Successful haploidentical PBSCT with subsequent T-cell addbacks in a boy with HyperIgM syndrome presenting as severe congenital neutropenia

Abstract

Jasinska A, Kalwak K, Trelinska J, Borowiec M, Piatosa B, Zeman K, Mlynarski W. Successful haploidentical PBSCT with subsequent T-cell addbacks in a boy with HyperIgM syndrome presenting as severe congenital neutropenia.

Abstract:  HIGM syndrome is a group of primary immunodeficiency disorders characterized by recurrent bacterial and opportunistic infections; it is also associated with normal to elevated serum IgM levels and a concomitant deficiency of IgG, IgA, and IgE. In this report, we give account of a boy with X-linked HIGM and a novel Y172C mutation within his CD40LG gene. He presented with severe neutropenia as the dominating symptom. His bone marrow showed maturation arrest at the promyelocyte/myelocyte stage, typical of congenital neutropenia. This boy suffered from life-threatening infections and required high doses of rhG-CSF, and a haploidentical PBSCT was also successfully performed, thus leading to reconstitution of CD40L expression on activated CD4+ T cells (as assessed with flow cytometry six months after the procedure). Two low-dose T-cell addbacks were required to re-establish full donor chimerism and clear CMV reactivation. The report demonstrates that in select cases, alternative donor allogeneic HSCT supported by DLI may be effective in correcting the defect in X-linked HIGM, and HSCT in HIGM children is not necessarily limited to matched sibling donor transplantation.

Abbreviation:

CMV

cytomegalovirus

DLI

donor lymphocyte infusion

GvHD

graft versus host disease

HIGM

HyperIgM

HLA

human leukocyte antigen

HSCT

hematopoietic stem cell transplantation

PBSCT

peripheral blood stem cell transplantation

Neutropenia is often times a hematologic manifestation of a genetically heterogeneous group of complex inherited syndromes; one of primary immunodeficiency syndromes presenting with congenital neutropenia is HIGM syndrome (1, 2). HIGM is a group of disorders characterized by normal to elevated serum IgM levels with a concomitant deficiency of IgG, IgA, and IgE. In the majority of cases, the disease shows an X-linked inheritance pattern (XHIGM, HIGM-1) (1, 3). Most affected males present in early infancy with upper and lower respiratory tract infections, otitis media, sinusitis, and diarrhea.

Long-term prognosis of patients with HIGM remains poor, despite supportive therapy including IvIg substitution and antimicrobial prophylaxis (2). An allogeneic HSCT from the best available donor remains the only curative option for children with HIGM (4). There is, however, a substantial risk of graft rejection and/or viral complications after haploidentical T-cell-depleted HSCT. In the setting of a haploidentical donor and non-malignant disease, the use of DLI remains extremely questionable, because of the risk of unwanted GvHD (5, 6).

Case description

We present a nine-month-old child who was referred to our outpatient clinic because of persistent severe neutropenia, with absolute neutrophil count consistently below 500/μL. Laboratory studies revealed decreased IgA (3 mg/dL) and IgG levels (32 mg/dL) with normal IgM value (93 mg/dL). No signs of protein loss were found. Bone marrow examinations repetitively showed myeloid maturation arrest at the pro-myelocyte stage with nearly complete lack of mature neutrophils. Peripheral blood lymphocyte subpopulations measured by flow cytometry were normal, and neutrophil oxygen metabolism assessed by the BurstTest showed normal oxidative burst activity. Despite monthly administration of intravenous immunoglobulins (IVIg) at a dose of 400 mg/kg, the child was repeatedly hospitalized because of recurrent bacterial infections of respiratory and gastrointestinal tract and skin abscesses; finally complicated by sepsis of Pseudomonas aeruginosa etiology and multi-organ failure. As the boy remained neutropenic, granulocyte colony-stimulating factor (rhG-CSF, Filgrastim) was started in addition to antibiotics. Normal neutrophil counts (ANC > 1500/μL) and reduction in the frequency and severity of infections were only achieved with the dose of >30 μg/kg daily; however, over time, the dose became insufficient, and the boy periodically required about 48 μg/kg rhG-CSF daily.

Cytometric evaluation of CD40L on peripheral blood lymphocytes showed severely decreased CD40L expression on PMA stimulated lymphocytes of about 1.1% of our patient, as well as in his healthy mother (10%). Therefore, a CD40LG direct sequencing was performed (ABI 3130 genetic analyzer; Applied Biosystems, Foster City, CA, USA) and revealed a novel A to G substitution within the exon 5, resulting in Y172C amino acid substitution. The same mutation in a heterozygous pattern was identified in the boy’s mother. Despite a very low CD40L expression, she remained asymptomatic. Such expression patterns were previously described in some immune-competent female HIGM carriers (7). The patient’s pedigree is presented in Fig. 1.

Figure 1.  (a) Pedigree of the patient with hyper-IgM syndrome (filled shape). The boy’s mother is a healthy carrier of CD40LG Y172C mutation, with only 10% upregulation of CD40L expression on activated CD4+ T-cells. Both her sisters are also carriers of the mutation. The patient’s sister is not affected and bears two wild type alleles, with 96% upregulation of CD40L expression on activated CD4+ T-cells. The patient bearing Xlinked Y172C mutation almost lacks CD40L expression (1.1% after stimulation). (b) DNA sequencing of CD40L gene in the patient, his sister and mother. The patient’s chromatogram shows A to G (Y172C) mutation in CD40LG gene. (c) Ribbon diagram of CD40L protein. Tyrosine 172 residue (red) is substituted with cysteine, potentially disturbing CD40L trimeric structure.

As this patient suffered from several life-threatening conditions related to severe neutropenia, the high-dose rhG-CSF failed to prevent the infections, but worked to limit their number and severity. The boy was then later referred to the bone marrow transplantation unit. The patient had neither a matched related nor unrelated hematopoietic stem cell donor available. As the best available cord blood unit was only matched in four of six alleles (incl. DRB1* allele mismatch), his 6/10 HLA-matched father was chosen as a haploidentical donor. The patient and the donor were matched within DRB1* alleles. The option of initial haploidentical T-cell-depleted PBSCT was discussed with the family, and consent to this procedure was obtained. The conditioning regimen consisted of treosulfan 36 g/m², fludarabine 150 mg/m², cyclophosphamide 120 mg/kg, ATG (Fresenius) 30 mg/kg, and OKT-3/steroids up to day +20. OKT3 up to 0.1 mg/kg was given as rejection prop-hylaxis. T-cell depletion in vitro (CD3+ CD19+ negative depletion, CliniMACS; Miltenyi, Bergisch-Gladbach, Germany) (8) was performed as a method of GvHD prophylaxis. The parental graft consisted of 17.45 × 10⁶ CD34+, 2.07 × 10⁵ CD3+, and 0.5 × 10⁶ CD19+ cells/kg recipient body weight. Hematologic recovery was prompt and sustained: WBC > 1.0 G/L and ANC > 0.5 G/L on day +14 and platelets >50 G/L on day +15. The patient’s post-transplant period was uneventful even after the immunosuppressive therapy was stopped on day +31. After achieving primary full donor chimerism, an increased mixed chimerism (up to 9.2% autologous content) was observed at three months post-transplant. Cyclosporin A was stopped and it was decided to give the T-cell addback (DLI) of 5 × 10⁴ CD3+ cells/kg on day +85. Another reason for the administration of the DLI was because of the delayed immune recovery (ca. 100 CD3+ cells/μL, including merely 20 CD3+ CD4+ cells/μL), resulting in recurrent ganciclovir-resistant CMV reactivations, which were treated with Foscarnet. As the autologous content in PB increased up to 16%, a second addback of T cells (1 × 10⁵ CD3+ cells/kg) was given four wk later on day +108. This immune manipulation resulted in a decreasing incidence of mixed chimerism, greater CMV clearance, and excellent immunological and clinical status. The patient became a full donor chimera seven months later and remains now in excellent clinical condition with no signs of GvHD three yr post-transplant. Figure 2 presents the correlation between T-cell-recovery post-DLI and chimerism. An expression of CD40L on activated CD4+ T cells assessed with flow cytometry six months after the transplantation reached around 72.5%. This result correlated well with the adequate immune reconstitution of both cellular and humoral immunity; since the time the patient remains off from IVIg substitution (IgG and IgM within normal range), he does not require rhG-CSF.

Figure 2.  Improved T cell recovery and chimerism after DLI. CD3+ – T cells; CD4+ – T helper cells; %AUTO – percentage of autologous nucleated cells in peripheral blood.

Discussion

Despite the rhG-CSF treatment, IVIg substitution, and antimicrobial prophylaxis, a significant proportion of children with XHIGM still die unless they are cured by allogeneic HSCT (2, 4, 9). Recombinant CD40L is capable of improving T-cell function in patients with XHIGM; however, the effect is only partial and transient (10).

We believe that even in the absence of cholangitis, only neutropenia and T/B cell combined immunodeficiency should be considered indications for any transplant procedures, including the most difficult haploidentical HSCT, which sometimes require a gentle immune modulation by T-cell addbacks to balance between the risk of GvHD and autologous recovery or graft rejection. In the setting of non-malignant disease, GVHD confers no advantage, and therefore, DLI with the associated high risk of GVHD, particularly in an HLA-mismatched setting, has been only used exceptionally (5, 6) Nevertheless, the present case demonstrates efficacy of DLI in CMV clearance, modulation of chimerism, and immune recovery. To our knowledge, this is the second case of immunomodulation by DLI in a patient with XHIGM syndrome. The first patient, however, underwent matched sibling donor PBSCT after non-myeloablative conditioning regimen and was given DLIs containing ≥2 log more T cells/kg recipient body weight (11). In contrast to our case, DLIs resulted in limited chronic GVHD.

To conclude, XHIGM syndrome should be considered in a male patient presenting with the symptoms of severe congenital neutropenia; in selected subjects, allogeneic HSCT may be indicated and successfully lead to reconstitution of CD40L expression, and finally, HSCT in XHIGM children not necessarily has to be limited to matched sibling donor transplantation. What is important, increasing mixed chimerism after haploidentical PBSCT can be eliminated by the gentle use of low-dose T-cell addbacks, which might also significantly improve immune reconstitution.

Acknowledgments

The work was supported in part by the grant from the Ministry of Science and Higher Education NN407146338 (BP) as well as by research funds from Wroclaw Medical University (KK, ST-395) and the Medical University of Lodz (AJ, MB, and WM). The authors thank the clinical and nursing staff of both clinics for their continuous and dedicated patient’s care.

Authors' contributions

AJ and KK were involved in the concept/design, data collection, analysis/interpretation, drafting the article, and approval. JT was involved in data collection, critical revision of the article, and approval of the article. BP and MB were involved in data analysis/interpretation, critical revision of the article, and approval of the article. KZ contributed to concept, critical revision of the article, and approval of the article. WM was involved in concept/design, critical revision of the article, and approval.