Patient Population and HSCT CharacteristicsPatient Population

A total of 18 patients were included in this study, 16 patients with homozygous or compound heterozygous mutations in either DNMT3B (ICF1, n = 6), ZBTB24 (ICF2; n = 4), CDCA7 (ICF3; n = 4) or HELLS (ICF4; n = 2) and two patients without a genetic diagnosis (based on clinical criteria (ICFX; ESID Registry criteria [21]). The median age at onset of symptoms was 0.6 years (range 0.3–14.3 years). Median age at (genetic) diagnosis was 2.0 years (range 0–17.2 years). Three patients (P4, P16, P18) were diagnosed in the first months of life via positive family history. Patient characteristics and pre-transplant disease manifestations are summarized in Tables 1 and 2.

Table 1 Patients’ and HSCT (baseline) charcteristicsTable 2 Clinical features and genetic variants of patients with ICF syndrome (pre-HSCT)Pre-Transplant Disease Manifestations

Pre-transplant infections were documented in all patients and represented the most common indication for HSCT (in 83% of patients). Viral gastro-intestinal infections, airway infections caused by both viral and opportunistic pathogens and systemic infections with EBV, CMV and/or adenovirus were most common. Two-thirds of patients (12/18) had opportunistic infections (PJP, EBV/CMV/adeno-viremia and/or severe candidiasis) and one-third (6/18) was documented to have pre-transplant bronchiectasis. All patients were on immunoglobulin substitution therapy (and, for patients with data available, serum IgG trough levels were within the normal range for age (Table 3)). Two-thirds of patients (12/18) were on antibacterial prophylaxis, one-third (6/18) received antifungal prophylaxis and 5/18 patients (28%) used antiviral prophylaxis (acyclovir). Two patients (P7 and P14) received valganciclovir to treat primary, persisting CMV infection.

Table 3 Immune parameters (pre-HSCT)

In addition to infectious diarrhea, four patients (P6, P8, P12 and P18) suffered from chronic non-infectious diarrhea/enteropathy. Overall, diarrhea and/or failure to thrive was documented in 13/18 (72%) patients, with need for nasogastric tube or parenteral feeding in 3/18 patients. Enteropathy was reported in 4/5 evaluable ICF1, 4/4 ICF3 and 2/2 ICF4 patients but in only 1/4 ICF2 patients. Enteropathy with or without failure to thrive was reported the second most common indication for HSCT (in 56% of patients).

Six patients (33%) were reported to suffer from manifestations related to immune dysregulation: 1- to 3-lineage cytopenia (n = 3; P2, P8, P17), enteropathy/colitis (n = 2; P6 and P17), hepatitis (n = 3; P8, P13-14), kidney- (n = 1; P13) and/or skin (n = 1; P17) disease. Of note, only one (P6) of four patients with non-infectious diarrhea (P6, P8, P12, P18) was categorized as ‘auto-immune enteropathy’, suggesting an underestimation of this disease manifestation in the current cohort. Three of six patients with immune dysregulation were treated with immunosuppressive drugs. In four patients (P6, P8, P13, P17; 22%), immune dysregulation was a major indication for HSCT.

Two patients, both ICF2, presented with a hematological malignancy: EBV-driven B cell lymphoma (P9) and diffuse large B cell lymphoma (P10), while another ICF2 patient (P7) presented with EBV-driven HLH. These three patients were treated with (immuno-)chemotherapy to achieve remission before HSCT. One patient (P3, ICF1) suffered from (transfusion-dependent) myelodysplasia.

Pre-Transplant Immunophenotype

Information about the pre-transplant immune parameters is summarized in Table 3. Consistent with existing data about humoral immunity in ICF syndrome, all patients suffered from moderate-severe hypogammaglobulinemia in the presence of normal numbers of circulating total B cells (according to age-adjusted reference values (Table 3)). Few patients (4/17) had decreased B cell counts at first analysis. Except for P18, B cell deficiency was likely secondary to myelodysplasia (P3), treatment of malignancy (P10) or immunosuppressive therapy (P17). In 3/3 patients with data available, switched memory B cells (CD19 + IgD-IgM-CD27-) were very low (≤ 1%).

Total numbers of CD3 + T cells, as well as CD3 + CD4 + and CD3 + CD8 + T cells, were in the normal age-adjusted range for 12/16 (evaluable) patients (Table 2). Three of four patients with reduced T cell counts suffered from either malignancy (n = 1, P10) or immune dysregulation (n = 2, P13 and P17) and two of these patients (P10 and P17) were treated with chemotherapy or immunosuppressants at the time of analysis. The two patients with T cell lymphopenia without chemo- or immunosuppressive therapy were both ICF3 patients, aged 5.5 (P11) and 15 years (P13). Information about pre-transplant naïve T cell numbers and/or mitogen-induced lymphocyte proliferation was unavailable for almost all patients. Absolute T cell counts did not correlate with occurrence of opportunistic infections and/or hematological malignancy. Due to a limited number of ‘older’ patients (age > 10 years) in this cohort, we were unable to evaluate the natural course of T cell lymphopenia over time.

NK cell counts were in the normal range for most (80%) patients. Two of 15 (evaluable) patients, both ICF1 (P2 and P4), had a mild reduction in NK cell counts. The single patient with significantly reduced NK cell counts was pan-lymphopenic due to chemotherapy for B cell lymphoma (P10).

HSCT Characteristics

A total of 18 HSCT procedures were performed in this cohort of 18 patients between 2003 and 2021 in 12 different centers (summarized in Table 4). Median age at HSCT was 4.3 years (range 0.5 – 19 years). Donors were MSD/MFD (n = 7), MMFD (n = 1), MUD (n = 9) and MMUD (n = 1). The sources of stem cells for HSCT were bone marrow (BM) or peripheral blood (PB) in n = 10 and n = 8 cases, respectively. Ex vivo TCRαβ/CD19 depletion of the graft was performed in three cases with PB grafts: one MMUD and two MUD cases. There was an equal distribution between MAC (n = 9) and RIC (n = 9), with a preference for RIC in patients with pre-transplant organ damage. Except for one MMUD TCRαβ/CD19 depleted HSCT, all patients received graft versus host disease prophylaxis with either a single (n = 2) or a combination of immunosuppressive drugs (n = 15). In 16 patients (89%), serotherapy was applied, with an equal distribution between ATG (n = 8) and alemtuzumab (n = 8).

Table 4 HSCT and patients’ post-HSCT characteristics

Age at transplant was remarkable high for patients with pre-HSCT bronchiectasis (mean age 12.4 years) and for patients transplanted for immune dysregulation (mean age 13.5 years) and hematological malignancy/HLH (mean age 13.1 years) as compared to patients with other indications for HSCT (mean age 2.0 years).

Engraftment, Chimerism and Post-Transplant Complications

Data about engraftment, chimerism and post-HSCT complications are summarized in Table 4. All evaluable patients (P17 excluded, death before graft infusion) engrafted successfully with a median time to engraftment of 17 days (range 10–22 days). With a median follow-up of 2.2 years (range 0.1–14 years), full donor chimerism (> 90% donor) was reported in all but two patients. Five patients received additional cell therapeutic interventions: P2 received a CD34-selected stem cell boost (at six months after HSCT) for mixed chimerism (pre-boost: 59% (T cells) and 84% (whole blood) donor) resulting in stable mixed chimerism at 2.5 years after HSCT (75% (T cells) and 44% (whole blood) donor); P9 received DLI (within the first month after HSCT) for persistent EBV- and CMV-viremia, leading to full clearance; P10 received DLI (at + 26 and + 71 days after HSCT; CD45RA-depleted T cells) for persistent adenovirus infection, but died at day + 145; P14 received virus-specific T cells for persistent CMV and EBV infection (within the first four months after transplant) and additional DLI for mixed chimerism (80% donor, pre-DLI) at 10 months, resulting in complete viral clearance and stable mixed chimerism (85% donor at 3 years after HSCT); P15 received a stem cell boost for mixed chimerism (25% donor, pre-boost) at 6 months resulting in full donor chimerism at 14 years after HSCT.

Acute graft versus host disease (aGVHD) was documented in 6/17 (35%) evaluable patients with skin-only grade I/II aGVHD in n = 4 patients (P6, P7, P12 and P18) and grade III (gut) aGVHD in n = 2 patients (P11 and P16, both suffering from pre-HSCT infectious diarrhea). Acute GVHD was successfully treated in all affected patients. There were no cases with chronic GVHD.

Infections were the most common post-HSCT complication: in 11/17 patients (65%) the infections were of viral origin and represented predominantly airway infections, gastro-intestinal infections and/or systemic infections with CMV/EBV/adenovirus. In 5/11 patients (P2, P9, P11, P13, P14), these viral infections originated pre-HSCT. Four patients demonstrated de novo post-HSCT infections of non-viral origin: bacterial (n = 3; S. pneumoniae (n = 1, P4) and Clostridium (n = 2, P10 and P14)) or fungal (n = 1, Aspergillus (P12)). All surviving patients recovered from infections without sequelae, except for two patients: pneumococcal meningitis resulted in severe hearing loss and precocious puberty in patient P4 and moderate-severe disability resulted from a pre-HSCT viral encephalitis in patient P14. At > 1 year post-HSCT, anti-microbial prophylaxis was continued in n = 4 patients (P6, P8, P11, P12), all with pre-transplant bronchiectasis.

Post-HSCT outcome of pre-existing immune dysregulation was evaluable in 5 survivors (P2, P6, P8, P13, P14). Immune-mediated cytopenia resolved in all evaluable cases (P2, P8) and recovery from hepatitis was documented for 3/3 patients (P8, P13, P14), with residual mild fibrosis without portal hypertension in P13. Data on recovery from pre-HSCT (non-infectious) enteropathy with or without failure to thrive was available for 9/13 survivors: diarrhea resolved in all patients, and 2/2 nasogastric tube and/or parenteral feeding dependent patients (P12 and P13) were off supplemental nutrition at latest follow-up. One patient (P11) was reported to be on PEG-feeding despite clearing Norovirus and gaining weight at 1 year post-HSCT. Two patients developed de novo post-HSCT immune dysregulation: (transient) thyrotoxicosis in P1 and thyroiditis plus vitiligo in P4. Concerning P4: although the HLA-identical sibling donor of this patient appeared healthy at time of stem cell donation, it was (retrospectively) demonstrated that she suffered from antibody-mediated (subclinical) hypothyroidism at time of donation. Therefore, P4 was diagnosed with donor-derived auto-immunity. At latest follow-up, none of the patients were on immunosuppressive drugs.

Survival Analysis

The mean follow-up after transplant was 51 months (range 0–185 months, median 58 months; Table 4). Overall survival was 83%, all deaths occurred within the first five months after HSCT (Fig. 1). Event-free survival (with events defined as death, GvHD > grade II (n = 2) and/or graft failure (n = 0)) was 72%, with resolution of GvHD in all patients. Three patients died during the HSCT trajectory: P3 (ICF1) died at day 41 after transplant, while being full donor, from respiratory failure most likely due to RS virus infection. P10 (ICF2) died at day + 145 from multi-organ failure after prolonged adenovirus infection, despite early engraftment, full donor chimerism, antiviral therapy and DLI (twice). P17 (ICFX) died one day before graft infusion, due to conditioning related toxicity and sepsis despite having received RIC (Table 4). All three patients who had been diagnosed by positive family history and were transplanted at young age (≤ 3 years), survived.

Fig. 1

Post-HSCT overall survival

Immune Reconstitution

Cellular immune reconstitution data were collected at 1 year and/or at latest follow-up after HSCT (Table 5). CD3 + T cells numbers were within the age-adjusted reference values at 1 year and at latest follow-up in 92% and 100% of patients respectively (n = 13 evaluable patients). A similar favourable pattern was found for CD4 + T cells numbers with 77% and 93%, and for CD8 + T cells counts with 100% and 93%, respectively (n = 14 evaluable patients). In 3/8 evaluable patients, naïve CD4 + T cell counts at latest follow-up were within age-adjusted reference values. Median age at HSCT for these three patients was 2.2 years (P14-16), as compared to 8.5 years for patients with naïve CD4 + T cell counts below age-adjusted thresholds (P1-2, P6, P12-13). NK cell reconstitution at 1 year and at latest follow-up showed values within the age-adjusted reference range in 92% of evaluable patients. Finally, B cell numbers were within the age-adjusted reference values in all (n = 15) evaluable cases at 1 year and at latest follow-up after HSCT. At latest follow-up, all patients had become immunoglobulin substitution independent, except for two: one patient treated pre-HSCT with high-dose rituximab for hematological malignancy (P9) and one patient with only 9 months post-HSCT follow-up (P12). In both patients, switched memory B cells were still absent (data not shown). Adequate responses to tetanus and pneumococcal (re)vaccination were reported in 8/8 evaluable patients, including a patient with stable mixed chimerism (P2).

Table 5 Post-HSCT immune reconstitution