Page 36 - Haematologica-5

768
V. Giudice et al.
file due to the presence of 1-3 immunodominant clones, and predominant classes in CDR3 size and DJ length pro- files. These findings were confirmed by TCR repertoire sequencing of CD8+CD57+ enriched cells from SAA patients. Similar deep sequencing features have been described in whole blood in T-cell large granular lympho- cyte leukemia (T-LGLL).17 T-LGLL, a chronic lymphopro- liferation of TCRaβ+CD3+CD5dimCD8+CD57+CD16+ cells with monoclonal TCRγ-chain rearrangement and preva- lence in the elderly, is frequently associated with autoim- mune diseases.16,17 Thus, similarities between our SAA cohort and T-LGLL patients suggest a common patho- physiology of expanded autoreactive T lymphocytes. Characterization of long-term Vβ usage has already been proposed as a biomarker of disease progression in T-LGLL and AA,8,16 given that immunodominant clones can remerge during relapse.16 However, high heterogeneity in the TCR repertoire during immunosuppressive therapies has been reported.8,16 In our study, immunodominant clones were longitudinally investigated in 3 AA patients. In Patient 4, expanded clones slightly decreased during treatment but did not disappear, as no hematologic improvements were observed. In Patient 34, clone 17 remained stable during the course of the disease, while clone 13.6 increased at three months and slightly decreased at six, concomitantly with a minimal partial response. In Patient 22, clone 2 completely disappeared at six months of treatment and the patient achieved hemato- logic remission; however, at the time of relapse the clone increased again. Our data suggest that Vβ typing of the CD8+ CD57+ T-cell population by flow cytometry might be a useful biomarker to monitor clonal kinetics during the course of AA, but a larger cohort of patients and a more sensitive technique, such as deep sequencing, are needed to validate the clinical usefulness.
Chronic antigen exposure is required to trigger T-cell acti- vation, as in persistent viral infections or with antigen spread during autoimmune and malignant diseases.17 For viral infections, CDR3 homology in public sequence reper- toires, and also cross-reactivity between viruses, is limited
by the number of possible epitopes.41,48 For autoimmune dis- eases, including T-LGLL, clonotypes can be private to a spe- cific disease because of the unlimited number of possible epitopes.17,41,49 Despite the large diversity of TCR CDR3 sequences, only 29 shared CDR3 sequences were found in our cohort; these were highly expressed in SAA patients and enriched in the CD8+ CD57+ T-cell population. By using sensitive techniques such as deep sequencing, we detected sequences at very low frequencies. However, the finding that a group of clonotypes is shared between SAA patients and healthy subjects suggests the existence of common epi- topes driving activation of T-cell autologous clones (as also described by Gargiulo et al. in PNH patients40). As chronic transfusion could be the source of antigen exposure, we investigated its effect on effector memory T-cell expansion in other hematologic diseases. A comparison between transfused patients and healthy subjects showed no signifi- cant variations in CD8+ CD57+ cell frequencies (P=0.216).
Oligoclonal expansion of effector memory CD8+ T cells is frequent in AA and may correlate with prognosis, con- sistent with a role of effector memory T cells in BM destruction during active disease. Deep sequencing tech- nologies allow in-depth characterization of the TCR reper- toire, and flow cytometric analysis of Vβ usage may be useful to determine diagnosis and prognosis of SAA patients, and to monitor their clinical course.
Acknowledgments
The authors would like to thank Sachiko Kajigaya and Keyvan Keyvanfar (Hematology Branch, NHLBI), Ying Rao (BGI Genomics), and Swee Lay Thein (Sickle Cell Branch, NHLBI) for assistance; Kinneret Broder (Hematology Branch, NHLBI) for assistance in obtaining healthy volunteer samples; and Barbara Weinstein (Hematology Branch, NHLBI) and Jim Nichols (Sickle Cell Branch, NHLBI) for obtaining patient sam- ples.
Funding
This research was supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute.
References
1. Young NS, Calado RT, Scheinberg P. Current concepts in the pathophysiology and treat-
ment of aplastic anemia. Blood. 6. 2006;108(8):2509-2519.
Rotoli B. Function and malfunction of hematopoietic stem cells in primary bone marrow failure syndromes. Curr Stem Cell Res Ther. 2007;2(1):39-52.
Maciejewski JP, O’Keefe C, Gondek L, Tiu R. Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones. Folia Histochem Cytobiol. 2007;45(1):5-14. Feng X, Scheinberg P, Wu CO, et al. Cytokine signature profiles in acquired aplastic anemia and myelodysplastic syn- dromes. Haematologica. 2011;96(4):602- 606.
Risitano AM, Maciejewski JP, Green S, Plasilova M, Zeng W, Young NS. In-vivo dominant immune responses in aplastic anaemia: molecular tracking of putatively pathogenetic T-cell clones by TCR beta- CDR3 sequencing. Lancet. 2004; 364(9431):355-364.
Young NS. Hematopoietic cell destruction by immune mechanisms in aquired aplastic anemia. Semin Hematol. 2000;37(1):3-14.
10. Sloand EM, Kim S, Maciejewski JP, et al. Intracellular interferon- in circulating and marrow T cells detected by flow cytometry and the response to imunosuppressive ther- apy in patients with aplastic anemia. Blood. 2002;100:(4)1185-1191.
11. Maciejewski JP, Selleri C, Anderson S, Young NS. Fas antigen expression on CD34+ human marrow cells is induced by interferon-gamma and tumor necrosis fac- tor-alpha and potentiates cytokine-mediat- ed hematopoietic suppression in vitro. Blood. 1995;85:(11)3183-3190.
12. Selleri C, Maciejewski JP, Sato T, Young NS. Interferon-γ constitutively expressed in the stromal microenviroment of human mar- row cultures mediates potent hematopoietic inhibition. Blood. 1996; 87:(10)4149-4157.
13. Hirano N, Butler MO, von Bergwelt-Baildon MS, et al. Autoantibodies frequently detect- ed in patients with aplastic anemia. Blood. 2003;102:(13)4567-4575.
14. Feng X, Chuhjo T, Sugimori C, et al. Diazepam-binding inhibitor-related protein
2. Young NS. Current concepts in the patho- physiology and treatment of aplastic ane- mia. Hematology Am Soc Hematol Educ Program. 2013;2013:76-81.
7.
3. Maciejewski JP, Selleri C, Sato T, Anderson
S, Young NS. A severe and consistent deficit
in marrow and circulating hematopoietic
cells (long-term culture-initiating cells) in acquired aplastic anemia. Blood. 8. 1996;88(6):1983-1991.
4. Maciejewski JP, Kim S, Sloand E, Selleri C, Young NS. Sustained long-term hematologic recovery despite a marked quantitative defect in the stem cell compartment of patients with aplastic anemia after immuno- suppressive therapy. Am J Hematol. 2000;65(2):123-131.
5. Risitano AM, Maciejewski JP, Selleri C,
9.
haematologica | 2018; 103(5)