Autoimmune Cytopenias Are Highly Associated with Inborn Errors of Immunity and They May Be the Initial Presentations in Cases without Severe Infections

Inborn errors of immunity (IEIs) are inherited disorders that lead to an increased risk of infections, autoimmunity, autoinflammation, lymphoproliferation, or malignancy [1]. Recently, IEIs were classified into 10 groups according to overlapping phenotypes: (1) immunodeficiencies affecting cellular and humoral immunity (combined immunodeficiencies [CIDs]), (2) CIDs with associated or syndromic features, (3) predominantly antibody deficiencies, (4) diseases of immune dysregulation, (5) congenital defects of the phagocyte number or function, (6) defects in intrinsic and innate immunity, (7) autoinflammatory diseases, (8) complement deficiencies, (9) bone marrow failure disorders, and (10) phenocopies of IEI [1].

Early diagnosis and management of IEI reduce the morbidities and mortalities of these patients [2]. Although increased proneness to infection is the hallmark of primary immunodeficiencies, around 25% of patients with IEI may initially present with a noninfectious manifestation [3]. Immune dysregulation diseases (IDDs), in particular, are characterized by diverse clinical phenotypes and aberrant inflammation and may present with normal vaccine response, lymphocyte subsets, and normal or increased immunoglobulin levels, which prevent early diagnosis [4].

Autoimmune cytopenia (AIC) and autoimmune hemolytic anemia (AIHA) are the most common manifestations of autoimmunity in patients with IEI, which have been reported to vary from 10.2% to 84.1% [5‒7]. It has also been identified that at least 65% of pediatric patients with Evans syndrome (ES) have a genetic predisposition [8]. Moreover, AIC is often refractory to treatment in patients with IEI, despite there being a self-limiting disease characteristic in the general population [9, 10].

It is important to report the atypical clinical and laboratory manifestation of IEIs to increase the awareness of physicians to help identify these rare diseases. We aimed to study the clinical and laboratory spectrum of AICs and their outcomes and associations in children with IEIs.

In this retrospective study (October 1994 to January 2023), the data of 562 patients with IEIs were evaluated from the database of the Departments of Pediatric Immunology and Pediatric Hematology in our center. All procedures were conducted according to the principles of the Declaration of Helsinki and human and animal rights. This study protocol was reviewed and approved by the Ege University Medical School Hospital Ethics Committee (approval number: 22-4T/36). The diagnosis of IEIs was based on the Human Inborn Errors of Immunity Committee Report of the International Union of Immunological Societies (IUIS) 2022 [1].

We recruited 34 patients who were diagnosed with IEIs before 18 years of age. These cases have an exact diagnosis of chronic refractory immune thrombocytopenic purpura (ITP) and/or AIHA, and they were differentiated from other causes (i.e., inherited bone marrow failure syndromes, congenital neutropenia, transient cytopenias due to infections or drugs were excluded).

AIHA was defined as the presence of clinical and laboratory evidence of hemolysis with a positive direct antiglobulin [11]. ITP was defined as isolated thrombocytopenia (platelet count <100 × 10³/µL in two distinct measurements) and the absence of other causes [12]. ES was defined as a combination of Coombs-positive hemolytic anemia and immune thrombocytopenia [9]. Since the definition of autoimmune neutropenias has been based on the demonstration of autoantibodies directed to various epitopes on blood neutrophils, we could not include these patients because of the unavailability of screening tests for neutrophil autoantibodies. Lymphoproliferation was defined as persistent (>6 months) lymphadenopathy and/or hepatosplenomegaly without any infections or malignancy.

Immunodeficiencies affecting cellular and humoral immunity result from defective development or function of T cells with variable B cell defects. These disorders include severe CIDs, defined by CD3 T-cell lymphopenia. CIDs without associated or syndromic features have generally less severe complications in the first year of life than severe CIDs [13]. CIDs with associated or syndromic features are characterized by unique clinical features in addition to immune deficiency [14]. Predominant antibody deficiencies refer to impaired antibody production due to molecular defects in B cell development [15]. IDDs due to IPEX syndrome, CTLA4 or LRBA defects, autoimmune lymphoproliferative syndrome, and STAT3 gain-of-function mutations are caused by monogenic defects in T-cell apoptosis, T-cell tolerance, or Tregs, characterized by the loss of self-tolerance, leading to autoinflammation, autoimmunity, and lymphoproliferation with less predominant infections [16]. Demographic details, clinical features, laboratory parameters (complete blood counts, lymphocyte subsets, serum immunoglobulin levels, antinuclear antibodies, direct antiglobulin test), genetic variants, immunomodulatory treatment (IMT) regimens, and outcomes were noted in a predesigned proforma.

In children with AIC, corticosteroids and/or intravenous immunoglobulin (IVIG) was used as the first-line therapy. Hence, refractory ITP and AIHA were treated with the second- and third-line therapies (mycophenolate mofetil [MMF], sirolimus, rituximab, and splenectomy). Corticosteroids (methylprednisolone 30 mg/kg/day, 3 days or 2 mg/kg/day prednisolone) or IVIG (at 1 g/kg/day for 2 days) were used as “first-line therapy” for AICs. MMF (1,200 mg/m²/day), rituximab (375 mg/m²/dose once per week, for 4 weeks), sirolimus (2–3 mg/m² once per day), and splenectomy were used as “second or further line therapy” for refractory AICs.

Response to IMT was defined according to the criteria for AIHA [11] and ITP [12], as shown in Table 1. We also classified the patients according to whether they achieved a complete response with IMT: “responders” (patients who had a complete response) and “nonresponders” (patients with partial and no response). Refractory AIC was identified as a complete failure of the first-line therapy (corticosteroids and IVIG).

Correlations between the demographic data, clinical manifestations, therapeutic outcomes, and patients in different IEI categories were also examined. The patients were also divided into two subgroups based on the presence of immune dysregulation: (1) presence of IDD and (2) absence of IDD (other IEI categories). All data were compared between these two groups. Additionally, immunologic findings were compared between patients who had AIHA, ITP, and ES.

Data analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 25.0 software package. Descriptive statistics are expressed using numbers and percentages. Numeric data are presented using median (IQR). Pearson’s χ² test, the independent samples t test, or the Mann-Whitney U test were used to compare the differences of variables between the groups.

Five hundred and sixty-two patients with IEI were enrolled in the study. A total of 6% (34/562) patients had AIC with a 2.4 ratio of males to females. The percentages of diagnosis of 562 patients and the study population according to IEI categories are shown in Figure 1. In this study, 8 patients (23.5%) had ITP, 12 (35.5%) had AIHA, and 14 (41.2%) had ES. Half of the patients (n = 17) presented with AIC as the first manifestation of IEI without any infection. The median (IQR) age at the initial AIC was 15.5 months (range: 20 days−190 months), whereas the median (IQR) age at IEI diagnosis was 21.5 (range, 2–225) months. The median (IQR) follow-up period from admission was 36 (range: 1–122) months. The most common initial clinical presentations were fatigue (75%), infection (50%), petechia (40%), and jaundice (40%). Lymphoproliferation was detected in 55.9% (n = 19) of the patients. The demographic data of the patients are shown in Table 2.

Patients were diagnosed according to IEI categories [1]: IDDs in 41.2% (n = 14), CIDs with associated or syndromic features in 23.5% (n = 8), immunodeficiencies affecting cellular and humoral immunity (n = 8, 23.5%), and predominantly antibody deficiency in 11.8% (n = 4) patients. None of the patients was diagnosed as having congenital defects of the phagocyte number or function, defects in intrinsic and innate immunity, autoinflammatory diseases, complement deficiencies. Distribution of all cohort and patients with AIC according to IEI categories (2022 IUIS classification) are shown in Figure 1. Diagnoses were confirmed through molecular genetic testing in 19 (55.8%) patients. Detailed evaluations including genetics, treatments, and outcomes of all patients are summarized in Table 3.

Among the patients with immune dysregulation (n = 14), infection and AIC initially manifested at a median (IQR) age of 102 (range: 6–204) and 100 (range: 6–190) months, respectively, which were significantly higher compared with other IEI categories (p < 0.05). The median (IQR) age of diagnosis was 144 (range: 10–225) months and the median (IQR) interval time between the first manifestation and diagnosis was 28 (range: 1–150) months. Delayed diagnosis was also more common in IDDs than in other IEI groups (Fig. 2).

When we compared the clinical characteristics of patients according to IEI categories, patients with IDDs were more likely to develop ES (n = 10, 71.4%) (p = 0.004) (Table 4). Patients with IDDs had a higher frequency of lymphoproliferation (n = 12, 85.7%) compared with other IEI groups (p = 0.005) (Table 4). AIHA was more frequently associated with CID (n = 6, 50%) and immunodeficiencies affecting cellular and humoral immunity (n = 4, 33%) (data not shown).

The distribution of lymphocyte subsets was compared in children with AIHA, ITP, and ES. The mean (±SD) percentage of CD8⁺.T lymphocytes was significantly higher in patients with ES compared with patients with ITP (40.77 ± 20.21% vs. 22.33 ± 12.48%, respectively; p = 0.011). There was no significant difference in other lymphocyte subsets, immunoglobulin levels, and laboratory findings between the AIC groups.

Of the 34 patients with AIC, 33 received the first-line IMT: IVIG (n = 10), corticosteroids (n = 5), or IVIG and corticosteroids (n = 18). Sixty-eight percent (n = 23) of patients failed to achieve remission with the first-line IMT. To investigate the factors that influenced the first-line IMT response in patients with AIC, we compared the pretreatment variables of patients between the first-line treatment responders (patients with CR) and nonresponders (patients with PR + NR). We found that the presence of lymphoproliferation, the diagnosis of immune dysregulation, white blood cell (WBC) counts, neutrophil counts, and the percentage of CD3⁺ T lymphocytes were significantly associated with the first-line treatment response. In the nonresponder group, 89.5% of patients had lymphoproliferation at admission (p = 0.007), 92.9% were diagnosed as having IDDs (p = 0.021), had lower WBC counts (p = 0.025), and lower absolute neutrophil counts (p = 0.013). Nevertheless, responders had lower CD3⁺ T lymphocytes than nonresponders (p = 0.028) (Table 5).

Seventeen patients received the second-line IMT: MMF (n = 9) (CR [n = 4], PR [n = 6]), rituximab (n = 4) with a complete response rate of 100%, sirolimus (n = 7) (CR [n = 6], PR [n = 1]), and splenectomy (n = 3) (CR [n = 2] NR [n = 1]). A comparison analysis of the outcomes of the second-line IMT was not possible because of the small number of cases.

Turkey has a high rate of consanguineous marriages, which is responsible for the increased frequency of IEI, with a prevalence of 30.5/100,000 [17]. Our data demonstrated that AIC was observed in 6% of all our IEI patients. IDDs were the most frequent IEI category diagnosed in 41.2% of the patients with AIC. Lymphoproliferation was the most common accompanying finding, especially in patients with IDDs. Moreover, the presence of lymphoproliferation and IDDs were significantly associated with the first-line treatment responsiveness, which was detected in 68% of our patients. It has been shown that non-IEI-associated AICs are curable with IVIG in around 80% of patients [10]. This finding emphasized the importance of treatment refractory AIC as a probable warning sign of the underlying IEI. To our knowledge, this is the first study to assess the treatment response of AIC from the immunologic perspective of patients with IEIs who are followed up with a multidisciplinary approach for hematology and immunology in a reference hospital in Turkey.

Autoimmunity, especially AIC, is increasingly recognized as a disease manifestation in many IEIs [18]. Westermann-Clark et al. [19] suggested that 11% of patients with AIC might have underlying IEIs. Additionally, the same authors reported that AIC was detected 3 years before IEI was diagnosed [19]. Our results showed that the median delay of diagnosis was 10 (range: 1–150) months after AIC had been detected. Diagnostic delay may be related to the presence of high amounts of patients with IDDs who had later onset of infection episodes than other IEI categories.

IDDs are characterized by the loss of self-tolerance mechanisms within the persistent immune stimulation, leading to autoimmunity [4]. Our results indicated that patients with IDDs were more likely to develop ES and lymphoproliferation. In addition, most of them had a refractory course to the first-line IMT, which should alert physicians for the suspicion of IEI. These findings are consistent with those recently reported in long-term follow-up studies. In related studies, ES is a marker for immunodeficiencies, especially immune dysregulation, particularly when refractory [20‒22].

The genetic variants associated with IEI disturb immune tolerance to self-antigens via different pathophysiological pathways and initiate autoimmunity [4]. Although the exact pathophysiology of ES remains unknown, the existing data have proposed that T-cell abnormalities, including chronic T-cell activation, reduced levels of helper T cells, and increased proportions of cytotoxic T cells, were all the involving mechanisms [23, 24]. Although the role of activated autoreactive cytotoxic CD8+ T cell is well established in autoimmunity, its role is still unknown in ES [25]. Our study showed an increased amount of CD8+ T cells in patients with ES compared to ITP patients, requiring deeper investigation in detailed phenotypic and functional T-cell profiling. Recently, Kumar et al. [24] revealed that pediatric ES patients exhibit a different immune profile rather than ITP. Hence, they propose a scoring system based on immunophenotyping (including cTfh frequency, CD4 effector memory T-cell activation, frequency of naïve CD4+ T cells, and CSMB cells), which would be available and feasible for most clinical flow cytometry laboratories.

Immunomodulators with targeted therapy rather than conventional IMT provide better disease control and better prevention from infections [26]. Corticosteroids and IVIG are accepted as the first-line treatment for AIC in patients with IEI; however, the relapse rate is higher in those patients [27, 28]. Different prognostic factors have been reported for IVIG and corticosteroids as the first-line treatment response of AIC [29, 30]. In our study, the presence of lymphoproliferation, having diseases of immune dysregulation, low WBC and neutrophil counts, and high CD3+ T-lymphocyte counts were negatively associated with better response (Table 5). However, no study in the current literature concerning the association between WBCs, neutrophils, CD3+ T-lymphocyte counts, and AIC treatment was found, and this subject remains to be fully elucidated in further studies with larger sample sizes. No consensus for the second-line therapy and the management of resistant AICs has been reached.

The limitations of our study are its retrospective nature, lack of post-treatment lymphocyte subset values, and the existence of a nonhomogeneous study population. Although we had these limitations, we concluded that AIC may be the initial feature of IEIs without infections. Our data revealed that the finding of an AIC warrants the investigation for the underlying IEI to avoid diagnostic delay, particularly when lymphoproliferation and resistance to first-line therapy co-occur. The presence of lymphoproliferation and IDDs, low WBC, low neutrophil, and high CD3+ T-lymphocyte counts should be considered negative predictors for the best response before giving the first-line treatment. Prospective studies are needed to determine some more predictor markers for AIC treatment response in patients with IEIs.

This study protocol was reviewed and approved by the Ege University Medical School Hospital Ethics Committee, approval number: 22-4T/36. Written informed consent was obtained from the presented patients and their parents to participate in the study.

The authors have no conflicts of interest to declare.

No funding was received for this study.

Raziye Burcu Taşkın wrote the paper. Ezgi Topyıldız collected the data. Neslihan Edeer Karaca and Güzide Aksu performed the analysis. Deniz Yılmaz Karapınar and Necil Kütükcüler designed the study.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.