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Keywords:

  • adult T-cell leukaemia/lymphoma;
  • CD4+CD25+ T cells;
  • regulatory T cells;
  • FoxP3

Summary

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

Adult T-cell leukaemia/lymphoma (ATLL) is an aggressive neoplastic disease that usually exhibits a CD4+CD25+ phenotype. Regulatory T cells (Treg), which suppress T-cell effector function, are characterized by the co-expression of CD4 and CD25. We analysed the expression of forkhead/winged helix transcription factor (FoxP3), a specific marker that is important for the function of Treg, on ATLL cells from 17 patients (peripheral blood, n = 8; lymph node, n = 9). Real-time polymerase chain reaction and immunostaining detected FoxP3 expression in 10 ATLL cases, but was relatively down-regulated compared with Treg from normal subjects. These results indicate the association of ATLL and Treg.

Adult T-cell leukaemia/lymphoma (ATLL) is a highly aggressive neoplastic disease (Uchiyama et al, 1977) and usually exhibits a CD4+CD25+ T-cell phenotype (Uchiyama et al, 1985). Human T-cell leukaemia virus type-1 (HTLV-1) is considered to be the aetiological agent of ATLL. The prognosis of ATLL is very poor; the disease shows rapid progress and drug resistance, and is often complicated with opportunistic infections (Uchiyama et al, 1977).

Recently, so-called regulatory T cells (Treg), have been described and characterized by the co-expression of CD4 and CD25 (Sakaguchi et al, 1995). Although Treg serve in vivo to protect the host against the development of autoimmunity (Shevach, 2001), in some malignant neoplasms, Treg suppress the reaction of lymphocytes to tumour antigens, inducing malignant neoplasm invasion (Liyanage et al, 2002). Recent studies also showed that Treg help malaria parasites escape from host immunity (Hisaeda et al, 2004). These findings indicate Treg sometimes account for immunodeficiency.

These immunological and phenotypical features of Treg and ATLL suggest that Treg could be viewed as a normal counterpart of ATLL cells. However, CD25 is not a specific marker of Treg, i.e. activated T cells usually express CD25 but the mere acquisition of CD25 expression does not induce the suppressor phenotype (Suri-Payer et al, 1998; Thornton & Shevach, 2000). The forkhead/winged helix transcription factor (FoxP3) is expressed on Treg and is associated with Treg activity and phenotype. Generally, FoxP3 has been shown to be expressed exclusively in Treg, and is not induced upon activation of CD25 T cells. However, when FoxP3 is introduced via retrovirus or enforced transgene expression, naive CD4+CD25 T cells transform to Treg (Hori et al, 2003). Recently, CD4+CD25 T cells were induced to express FoxP3 expression and achieve regulatory function by special stimuli through the T-cell receptor (TCR) in humans (Walker et al, 2003) and transforming growth factor-β (TGF-β) in mice (Chen et al, 2003). From these findings, FoxP3 is considered to be a critical regulator of CD4+CD25+ Treg development and function in mice and human. The goal of this study was to examine FoxP3 expression on ATLL cells to evaluate the potential for immunosuppressive activity.

Cases

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

All 17 ATLL cases were obtained from the files of the Department of Pathology, Fukuoka University, Fukuoka, Japan (all cases were confirmed to be positive for HTLV-1 provirus integration) (Table I). This study was approved by each participating institute or hospital, and written informed consent was obtained from the patients or their legal guardians. Blood samples were also obtained from five normal healthy volunteers as controls. Nine cases (cases 9–17, Table I) showed lymphadenopathy, and enlarged lymph nodes (LNs) were excised and examined histopathologically. These cases were diagnosed by examination of representative haematoxylin and eosin-stained sections prepared from paraffin-embedded, formalin-fixed material by two specialist haematopathologists. In all samples, tumour cells obliterated the entire structure of the LN. Frozen sections and suspensions were prepared from each sample.

Table I.  Patient data.
Case numberSexAge (years)Clinical typeSampleProportion of atypical cells in white blood cells (%)FoxP3/GAPDH (quantitative RT–PCR)FoxP3 (immunostaining)
  1. M, male; F, female; PB, peripheral blood; LN, lymph node.

  2. −, no detectable expression; ±, >5% positive cells; +, >30% positive cells; 2+, >50% positive cells; RT–PCR, reverse transcription polymerase chain reaction.

1F68ChronicPB930·336+
2F64AcutePB960·094
3F69AcutePB940·345±
4F59AcutePB930·0154
5F68AcutePB870·865+
6M74AcutePB690·0283
7F62AcutePB870·9362+
8M56ChronicPB950·5142+
9M66LymphomaLN00·2952+
10F60LymphomaLN00·2022+
11F85LymphomaLN00·107+
12M65LymphomaLN00·0761±
13M50LymphomaLN00·00364
14F48AcuteLN 1·50·00139
15F41LymphomaLN00·2822+
16M75LymphomaLN00·0000923
17M63LymphomaLN00·000542
MT-1     0·000254
MT-2     0·164

Cell purification

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

CD4+CD25+ cells were isolated by using the AutoMACS® magnetic separation system with the human CD4+CD25+ isolation kit (Miltenyi Biotec, Auburn, CA, USA).

The purity of CD4+CD25+ cells was more than 90% and almost all CD4+CD25+ cells from the peripheral blood mononuclear cells (PBMC) and suspension from each patient were abnormal lymphoid cells (Fig 1A) by Giemsa stain.

image

Figure 1. Analysis of FoxP3 expression. (A) Magnetic-activated cell sorter (MACS)-isolated CD4+CD25+ T cells from adult T-cell leukaemia/lymphoma (ATLL) patients were atypical with irregular with ‘flower-like’ nuclei. Giemsa stain ×200. (B) Quantitative reverse transcription polymerase chain reaction (RT–PCR) for FoxP3 in each sample. CD4+CD25+ and CD4+CD25 are the mean data from each of five normal donors. (C) Immunohistochemistry for FoxP3 in a representative ATLL sample. Lymphoma cells are diffusely and strongly positive for FoxP3.Original magnification ×200. (D) Immunohistochemistry for FoxP3 in a representative normal tonsil as control. Note the presence of a few scattered positive cells, considered as regulatory T cells (Treg). Original magnification ×200. (E) Immunocytochemistry for FoxP3 in a representative ATLL sample. Leukaemic cells are diffusely positive for FoxP3. Original magnification ×100. (F) Immunocytochemistry for FoxP3 in peripheral blood mononuclear cells (PBMC) from a representative normal donor. Almost cells are negative with a few scattered positive cells, considered as normal Treg. Original magnification ×200.

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Quantitative reverse transcription polymerase chain reaction

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

The RNA was extracted from isolated CD4+CD25+ cells by the guanidinium thiocyanate-phenol-chloroform method using the Total RNA Separator kit (Clontech, Paolo Alto, CA, USA). Quantitative reverse transcription polymerase chain reaction (RT–PCR) was performed with a TaqMan assay on an ABI 7000 system (Applied Biosystems, Foster, CA, USA), on RNA isolated from the CD4+CD25+ ATLL cells from eight PBMC samples of ATLL patients, nine tissue suspensions from LNs, CD4+CD25+/−T cells from five normal donors and representative ATLL cell lines, MT1 and MT2, as described previously (Haslett et al, 2002). To detect FoxP3, Assays-on-Demand Gene Expression probes for FoxP3 (Hs 00203958; Applied Biosystems) were used. To detect GAPDH, Taqman probe and primer was used. Probe sequences were as follows: FAM-5′-AAG GTG AAG GTC GGA GTC AAC GGA TTT G-3′-TAMRA. Primer sequences for GAPDH were as follows: 5′-CCACATCGCTCAGACACCAT-3′ and 5′- CCAGGCGCCCAATACG-3′.

In each reaction, GAPDH was amplified as a housekeeping gene to calculate a standard curve and to correct for variations in target sample quantities. Relative copy numbers were calculated for each sample from the standard curve after normalization to GAPDH by the instrument software.

Immunohistochemistry or -cytochemistry

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

The FoxP3 expression was analysed by immunostaining using anti-FoxP3 polyclonal antibody (Abcam, Cambridge, UK). We also analysed other T-cell and B-cell lymphomas.

Results and discussion

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

The proportion of CD4+CD25+ cells in the PBMC of ATLL patients was 81·4% (SD: 7·97) compared with 5·64% (SD: 3·62) in normal donors. We used quantitative RT–PCR of the corresponding transcripts in CD4+CD25+ and CD4+CD25 T cells in normal donors and ATLL patients. CD4+CD25+ T cells from normal donors showed definitively higher FoxP3 expression (FoxP3/GAPDH: 0·53–1·58) than CD4+CD25 T cells (FoxP3/GAPDH: 0·061–0·081). FoxP3 expression (FoxP3/GAPDH > 0·2) was detected in eight ATLL cases (PB: 5, LN: 3), but was relatively down-regulated compared with Treg from normal subjects (Fig 1B). The ratio of FoxP3/GAPDH tended to be relatively low compared with normal CD4+CD25+ T cells, especially in LNs. The ATLL cell lines, MT-1 and MT-2, showed low FoxP3 expression. We detected strong expression of FoxP3 mainly in the nuclei of atypical lymphoid cells of 10 cases with relatively high mRNA expression (FoxP3/GAPDH: PB: >0·336, LN: >0·07) by immunocytochemistry or -histochemistry (Fig 1C–F, Table I). MT-1 and MT-2 did not show definite positivity. To compare the expression level of other lymphomas, we analysed FoxP3 expression in diffuse large B-cell lymphoma (DLBCL) (n = 10) and peripheral T-cell lymphoma (n = 15) by immunostaining; all of these samples were negative. RNA was isolated from seven cases of DLBCL, all of which showed low-FoxP3 expression (FoxP3/GAPDH: <0·05) by quantitative RT–PCR (data not shown).

In this study, even in those cases that showed FoxP3 expression, the expression level was relatively lower than normal CD4+CD25+ Treg. Although there have not been any reports about the effects on FoxP3 expression and immunosuppressive activity of regulatory T cells by HTLV-1 infection, it is possible that the transformation induced by HTLV-1 infection or the neoplastic process results in the reduction of FoxP3 expression in ATLL. Our results also showed differences in FoxP3 expression in lymphoma cells from PB and LN samples. While the exact reason for this difference is not clear at present, it may be related to differences in clinical type. Analysis of further cases is necessary in future.

In conclusion, the present study has demonstrated FoxP3 expression on ATLL cells, suggesting that such expression may have a regulatory function in at least some cases of ATLL. This is the first report of FoxP3-expressing neoplastic cells.

Acknowledgments

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References

Authors thank Dr Sakiko Shimizu and Hitoshi Nakajima for their kind technical advice and Dr Kenji Furuno for special advice. Authors appreciate the co-operation of Mrs Konomi Takasu, Ms Kaori Saga and Ms Kanoko Miyazaki.

References

  1. Top of page
  2. Summary
  3. Study design
  4. Cases
  5. Cell purification
  6. Quantitative reverse transcription polymerase chain reaction
  7. Immunohistochemistry or -cytochemistry
  8. Results and discussion
  9. Acknowledgments
  10. References
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