Autoimmune-mediated pathogenesis of acquired aplastic anemia: Roles of T lymphocytes

INTRODUCTION

Acquired aplastic anemia (aAA), an autoimmune bone marrow (BM) failure syndrome, is characterized by peripheral blood (PB) pancytopenia and a hypocellular BM. In 1888, Ehrlich described the first ever case of aplastic anemia in a young pregnant woman. The patient died of severe anemia, neutropenia, hemorrhage, high fever, and BM failure. Her autopsy featured a “strikingly hypocellular” fatty marrow.^[1] The term “Aplastic Anemia” was coined by Vaquez and Aubertin in 1904, emphasizing the pathophysiology as “anhematopoiesis.” The disease was strongly associated with radiation, chemicals, and drugs causing severe depletion of hematopoietic progenitor stem cells.^[2] Thus, aplastic anemia can rightly be reckoned as a historical disease. Numerous pathways have been suggested to drive the pathologic nature of the disease and the accumulating evidence hypothesizes a T-cell-mediated autoimmunity with BM destruction and defective hematopoiesis.^[3]

Worldwide reported incidence of aplastic anemia ranges from 1 to 4 cases/million population in all age groups with extensive geographic heterogeneity. The geographical differences are due to the environmental and genetic factors of the respective area. Asian countries have higher incidence rates of aplastic anemia (3–7.5 cases/million) as compared to rest of the world.^[4] Moreover, there are limited resources and many constraints in Asian region such as cost of therapy, lack of awareness about the disease, resulting in delayed diagnosis and treatment. A study depicted lower socioeconomic factors to be connected with higher incidence of aplastic anemia in India.^[5] BM aspiration and trephine biopsy are the diagnostic tools to confirm aplastic anemia. Based on these tools, the disease can be categorized as non-severe aplastic anemia (NSAA), severe aplastic anemia (SAA), and very SAA (VSAA).^[6] In addition, a careful drug history, infection, any occupational exposure, and family history must be taken into account. Patients should be ruled out for congenital marrow failure, myelodysplastic syndrome, leukemia, and paroxysmal nocturnal hemoglobinuria (PNH). Flow cytometric analysis for PNH screening of glycosylphosphatidyl-inositol anchored proteins: Cluster of differentiation 14 (CD14), CD16, CD24, CD55, and CD59 for red blood cells and fluorescent aerolysin for white blood cell needs to be done.^[7]

The treatment goals in aAA aim to halt the self-directed autoimmune attack on hematopoietic stem cells (HSCs) in BM. The standard treatment consists of immunosuppressive therapy (IST) and allogenic BM transplantation (BMT) in transplant-eligible patients. Allogenic BMT is the first line of treatment in children and young adults (<40 years of age), if human leukocyte antigen (HLA) matched sibling donor is available. The rates of cure for BMT are 70–90% with survival rate of 65% for high-risk patients and 95% for low-risk groups. IST involving anti-lymphocyte globulin/anti-thymocyte globulin (ATG) and cyclosporine A (CsA) show hematological recovery in 70% of the cases and excellent long-term survival in responders. ATG and CsA produce tolerance by inhibition and depletion of activated T cells.^[8,9] In 2015, the US Food and Drug Administration approved eltrombopag (an oral thrombopoietin-receptor agonist) for the treatment of SAA refractory to IST. Eltrombopag, when used in combination with standard IST, might salvage and expand residual HSCs in aAA.^[10] There is high mortality rate of 70% in patients not receiving any kind of treatment.^[11]

ETIOLOGIES

Aplastic anemia can be inherited or acquired, affecting young people between the ages of 15 and 25 years and later after the age of 60 years. The inherited BM failure syndromes (IBMFSs) are rare and account for up to 15– 20% of all aplastic anemia cases. IBMFS include Fanconi anemia, congenital keratosis [dyskeratosis congenita (DKC)], congenital pure red cell aplasia [diamond-blackfan anemia (DBA)], and Shwachman-Diamond Syndrome. Majority of the patients not belonging to the IBMFS group suffer from aAA, which is immunological in pathophysiology.^[12] A multitude of etiological factors, genetic predisposition, epigenetic modifications, and environmental factors play a predominant role in aAA development. Genetic factors, human HLA alleles (class I and II), germline genetic variants occurring as loss on chromosome 6 of HLA region, mutations in telomere complex genes (dyskerin pseudouridine synthase 1 [DKC1], telomerase RNA component [TERC], telomerase RNA component [TERT], nucleolar protein 10 [NOP10], ribonucleoprotein [NHP2], TERF1-interacting nuclear factor 2 [TINF2]), short telomeres, somatic mutations, genetic polymorphisms, influence disease predisposition, and treatment outcomes in aAA, in distinct racial and ethnic populations.^[13] To rule out any genetic related predisposition to aAA, a complete genetic evaluation of the patients at the time of diagnosis should be carried out.

External environmental factors and occupational hazards increase the susceptibility toward aAA. Occupational exposure to radiations, chemicals, exotoxins, drugs (antiepileptics carbamazepine, valproic acid, anti-inflammatory, antimicrobial, anticonvulsant, and chemotherapeutic agents), pesticides, insecticides, viral infections, consumption of non-bottled water, non-medical needle exposure, and pregnancy are linked to a significant risk of developing aAA. Pregnancy with aAA is relatively rare and relationship between the two remains uncertain. Nevertheless, pregnancy complicated by aAA should be considered a serious condition, bearing substantial maternal and fetal risks. The degree of seriousness relates to the BM suppression. However, through multidisciplinary approach, improved treatment modalities, optimal use of chemotherapeutic agents, prophylactic transfusions, meticulous follow-up, and ante-natal care, these cases are showing favorable outcomes.^[14]

Radiation from radioactive materials, like radium, damages the HSCs in the BM. Several isotopes of radium exist in the environment. Radium is present in groundwater, soil, minerals, man-made materials, and construction material. Emerging knowledge illustrates an increased risk of aplastic anemia, leukemia, and cancers on exposure to radium over a period of time.^[15] The BM microenvironment consists of HSCs and mesenchymal stromal cells (MSCs) that support normal functioning of BM.^[16] Hazardous exposure to ionizing radiation disturbs the BM architecture, rendering it hypocellular and unable to sustain hematopoiesis. Radiation-stimulated hematotoxicity triggers cell apoptosis, DNA damage, alterations in signaling pathways, humoral immune suppression, deficit in hematopoietic pluripotent and multipotent stem cells, and raised adipocytes in BM. The deterioration of HSCs obstructs hematopoiesis, which culminates in BM aplasia.^[17] The classical example of radiation-induced aAA is that of Madam Marie Curie, who suffered from the radiation effects of radium, had multiple medical problems and lost her life.^[18]

Contact with chloramphenicol, phenylbutazone, oxyphenbutazone, benzene, organochlorines, organophosphates, and fertilizers causes progressive deterioration of BM. The metabolites deteriorate BM by diminishing the production of proteins and enzymes through ultrastructural microsomal changes. For instance, benzene metabolites hamper the ability of progenitor cells to produce sufficient cytokines and response toward cellular adhesion molecules required for HSC survival and proliferation. The mechanism of this interaction forms the basis of chemical toxicity.^[19] The toxicity has a dose response relationship with exposure to chemicals or drugs. In the long-term, toxic exposure leads to disruption of hematopoietic commitment, maturation, suppression of BM progenitor cells, and potential induction of aAA.^[20] Conducting studies on mechanisms linking environmental triggers to BM failure will strengthen the understanding of various agents responsible for inducing aAA.

Infections with Epstein-Barr, cytomegalo, varicella zoster, hepatitis, dengue, rubella, herpes simplex virus (HSV), parvovirus, and human immunodeficiency virus are incriminated as putative cause of aAA. T lymphocytes reactive toward epitopes derived from viral infection, undergo clonal expansion and reduce HSCs in aAA by cross reactivity. The resultant marrow failure can be severe if left untreated.^[21] The most well recognized virus associated aAA is hepatitis-associated aplastic anemia (HAAA); first reported by Lorenz and Quaiser in 1955. Several hepatitis viruses, namely, hepatitis A, B, C, E, and G are responsible for HAAA. An acute attack of hepatitis leads to BM failure and pancytopenia. The incidence of HAAA is higher in hepatitis prevalent countries.^[22] Another evidence of viral induced aAA is due to HSV-1, where antiviral treatment, restored cell counts in PB and BM of HSV-1-positive aAA patient.^[23] Therefore, it is advised to test aAA patients for any viral infections and manage accordingly.

T-CELL-MEDIATED PATHOGENESIS

Aplastic anemia as a hematological syndrome is an umbrella term clustering various clinical phenotypes which are an immuno-pathogenetic outcome of differently converging pathophysiologies, ranging from impaired BM niche, deficiencies in HSCs, dysfunctional MSCs, overproduction of BM inhibiting cytokines, and T-cell-mediated autoimmune attack against HSCs.^[24] The clinical observation of a significant fraction of patients responding to IST has established the consensus on autoimmune basis of aAA. Consequently, aAA represents an autoimmune hematological disorder.^[25] Alterations in gene pathways related to autoimmunity, immune response, cell proliferation, and activation of T-cells contribute toward effective organ-specific destruction in aAA. Studies have recognized and confirmed the involvement of aberrantly activated immune cells and T-cell subsets in the suppression of hematopoiesis in aAA, with BM being the target-organ.^[26]

T lymphocytes are the most important cells in adaptive immunity. T cells express antigen receptors (TCR) of unique specificity and recognize antigens bound to major histocompatibility complex molecules on the surface of antigen-presenting cells. Antigen recognition activates a cascade of events right from transduction of TCR signals leading up to T-cell effector functions, proliferation and differentiation. Defective TCR signaling results in an unbalanced immune system and autoimmunity. A population of clonally expanded T cells with altered TCR is often encountered in aAA patients, that correlates with disease activity and drives the disease process.^[27,28] Characterization of respective TCR repertoire by high-throughput technologies may help in diagnosis and monitoring of disease in patients. TCR repertoire can also identify pathogenic T-cell clones and avert their action by guided inhibition of T-cell activity and achieve immune tolerance.^[3]

T cells perform their effector responses with massive proliferation and release of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukins (IL), inducing apoptosis of progenitor cells in BM.^[29] Cytokines are soluble proteins that act as inflammatory signals and regulate hematopoiesis by modulating BM microenvironment. In tandem, antigen receptor stimulation and cytokine levels tightly regulate T-cell homeostasis. Any disturbances due to deranged cytokine level can cause T-cell reactivity toward self-tissues and disruption of immune homeostasis in aAA.^[30] Estimating cytokine levels, T-cell functionality at diagnosis and after therapy can help in understanding the prognostic and predictive utility of disease biomarkers with severity and treatment response.^[31]

CD4⁺ T cells known as T helper (Th) cells are characterized as per the secretion of particular cytokines. CD4⁺ T cells producing IFN-γ are designated as Th1, IL-4 as Th2, and IL-17A as Th17. CD4⁺ T cells expressing surface CD25 and intracellular transcription factor fork head box P3 (FOXP3) are designated as regulatory T cells (Tregs). Th cells assist cytotoxic T cells, B cells, macrophages in performing their respective functions of killing target cells, secreting antibodies, destroying microbes, prevent graft-versus-host disease, and offer long-term immunity to the host.^[32]

An affinity toward Th1 derived response is a recurring theme in aAA. Th1 cells negatively correlate with clinical parameters and create an inflammatory environment in aAA.^[33] Upregulated transcription factor T-bet (T-box protein expressed in T cells), induced by TCR signaling is crucial for Th1 cell differentiation. T-bet promotes elevated production of IFN-γ, modulating the BM milieu toward Th1.^[34] In case of Th2 cells, with the exception of a few studies, most of the studies have either shown lower frequencies or no significant difference in aAA.^[33,35,36] GATA-binding protein 3 (Gata-3) is required for stabilizing the Th2 phenotype, but, in aAA an upregulated T-bet, inhibits Gata-3 and prevents Th2 differentiation.^[37] Consequently, the major idea in aAA pathogenesis is toward an unfavorable tilt in Th1/Th2 balance. Re-establishment of Th1/Th2 balance can be a treatment strategy in aAA.

Apart from Th1 and Th2 cells, abnormality of Th17 cells is also found in aAA. Th17 cells promote inflammation, infection, and autoimmunity. A balanced Th17 cell activity is critical for maintaining health. There are six members of IL-17 cytokine family (IL-17A–IL-17F), regulated by their specific transcription factor retinoic acid receptor-related organ receptor gamma t (ROR-γt). Among these, IL-17A is the most studied cytokine and induces devastating tissue damage.^[38] De Latour et al., in 2010, for the first time studied Th17 cells in aAA and reported increased number of Th17 cells in PB of newly diagnosed aAA patients as compared to healthy donors. Treatment with anti-IL-17 antibody abrogated the hypocellular BM in mouse model.^[39] This work paved the way for subsequent research on Th17 cells in aAA. In other study, increased number of Th17 cells accompanied by raised level of IL-17 cytokine and ROR-γt in PB mononuclear cells was observed in SAA and VSAA patients.^[40] Th17 specific T-cell subset, cytokine and transcription factor, generates the immune disorders in aAA patients. Polarization of CD4⁺ T cells toward Th17 and a predisposition to inflammatory pathogenesis in BM mononuclear cells of pediatric aAA patients is highlighted by single-cell mass cytometry. γδ T cells producing IL-17A from SAA patients could be used as biosignatures for surveillance of T-cell activation during disease progression.^[41] Along these lines, development of newer treatment strategies incorporating anti-IL-17 antibodies in aAA should be explored.

Follicular Th T cells (TFH) expressing CXCR5, ICOS, PD1, Bcl-6, and IL-21 regulate B-cell proliferation and antibody responses. Other category of T follicular regulatory (TFR) T cells are characterized by the additional expression of CD25, FOXP3, and inhibition of B-cell responses. In autoimmune diseases, the frequency and phenotype of TFH and TFR cells is altered.^[42] Increased numbers of circulating TFH cells and decreased number of TFR cells, correlating with disease progression and response to treatment, are found in patients with aAA.^[43] Unbalanced TFH/TFR ratio might be promoting production of excessive antibodies, mediating the HSC destruction and promoting disease evolution in aAA. Comparative gene expression profiling for elucidating the molecular mechanisms of TFH and TFR cells will shed light on their actions in aAA pathogenesis.

Participation of unconventional T cells, referred to as mucosal associated invariant T (MAIT) cells, has been reported in aAA. MAIT is innate-like T cells enriched in mucosal tissues; produce high amount of IFN-γ and TNF-α, partaking in hematopoietic progenitor cell destruction. MAIT cells thereby act as bridge between innate and adaptive immune responses.^[44] A recent study demonstrated close links between T-cell-related factors and an increased risk of aAA. The signatures of T–cell-related factors diverge according to the disease severity stages and treatment methods.^[45] Elucidation of interactions among T-cell-related factors and innate immune cells in aAA will guide the succeeding research and help the clinicians.

Cytotoxic T lymphocytes (CTLs) or CD8⁺ T cells perform cytolytic activities through perforin/granzymes/fas-mediated pathway to kill target cells and recognize auto-antigens presented through class I HLA molecules. CTLs are involved in apoptotic pathways in aAA and an increased percentages of CTLs may leave patients unresponsive to IST.^[46,47] Linker for activation of T cells (LAT) plays a central role in TCR signal transduction pathway. LAT overexpression in aAA results in activation of CTLs with a propensity toward increased apoptosis and hematopoiesis failure.^[48] Acetylation level of histone H3 might be causing activation of CD8⁺ T cells in aAA, which also negatively correlate with clinical parameters.^[49] This implies that an increase in number of CD8⁺ T cells leads to decrease in hematological parameters such as red blood cells, white blood cells, and platelets. Highly activated CD38⁺ CD8⁺ T cells bearing proinflammatory and proliferative capacity might be contributing to the pathologic progression in aAA.^[50] Patients having elevated frequency of CD8⁺ Memory stem T cells (TSCM) at the time of diagnosis respond to IST. Whereas, increased frequency of CD8⁺ TSCM cells after IST correlates with relapse or treatment failure, suggesting the considerable role of CTLs as disease biomarkers in aAA.^[51] Similarly, increased presentation of CTL antigen 4 (CTLA-4) on CD8⁺ T cells predicted a worse response to IST in aAA patients.^[52] The abnormal effector functions of CTLs may amplify the damage to HSCs in aAA.

Natural killer (NK) cells, defined as per the expression level of CD56, directly lyse the target cells through IL-2, IL-18 cytokines during the early phase of immune responses.^[53] Investigations on peripheral circulating NK cells in severe aAA patients revealed significantly decreased populations of CD56bright/dim subsets, which increased after IST.^[54] Exhaustion of NK cells might cause over-production of T cells and BM failure in SAA, which might be mitigated by replenishing NK cells. Meanwhile, same authors in another study reported an increased percentage of CD56 bright NK cells and higher concentrations of IL-2 and IL-18 in newly diagnosed NSAA patients.^[55] Hence, in NSAA patients, increased proportions of NK cells might have a protective role.

Single-cell analysis in aAA revealed a dysregulated population of terminally differentiated effector CD8⁺ T (T_EMRA) cells expressing NK receptors in BM. In co-culture experiments, NK cells could not kill HSCs, whereas, HSCs induced activation of T-cell clones with CD8⁺ T_EMRA NK-like phenotype. The resultant convergent phenotype of CTLs and NK cells may drive aAA pathogenesis.^[56] Restituting the balance of NK cells in aAA could be viewed as another therapeutic approach.

CD4⁺ CD25⁺ Tregs subdue autoreactive T-cell stimulation, function, and maintain peripheral tolerance. Tregs release anti-inflammatory cytokines, namely, IL-10, IL-35, and TGF-β to control unfavorable immune responses. An impaired Treg number or function leads to autoimmunity, while optimum function and number offers self-tolerance and defense against autoimmune diseases.^[57] In aAA, deficient number and function of Tregs depicts inferior response to IST.^[23] However, in patients responding to IST, a normal Treg/Th1 ratio can be achieved, implying that a decreased Treg count is related to immune-mediated disease activity.^[58] In vitro and in vivo studies with Dioscin (a natural steroid saponin) on aAA murine model promoted Tregs by increasing Foxp3, IL-10, IL-35, TGF-β secretion, and attenuated BM failure.^[59] Hence, employing Tregs as cellular therapy in aAA might prevent T-cell-mediated destruction. Treg subpopulations with distinct immunological phenotypes, known as Treg-A (naive) and Treg-B (memory), have been described in aAA and can be used as potential targets to overcome immune dysregulation.^[60] Tregs offer new immunotherapeutic options and therapies involving expansion of Tregs could be used in aAA.

In further investigations, many newer immunophenotypes of cells might be discovered, which would offer another perspective on the complex mechanisms of immune-mediated pathogenesis of aAA [Figure 1].

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Figure 1:

Autoimmune - mediated pathogenesis of acquired aplastic anemia.

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Most of the studies on aAA are standalone studies measuring different T lymphocyte subsets in either blood or BM. We suggest that in future, a comprehensive study involving all the T lymphocyte subsets and other newly discovered immune cells should be accomplished both on blood and BM profiles of aAA patients. For a deeper analysis, clinical history of patients, details of viral infections, and occupational exposure needs to be collected. Together, these data can identify cellular and molecular drivers of aAA. We may find immune cells that might serve as an immune biomarker for aAA and its severity estimation. Alongside scrutiny of dysregulated genes, molecular signaling pathways involved in immune perturbations ought to be done. This would give an overall picture of the various immunological and molecular changes occurring in aAA. Experiments involving evaluation of therapy on T lymphocytes obtained from individual patients can also be recommended for pinpointing the effect of therapy on underlying immune profiles of aAA patients.

In summary, a combination of abnormal increase in different activated Ths, CTLs, and a skewed Treg immunophenotype and function depicts impaired immunity in aAA. Thus, it can be postulated that an imbalance and dysfunction of different T-cell subsets inhibit immune homeostasis and leads to autoimmunity and intolerance in aAA. Evaluation of different T-cell signatures in aAA for developing potential therapeutic targets should be considered for future investigations, as well as to distinguish patients who could benefit from IST or BMT. The clinical course and complications in aAA patients are variable due to infections, bleeding, relapse, and clonal evolution. In under developed and developing counties, most of the patients cannot afford expensive treatments. Studies on development and accessibility of effective treatment algorithms for aAA patients are required that can circumvent delayed transplant and repeat IST.

CONCLUSION

There is significant burden of aAA. Studies delineating the complexity of T-cell subsets and their impact on the disease development at immunological and molecular levels are required. Alongside a detailed history of infections, any exposure should be included for identifying the putative cause of aAA. May be these studies can answer why the profiles of aAA patients vary geographically, anthropologically, and why some patients do not respond to therapy. Poor survival in patients is attributed to immune cell exhaustion and refractoriness to treatment. Thus, researching the prognostic relevance of immune cells is crucial for treatment. A well-defined approach for understanding and unraveling the flawed processes leading to autoimmunity with considerable importance to any kind of environmental exposure, influence of external agents should be undertaken. Genome-wide association studies, single-cell sequencing should be employed for deciphering the diversity of imbalanced T lymphocytes. In-depth analysis on the immune-mediated pathogenesis with effects on disease progression and optimal management of aAA is recommended as research direction.