DISCUSSION
This study found a strong association between immune cytopenias and IEI, demonstrating the feasibility and usefulness of an extended lymphocyte immunophenotyping to detect IEI signs in immune cytopenic pediatric patients. In this context, we identified T and B lymphopenia, a reduction in CD4+ and CD8+ naїve T-cells%, T-reg cells% and switched memory B-cells%, and an increase in CD4+ effector memory T-cells% and CD21low B-cells% along with the presence of hypogammaglobulinemia as variables significantly associated with IEI.
Mean lymphocyte, CD4+ and CD8+ T-cell count reached lower mean levels in IEI+ group. Lymphopenia is indeed one of the factors ofImmunodeficiency related score , a diagnostic algorithm developed for an early identification of IEI13; thus, it is a suggestive sign of CID11, described also in individuals with CVID14.
A general reduction in CD4+ T-cells probably led to a decline in T-reg cells, more prevalent in IEI+ group and responsible for the impairment of self-tolerance mechanism that frequently occurs in subjects with IEI15.
T-cell phenotype in IEI+ group showed a decrease in CD4+ and CD8+ naїve T-cells% against an expansion in CD4+ and CD8+ effector memory T-cells%, being common features of CVID16, reported also in DGS17, likely due to persistent immune system stimulation by chronic/recurrent infections. Diverging from literature, we found increased CD4+ central memory T-cells% in IEI+ group, as a potential consequence of their abnormal interaction with antigen-presenting cells and subsequent failed differentiation into effector memory T-cells18.
B-cell phenotype was characterized in IEI+ group by an accumulation of CD21low B-cells% against a decrease in PAN-B%, switched memory B-cells% and plasmablasts%, whose lack resulted in a defective production of IgG, IgA and IgM. This is in line with the fact that switched memory B-cells%, IgG and IgA deficiency and CD21low B-cells% expansion are diagnostic criteria of CVID11, which is the most frequent IEI in literature19 as well as in our cohort.
In IEI+ group, CD4+ naїve T-cells% showed a positive correlation with CD8+ naїve T-cells% and a negative correlation with CD4+ effector memory T-cells% and CD21low B-cells%. The negative correlation between CD4+ naїve T-cells% and CD21low B-cells% maintained significance in our CVID subgroup, as reported by Warnatz20. All these associations probably reflect the role of persistent/chronic inflammation- and infection-driven immune activation in the impairment of T-cell regulation of B-cells20, resulting in an immunosenescent-like phenotype typical of some IEI forms21. These features were also found in patients suffering simultaneously from both CVID and ITP22, characterized by an accumulation of auto-reactive CD21low B lymphocytes, responsible for IEI-related autoimmune disorders8, along with defects in PAN-B cells bone marrow production and CD4+ naїve T-cells differentiation. These findings suit also with the individual of the cohort with DGS, showing low CD4+ naїve T-cells%, switched memory B-cells% and IgM levels, along with an inverted CD4+/CD8+ ratio, all biomarkers configuring an immunophenotype associated with autoimmunity, lymphoproliferation and a severe clinical course, characterized in our case by invasive and recurrent infections23–25. Memory and transitional B-cells% did not show a significant difference between the two groups. This result could be indicative of continuous autoantigen-driven stimulation and defective immune-regulatory networks, previously described in subjects with autoimmune manifestations, including ITP26,27, which was the most represented immune cytopenia in our two groups.
We identified a high rate of correlation between immune cytopenias and IEI, probably due to the inclusion criteria of the cohort, in which persistent/relapsing forms of immune cytopenias affected 97% of patients. Our hypothesis is supported by Hadjadj, assessing the pathogenic role and poor prognostic significance of proven and/or potential IEI-related genes in 65% of pediatric ES cases28.
We found two individuals with CVID carrying TNFRSF13B variants. The frequency of TNFRSF13B gene mutations was reported to be significantly higher in CVID compared to healthy controls and TACI biallelic mutations were detected only in CVID. Patients with biallelic TACI mutations had a similar incidence of autoimmunity and lymphoproliferation compared to wild-type TACI subjects. CVID individuals carrying monoallelic mutations had a severe clinical course with the highest prevalence of immune-dysregulation disorders29. Interestingly, the Pt.2 patient carried also three other alterations, the pathogenic variants CR2:c.826delT and PRF1:A91V and the VUS TCF3:G444E (Table 1-A). Further studies are needed to validate these additional variants and to understand if they act in concert with biallelic genetic alterations in TNFRSF13B  to give rise to complex IEI-related phenotype.
This study’s primary limitations result from the sample size and the retrospective nature of the review. The high diagnosis rate of IEI in such a relatively limited cohort could be related to subjects’ enrollment at a tertiary care center, where immunodeficiency suspicion may be elevated.
The strength of this study consists in an in-depth clinical/immunological characterization of the cohort highlighting correlations useful in the prompt identification and management of immune cytopenic children with higher risk of IEI.
In conclusion, IEI diagnosis in patients with immune cytopenias was significantly associated with specific clinical signs and immunophenotypical anomalies, namely T/B lymphopenia, decrease in naїve T-cells%, switched memory B-cells%, plasmablasts% and immunoglobulins, increase in effector/central memory T-cells% and CD21low B-cells%. Thus, an extended lymphocyte typization can identify subjects worthy of IEI-related molecular analysis, useful for validating new targeted-gene variants and unveiling their correlation with IEI phenotype, which in some cases is the expression of a complex genotypic interaction between a small number of mutant genes rather than of a single-gene inherited Mendelian disorder.
Authorship Contributions Drs Zama, Conti, Moratti and Cantarini conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript.
Dr Rivalta designed the data collection instruments, collected data and critically revised the manuscript.
Dr Rondelli carried out statistical analyses and critically revised the manuscript.
Drs Facchini, Prete and Ferrari contributed to interpretation of data and critically revised the manuscript.
Prof Seri carried out molecular analyses and critically revised the manuscript.
Prof Pession critically reviewed the manuscript for important intellectual content and contributed to the design of the study.
All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.