Gut immune reconstitution in immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome after hematopoietic stem cell transplantation.

To the Editor:

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is an inherited syndrome of early-onset systemic autoimmunity and the prototype of immune dysregulatory disorders. It is caused by mutations of forkhead box p3 (FOXP3) gene (Xp11.23), encoding a key transcription factor for natural regulatory T (nTreg) cells. Treg cell dysfunction leads to severe multi-organ autoimmune phenomena including enteropathy, dermatitis, endocrinopathy, and other organ-specific diseases. Patients often present early in infancy and, without treatment, usually die in the first years of life. The only effective cure is hematopoietic stem cell transplantation (HSCT). However, the outcome is variable. Early HSCT with non-myeloablative conditioning provides the best outcome, before organs are damaged by autoimmunity and/or adverse effects of therapy. Complete donor engraftment in all hematopoietic lineages may not be necessary, because the preferential engraftment of donor Treg cells seems to be sufficient to control the disease.

With parental informed consent and Newcastle upon Tyne Hospitals National Health Service Foundation Trust approval, we report the case of an IPEX patient presenting with severe enteropathy, dermatitis, and other signs of autoimmunity since 3 to 4 weeks of age. The identified FOXP3 gene mutation was associated with a reduced protein expression in Treg cells (Figs E1 and E2 available in this article's Online Repository at www.jacionline.org). Therefore, at 6 months of age, he received an unmanipulated, unrelated donor cord blood stem cell transplant (1 DP mismatch) with sub-myeloablative conditioning and graft-versus-host disease (GvHD) prophylaxis (see this article's Methods section and Fig E3 in the Online Repository at www.jacionline.org). He had an uneventful engraftment and a reasonable immune reconstitution (Fig E4 in this article's Online Repository at www.jacionline.org). Chimerism analysis in peripheral blood showed 90% donor T lymphocytes during the first 6 months after the transplant, with a decrease and stabilization at 70% 1 year post-transplant (Fig E5 in this article's Online Repository at www.jacionline.org). Consistent with the sub-myeloablative conditioning regimen the patient has received, mixed myeloid chimerism was also observed; however, as previously reported, donor myeloid chimerism is not necessary to control the disease. Moreover, FOXP3 protein expression by Treg cells increased over time (Fig E2).

Fig E3

Timeline representing experiments and analysis performed during this study. HE, Hematoxylin-eosin.

Fig E4

Absolute count of T, B, and natural killer (NK) cells (per uL blood) in peripheral blood pre- and post-HSCT (3, 6, 9, and 12 months).

Fig E5

Donor cell chimerism in peripheral blood T cells (CD3+), B cells (CD19+), and monocytes (CD15+) during 1 year post-transplant, shown in months (m).

Fig E2

Intracytoplasmic FOXP3 expression in peripheral blood cells before and at indicated time points after transplant. Gates for analysis were set on CD4+CD25+bright T cells. x axis/arrow, Fluorescence intensity; y axis, cell counts.

Despite good post-transplant immune reconstitution, the patient continued to suffer from diarrhea and malabsorption and was dependent on parenteral nutrition. He developed episodes of upper intestinal obstruction and, despite anti-inflammatory therapy with an anti-TNF-α agent, required jejunal resections at 3, 4, and 6 months. Histopathology of resected areas revealed severe chronic mucosal injury without histological signs of GvHD, with an improvement of the architecture over time (see Fig E6 in this article's Online Repository at www.jacionline.org). The gut dysfunction improved progressively from month 6 to 9, and at 1 year post-transplant, the patient was independent of parenteral nutrition and thriving on enteral feeding.

Fig E6

Histopathological findings of the resected small bowel specimens. Hematoxylin-eosin (HE) staining showed loss of mucosal epithelium with total villous atrophy at both 3 months (A) and 4 months (B) post-transplant and long slender villi with normal brush borders at 6 months post-transplant (C). No histologic evidence of GvHD.

The intestine has a major interface with the external environment, and its integrity is important in the maintenance of immune homeostasis. The intestinal mucosa contains an extensive network of secondary lymphoid tissues and is home to several lymphocyte subsets, including intestine-specific subpopulations. The beta-7 integrins (α4β7 and αEβ7) are selective mediators of lymphocyte homing to the gut-associated lymphoid tissue. In particular, α4β7 is expressed at low levels on naive T and B cells and at high levels on effector and memory T (mainly CD4+) cells.

Because the persistence of enteropathy in the patient was inconsistent with the transplant outcome, we explored the hypothesis that intestinal immune reconstitution proceeded at a different pace to that in the peripheral blood with a delay in the re-establishment of homeostasis within the gut immune system. We therefore investigated the engraftment of donor lymphocytes in the gut mucosa to evaluate any differences between peripheral blood and gut immune reconstitution that might explain the clinical course.

To study gut immune reconstitution, we probed for FOXP3+ and CD4+ T cells on tissue sections of small bowel mucosa at different times after transplant (3, 6, and 9 months). We observed the presence of lymphoid nodules numerically decreasing over time, with an increased proportion of FOXP3+ cells both within nodules and the mucosal area (Fig 1), suggesting a reduction of the small bowel inflammatory state.

Fig 1

FOXP3 “reactive nuclei” (arrow) in and around lymphoid follicles in small bowel mucosa and submucosa on immunohistochemically stained sections at 3 months (A), 6 months (B), and 9 months (C) post-transplant. (FOXP3 reactive nuclei are stained black, and the sections are counterstained with hematoxylin.) D, Quantitative analysis of FOXP3+nuclei in the examined total small bowel mucosal and submucosal tissue.

We isolated CD4+ cells from small bowel tissue sections obtained at 3 months post-transplant and investigated their origin by genotyping FOXP3 (donor or recipient) to evaluate the donor chimerism within the relevant cellular compartment. Both wild-type and mutated nucleotides were present at the c.1037 position on the FOXP3 gene, suggesting a mixed population of lymphocytes in the gut. This was confirmed by chimerism analysis of polymorphic markers on the same cell population showing 60% donor, 40% recipient origin (Fig 2, A), while donor chimerism in the blood CD3+T cells was 91% (Fig E5). We set out to study circulating gut-homing lymphocytes in a patient blood sample, which was available at only 9 months after HSCT. We recovered CD4+CD31+α4/β7low naive and CD4+CD31−α4/β7high memory T cells by cell sorting. Sequence analysis showed wild-type FOXP3 sequence and 90% donor chimerism on CD4+CD31−α4β7high gut-homing lymphocytes, whereas wild-type and mutated FOXP3 genes along with a 50% donor chimerism were found on CD4+CD31+α4/β7low naive T cells not specifically committed to the intestine (Fig 2, B and C).

Fig 2

Exon 9 FOXP3 sequencing and chimerism analysis on laser microdissected CD4+ T cells from small bowel biopsies at 3 months post-transplant (A) and on CD31−CD4+α4β7high memory T cells (B) and CD31+CD4+α4β7low naive T cells (C) sorted from peripheral blood at 9 months post-transplant.

Our results suggest that, in this patient, the gut immune system took longer to recover and function compared with the peripheral immune system (Fig E3). FOXP3 expression assessed in small bowel biopsies taken at different times post-transplant was found to correlate with the patient's clinical condition. Gut function and tolerance of enteral nutrition progressively improved in parallel with the increased FOXP3 expression within the gut mucosa and the appearance of donor CD4+CD31−α4/β7high cells. Furthermore, cells of donor origin were mostly present in the periphery rather than in the gut early after transplant, possibly explaining poor gut function. The increase in donor chimerism within gut-homing lymphocytes was associated with a progressive rise of FOXP3+ cells within the small bowel later on, possibly suggesting that a preferential homing of donor Treg cells to the gut is associated with disease recovery. Further studies in additional patients will be required to determine if this is applicable to other patients with IPEX.

To our knowledge, this is the first study of gut immune reconstitution in a patient affected with an inherited disorder of immune tolerance, and we believe it could be a unique case study that sheds light on the role of the intestine in reconstituting the immune system after HSCT. Further investigation of function of external factors (such as microbiota) in influencing the imprinting of mucosal immunity will help to identify new therapeutic approaches.