Page 15 - Haematologica-5

(Auto-)immune signature in aplastic anemia
Antonio M. Risitano
Hematology, Department of Clinical Medicine and Surgery, Federico II University, Naples, Italy
E-mail: amrisita@unina.it doi:10.3324/haematol.2018.190884
Acquired idiopathic aplastic anemia (IAA) is a rare hematologic disorder characterized by the failure of hematopoiesis secondary to an immune-mediated damage of the bone marrow. IAA is bona fide considered an auto-immune disease with a T-cell-mediated pathophysiolo- gy.1 In this issue of the Journal, Giudice et al.2 describe the oligoclonal pattern of effector memory CD8+ CD57+ T cells in IAA patients by using a combined deep sequencing and flow cytometry approach. Indeed, Giudice et al. show that clonally expanded T-cell populations are frequently detectable even within the effector memory compartment, and that they tend to correlate with disease activity. Thus, the characterization of the T-cell receptor (TCR) repertoire by high-resolution tech- niques may play a role in confirming the diagnosis of immune-mediated IAA and to monitor affected patients dur- ing their disease course.
There is widespread clinical and experimental evidence to support the autoimmune pathophysiology of IAA.1 The most striking is that patients with IAA may respond to T- cell-targeted immunosuppressive therapies (IST), with rates of hematologic responses ranging between 50% and 70%.3 Almost four decades of investigations have provided us with a plethora of experimental data corroborating the hypothesis of an immune-mediated pathophysiology. Increased circu- lating activated T cells were described in IAA patients in the ‘80s.4 These T cells may suppress hematopoiesis through the secretion of different inflammatory cytokines5,6 and/or via cell-mediated direct killing. Among the different inhibito- ry cytokines, interferon-γ (IFN-γ) plays a major role in sup- pressing human hematopoietic stem cells (HSC) in vivo, as suggested by in vitro inhibition of cell cycle progression and induction of apoptosis of hematopoietic progenitors.7 More recently, it has been suggested that IFN-γ may also exert its inhibitory effect on HSC impairing the homeostatic survival signal delivered by thrombopoietin through its cognate receptor c-MPL.8 This inhibitory milieu is generated by immune cells, and mostly by T cells that become activated and proliferate in response to an antigen-driven stimulation. While the search for these putative antigens has remained unsuccessful, the demonstration of clonal expansion of T-cell populations identified by their TCR has been considered robust proof of a T-cell-mediated pathophysiology in IAA.9,10
Our growing understanding of the immune system and the availability of powerful novel techniques has nurtured continuous research in the field of IAA. On the one hand, investigators have tried to further dissect the abnormalities of the immune system in patients suffering from IAA. Indeed, looking at specific functional T-cell subsets, recur- rent immune derangements have been found, such as increased T-helper type 17 cells (Th17)11 and reduced regula- tory T cells (Treg).12,13 However, irrespective of the deep phe- notyping of CD4+ T cells [i.e. by multiparameter mass cytometry, termed cytometry by time-of-flight (CyTOF)],14
only limited data are available about the characterization of specific functional CD8+ T-cell subsets. On the other hand, novel techniques of deep DNA sequencing have become available, and their application in IAA has led to the descrip- tion of somatic mutations in myeloid cells,15 with the subse- quent ongoing debate about their actual meaning,16 but also to high throughput TCR analysis.17 In their study, Giudice et al. specifically investigated the compartment of effector memory T cells (TEM) in IAA patients, looking for possible clonality as assessed by flow cytometry analysis of Vβ usage and sequencing of the hypervariable complementary deter- mining region 3 (CDR3) of the TCR.
In agreement with previous reports,1,10 Giudice et al. con- firm that AA patients often exhibit a skewed usage of Vβ families, usually within the CD8+ T-cell compartment; these oligoclonal expansions are more frequent in patients with increased percentage of TEM (as defined by co-expression of CD8 and CD57), which are found in approximately 70% of AA patients. These gross abnormalities of the TCR Vβ usage were dissected at the clonal level by deep sequencing of the TCR, which allows the comparison of more than 107 reads corresponding to TCRs harbored by individual T cells. Indeed, clonal expansions were identified by repetitive use of TCR β variable (TRBV) and joint (TRJV) genes (i.e. redun- dant TRBV/TRBJ combinations), as well as by CDR3 size and DJ length profiles. Immunodominant clones within dif- ferent T-cell subsets were invariably detected in all AA patients with increased CD8+ CD57+ TEM; however, the actu- al magnitude of these clonal expansions was extremely het- erogeneous in the different T-cell subsets. Indeed, these clones remain minimally expanded (approx. 3%) within the CD4+ compartment, while they become largely dominant (approx. 18%) in the CD8+ compartment; the expansion appears even larger when the CD8+ CD57+ TEM is analyzed. This difference in behavior of TCR heterogeneity in differ- ent T-cell subsets of AA patients was further confirmed by analysis of Simpson’s diversity score, which shows how TCR diversity decreased from CD4+ to CD8+ T cells, and even more from both CD4+ and CD8+ T cells to CD8+ CD57+ TEM. In summary, the work performed by Giudice et al. con- firms that clonal CD8+ T-cell expansions are common in AA patients, in agreement with the well-established T-cell-medi- ated immune pathophysiology of AA.1 But for the first time, here the Authors provide evidence that the clonal expan- sions are not limited to the CD8+ effector T cells, since they can be found even in the TEM compartment. Very interesting- ly, with the caveat of a limited sample size, Giudice et al. show that abnormalities of the TEM compartment (i.e. increased TEM, with possible clonal expansions) may be asso- ciated with a dismal outcome in AA, due to refractory or relapsed disease. This is further supported by the observa- tion of concordance between the expansion of the immun- odominant CD8+ CD57+ TEM clone and disease activity (sim-
haematologica | 2018; 103(5)
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