor a skewed TCRBV repertoire consisting of large clonal expansions as well as gaps in many BV families. Induction of a hematologic remission by IFN-α is followed by a restoration of the T-cell abnormalities after a time period of at least several years. Functional immunological studies confirm the severe T-cell immune deficiency and also show improvement in parallel to the genotypical and phenotypical normalization of the TCRBV repertoire. These observations give more insight into the mechanisms of acquired T-cell immune deficiency in relation to malignant diseases.

The authors thank A. van de Marel for his help with cell sorting and are grateful to Immunotech, T Cell Diagnostics, and T Cell Sciences for providing the TCRBV antibodies.