Delayed Reconstitution of Thymopoiesis After a Thymus Transplant

To the Editor: Defects of thymus development present a phenotypically highly variable group of inborn errors of immunity. At their most severe, they manifest as severe combined immunodeficiency (SCID) with complete lack of thymopoiesis resulting in absent circulating T cells. Fortunately, such severe phenotypes may be identified early through neonatal SCID screening programmes due to absent T-cell receptor excision circles (TRECs) in neonatal dry blood spots, allowing for prompt prophylactic measures and urgent referral for a curative treatment. In the case of defects of thymus development, thymus transplantation is the only viable long-term treatment option [1].

We have, through the neonatal TREC/kappa-recombination excision circle (KREC) SCID screening programme implemented in Czechia in 2024 [2], identified a boy with absent TRECs (0 copies/100 000 cells) and normal KRECs. He was a first child, born at term to a mother with essential hypertension and type 2 diabetes, with a prenatally diagnosed congenital heart defect (pulmonary valve aplasia, ventricular septal defect and double-outlet right ventricle). Postnatal imaging uncovered multiple defects of the spinal column, including hemivertebrae, butterfly vertebra and atypical rib attachments. The boy additionally suffered from hypoparathyroidism with hypocalcaemia, but had no seizures.

Upon positive screening recall, peripheral blood immunophenotyping was performed, revealing absent circulating T cells, with normal B and NK cell compartments. Maternal engraftment was not detectable. Imaging of the mediastinum was performed using magnetic resonance imaging (MRI) and ultrasound, failing to visualise thymic tissue. The patient was started on cotrimoxazole, fluconazole and acyclovir prophylaxis, immunoglobulin replacement therapy and increased hygienic precautions were adopted.

Genetic evaluation of microdeletions, next generation sequencing data of a panel of genes associated with inborn errors of immunity and agnostic whole exome sequencing did not identify a genetic aetiology. Given his clinical phenotype we concluded that a suboptimally compensated diabetes of the mother (glycated hemoglobin A1c 45–49 mmol/mol) during the first trimester of the pregnancy induced a diabetic fetopathy, resulting in the DiGeorge sequence, including congenital athymia. Successful ex vivo differentiation of the patient’s CD34⁺ haematopoietic stem cells into T cells in artificial thymic organoids further strengthened a likely diagnosis of athymia (P11 in Golwala et al.) [3].

Following corrective cardiac surgery at 1 month of age, which found no thymus upon median sternotomy, the patient was referred for a thymic transplant at the Great Ormond Street Hospital (GOSH) in London, UK. His pre-transplant period was complicated by transient elevation of liver enzymes and development of bile stones, presumed to be caused by the early administration of cotrimoxazole, an episode of suspected catheter sepsis (Staph. hominis + epidermidis), as well as acquisition of extended spectrum beta-lactamase (ESBL)⁺ E. coli and norovirus colonization. A thymus transplant was performed at 3 months of age, with cyclosporin alone as immunosuppression (Fig. 1A).

Fig. 1.

(A) Patient timeline indicating major life events and treatments. (B) histopathology of transplanted thymic tissue embedded in skeletal muscle, 3 months post-transplant, a-d are representative images from a patient with evidence of active thymopoeisis and early T-cell reconstitution (positive control), e-h are representative images from the patient described, a/e hematoxylin and eosin staining, b/f pan-cytokeratin staining, c/g CD3 staining, d/h Ki67 staining. (C) Longitudinal development of total T cell, CD4 T cell and CD8 T cell numbers and (D) the proportion of naïve CD4 and CD8 T cells, shown as % of total CD4 and CD8 T cells respectively. (E) CD4 T cell phenotype at 11, 24 and 33 months of age assessed using flow cytometry, showing naïve, central memory (CM), effector memory (EM) and effector memory cells re-expressing CD45RA (TEMRA) CD4 T cells and the proportion of HLA-DR⁺ activated CD4 T cells. (F) TCR beta (TRB) clonality shown as CDR3 nucleotide length histograms, where each color represents a distinct clone and column height indicates the percentage of total TRB rearrangements, from 11 and 30 months of age

At 3 months post-transplant, a routine biopsy of the graft was performed at GOSH. Histopathology demonstrated suboptimal graft vitality, with condensed thymic epithelial cells and reduced T-cell density and proliferation (Fig. 1B). The appearances were more in keeping with an “empty thymus” seen in cultured pre-implantation slices.

Usually, following a thymus transplant, naïve T cells appear in circulation gradually starting at 4–6 months post-transplant, although a recent study has shown a wide heterogeneity with the timing of the reconstitution, with ~ 60% of patients recovering naïve CD4 T cell production by 12 months post-transplant and 79% reconstituting by 24 months [4]. Our patient showed absent T cells until 9 months of age (6 months post-tx), whereupon a precipitous T cell expansion became apparent, reaching 1500 cells/µl within less than 30 days (Fig. 1C). Notably, all of the newly present T cells had the memory phenotype (CD45RO⁺) with signs of activation (HLA-DR⁺) (Fig. 1D, E). This development was accompanied by whole-body rash and alopecia (Figure S1), as well as eosinophilia (max 3000 cells/µl). The T cells were not monoclonal based on TCRB sequencing, although their diversity was limited, with 538 clonotypes identified and diversity index of 0.65 (Fig. 1F, top). There was no evidence of thymic donor engraftment in T cells or peripheral blood mononuclear cells on short tandem repeat chimerism analysis. We hypothesized that this was an Omenn-like expansion of oligoclonal autoreactive T cells and treated it with increased cyclosporin dosage (targeting trough levels of 150 ng/ml), with gradual effect. After this flare-up, the patient’s T cells continued decreasing, reaching the nadir at 15 months of age, at 220 T cells/µl, with no CD8 T cells and no naïve forms.

Due to stagnating weight (body weight 6,38 kg (−5.4 SD) at age 16 months), persistently poor absorption of orally administered medication which additionally compromised the maintenance of calcaemia and cyclosporin trough levels, chronic diarrhoea and highly positive norovirus in the stool, we decided to treat the patient with favipiravir, following previous positive reports in immunocompromised patients [5]. Treatment with 200 mg favipiravir 2x/day was initiated at 16 months of age, 7-days after initiation achieved plasma favipiravir trough levels of 17.5 µg/ml, peaking at 67 µg/ml one hour after administration. The treatment led to substantial increase in weight over the first 3 months, fewer stools and no noted adverse effects. There was no consistent reduction in norovirus load during the favipiravir treatment, which was discontinued at 25 months of age due to no perceived further effect on stool frequency, consistency, or virus load. The patient’s stool became PCR negative by 29 months of age (26 months post-tx), 4 months after favipiravir discontinuation. This mirrors the previous experience that favipiravir increases mutagenesis and lowers pathogenicity, but not viral load in chronic norovirus carriers.

The unsatisfactory reconstitution of thymopoiesis was interrupted at 17 months of age by a Panton Valentine leukocidin (PVL)⁺ Methicillin-resistant Staphylococcus aureus (MRSA) sepsis with a concomitant rhinovirus respiratory infection, necessitating admission and 1 week of artificial ventilation. This was associated with a massive expansion of T cells, reaching 2400 cells/µl, 95% of which were TCRαβ⁺, 75% CD4⁺, 13% CD8⁺, with 0.5% and 8.7% naïve and 78% and 93% HLA-DR⁺ CD4 and CD8 T cells, respectively, but crucially, in the following months led to first appearance of naïve T cells and recent thymic emigrants with the CD4⁺CD45RO⁻CD62L⁺ or CD4⁺CD45RA⁺CD27⁺ and CD4⁺CD45RA⁺CD31⁺ phenotypes respectively, as well as limited but notable appearance of CD8 T cells, including naïve forms at 24 months of age (Fig. 1C, D). Naïve CD4 T cells continue increasing and reached 310/µl (50.6%), recent thymic emigrants attained 280/µl (45.8%) and TCRB clonality is significantly improved with 2322 clonotypes identified and diversity index of 0.85 (Fig. 1F, bottom). The patient remains free of severe infectious complications at 33 months of age, on 3 day/week cotrimoxazole and 1x/day fluconazole prophylaxis, as well as 400 mg/kg subcutaneous immunoglobulin replacement, and makes substantial developmental strides.

This case illustrates the complex journey of patients with congenital athymia in whom the 3-month post-transplant graft biopsy demonstrated limited thymopoiesis and as a result, the reconstitution of thymopoiesis was delayed. Even faltering graft may on rare occasions preserve the capability of reconstituting immune function, and the window of immune reconstitution is wide, with some patients achieving thymopoiesis almost 2 years after the transplantation [4, 6]. In our patient this reconstitution temporally followed an infectious stimulus. While infections generally lead to transient suppression of thymic output, mechanisms for its regeneration are equally important and well-described [7], with the thymus being able to recover after damage particularly well in young patients.

We also note a positive response of our patient to favipiravir, which should be considered a therapeutic option in immunocompromised patients with chronic norovirosis.

Close monitoring of immune phenotype is paramount in post-thymus transplant patients, allowing for therapeutic fine-tuning as well as family counselling.

Author Contributions

A.K. wrote the manuscript, attended the patient and performed cytometric analysis. B.O. attended the patient, provided the auxological data and co-wrote the manuscript. O.V. attended the patient, performed cytometric analysis and reviewed the manuscript. A.S. attended the patient, provided consultations and reviewed the manuscript. C.S. and E.K. set up and performed favipiravir level monitoring and reviewed the manuscript. S.L. performed histopathological analysis and reviewed the mansucript. E.F. performed TCRB sequencing and reviewed the manuscript. T.F. and V.B. performed genetic sequencing and analysis and reviewed the manuscript. Z.G., A.Y.K. and A.W. attended the patient, performed thymic transplant and co-wrote the manuscript.

Funding

The creation of this work was supported by the funding from the Ministry of Health of the Czech Republic AZV NU23-05-00097.

Data Availability

The data is available in processed form from the authors upon reasonable request. Raw data is not available.

Declarations

Ethics Approval and Consent to Participate

The study was approved by the Ethical Committee of University Hospital in Motol, ref. no. EK-602.1/22. The legal representatives of the patient signed an informed consent with the patient’s inclusion in the study in accordance with the Declaration of Helsinki.

Competing Interests

The authors declare no competing interests.