Alessia Scarselli

In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells

Genetically engineered T memory stem cells preserve differentiation activity for decades after patient infusion. Sealing T cell fate Clinical trials are a relatively untapped source of experimental data that can be leveraged to explore both basic and pathological biology in humans. Now, Biasco et al. take advantage of two different gene therapy trials for inherited immunodeficiency to track in the long term T cell fate in humans. They find that the recently described T memory stem cells (TSCM) are able to persist and preserve their precursor potential in human recipients for up to 12 years after genetic correction and infusion into patients. The safety and long-term survival of these cells not only strengthen our knowledge of human immunology but also support the use of TSCM cells for adoptive immunotherapy. A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow–transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.

The case of an APDS patient: Defects in maturation and function and decreased in vitro anti-mycobacterial activity in the myeloid compartment

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patients B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.

In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency.

BACKGROUND Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies. OBJECTIVES Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells. METHODS Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT). RESULTS We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles. CONCLUSION These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.

Cutaneous granulomatosis and combined immunodeficiency revealing Ataxia-Telangiectasia: a case report

Ataxia-telangiectasia (A-T) is a complex multisystem disorder characterized by progressive neurological impairment, variable immunodeficiency and oculo-cutaneous telangiectasia. A-T is a member of chromosomal breakage syndromes and it is caused by a mutation in the ataxia-telangiectasia mutated (ATM) gene. Because of a wide clinical heterogeneity, A-T is often difficult to diagnose in children.We report an unusual case of a 3-year-old boy affected by A-T who presented exclusively with extensive cutaneous granulomatosis and severe combined immunodeficiency, without neurological abnormalities, at the time of diagnosis. This case clearly emphasizes the variable presentation of A-T syndrome and highlights the difficulties in the early diagnosis of A-T.A-T should be considered in children with evidence of combined humoral and cellular immunodeficiency associated with unexplained skin granulomatous lesions, even in the absence of the classic features of this syndrome.

Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID

The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.

B‐cell activation with CD40L or CpG measures the function of B‐cell subsets and identifies specific defects in immunodeficient patients

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B‐cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B‐cell functions in infancy and throughout childhood. We show that T‐independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T‐dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

Relapsing Campylobacter jejuni Systemic Infections in a Child with X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency of the humoral compartment, due to a mutation in the Bruton tyrosine kinase (BTK) gene, characterized by a severe defect of circulating B cells and serum immunoglobulins. Recurrent infections are the main clinical manifestations; although they are especially due to encapsulated bacteria, a specific association with Campylobacter species has been reported. Here, we report the case of a boy with XLA who presented with relapsing Campylobacter jejuni systemic infections. His clinical history supports the hypothesis of the persistence of C. jejuni in his intestinal tract. Indeed, as previously reported, XLA patients may become chronic intestinal carriers of Campylobacter, even in absence of symptoms, with an increased risk of relapsing bacteraemia. The humoral defect is considered to be crucial for this phenomenon, as well as the difficulties to eradicate the pathogen with an appropriate antibiotic therapy; drug resistance is raising in Campylobacter species, and the appropriate duration of treatment has not been established. C. jejuni should always be suspected in XLA patients with signs and symptoms of systemic infection, and treatment should be based on antibiogram to assure the eradication of the pathogen.

A prospective study on the natural history of patients with profound combined immunodeficiency: An interim analysis

Background: Absent T‐cell immunity resulting in life‐threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and “atypical” SCID show reduced, not absent T‐cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P‐CID), for which outcome data are insufficient for unambiguous early transplant decisions. Objectives: We sought to compare natural histories of severity‐matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. Methods: In this prospective and retrospective observational study, we recruited nontransplanted patients with P‐CID aged 1 to 16 years to compare natural histories of severity‐matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. Results: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of “atypical” SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T‐cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched‐pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T‐cell immunity were good predictors of disease evolution. Conclusions: The P‐CID study for the first time characterizes a group of patients with nontypical SCID T‐cell deficiencies from a therapeutic perspective. Because genetic and basic T‐cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P‐CID.

Disease Evolution and Response to Rapamycin in Activated Phosphoinositide 3-Kinase δ Syndrome: The European Society for Immunodeficiencies-Activated Phosphoinositide 3-Kinase δ Syndrome Registry

Activated phosphoinositide 3-kinase (PI3K) δ Syndrome (APDS), caused by autosomal dominant mutations in PIK3CD (APDS1) or PIK3R1 (APDS2), is a heterogeneous primary immunodeficiency. While initial cohort-descriptions summarized the spectrum of clinical and immunological manifestations, questions about long-term disease evolution and response to therapy remain. The prospective European Society for Immunodeficiencies (ESID)-APDS registry aims to characterize the disease course, identify outcome predictors, and evaluate treatment responses. So far, 77 patients have been recruited (51 APDS1, 26 APDS2). Analysis of disease evolution in the first 68 patients pinpoints the early occurrence of recurrent respiratory infections followed by chronic lymphoproliferation, gastrointestinal manifestations, and cytopenias. Although most manifestations occur by age 15, adult-onset and asymptomatic courses were documented. Bronchiectasis was observed in 24/40 APDS1 patients who received a CT-scan compared with 4/15 APDS2 patients. By age 20, half of the patients had received at least one immunosuppressant, but 2–3 lines of immunosuppressive therapy were not unusual before age 10. Response to rapamycin was rated by physician visual analog scale as good in 10, moderate in 9, and poor in 7. Lymphoproliferation showed the best response (8 complete, 11 partial, 6 no remission), while bowel inflammation (3 complete, 3 partial, 9 no remission) and cytopenia (3 complete, 2 partial, 9 no remission) responded less well. Hence, non-lymphoproliferative manifestations should be a key target for novel therapies. This report from the ESID-APDS registry provides comprehensive baseline documentation for a growing cohort that will be followed prospectively to establish prognostic factors and identify patients for treatment studies.

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency

Background: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA‐ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N‐terminal truncations in I&kgr;B&agr; that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor &kgr; light chain enhancer of activated B cells (NF‐&kgr;B) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA‐ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in I&kgr;B&agr; have impaired noncanonical NF‐&kgr;B activity and defective lymphorganogenesis. Objective: We sought to establish genotype‐phenotype correlation in patients with AD EDA‐ID. Methods: A disease severity scoring system was devised. Stability of I&kgr;B&agr; mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF‐&kgr;B signaling in skin‐derived fibroblasts. Results: Disease severity was greater in patients with I&kgr;B&agr; point mutations than in those with truncation mutations. I&kgr;B&agr; point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF‐&kgr;B–dependent IL‐6 secretion and upregulation of the NF‐&kgr;B subunit 2/p100 and RELB proto‐oncogene, NF‐&kgr;B subunit (RelB) components of the noncanonical NF‐&kgr;B pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF‐&kgr;B–driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus &agr;‐lymphotoxin &bgr; receptor–stimulated fibroblasts from patients with point mutations compared with those with truncations. Conclusions: I&kgr;B&agr; point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF‐&kgr;B activity in patients with AD EDA‐ID.