Welcome to the ESID 2022 Meeting Interactive Programme

Background and Aims

PDLIM1, is a member of LIM proteins, that negatively regulates NF-κB-mediated signaling by sequestering p65 in the cytoplasm and suppressing its nuclear translocation. The role of PDLIM1 in human and murine T cell biology remains elusive.

Methods

Whole Exome sequencing was performed in a patient with early onset immune dysregulation and recurrent bacterial infections, born to healthy consanguineous parents, which identified a homozygous loss-of-function mutation in PDLIM1. The immunological phenotype was recapitulated in a PDLIM1 loss-of-function mouse model.

Results

The patient initially presented with immune dysregulation and enhanced proinflammatory cytokine production during infancy and early childhood. At older age, the patient’s phenotype shifted to recurrent staphylococcal infection due to a loss of peripheral Th17 cells. Similarly, young Pdlim1^-/- mice showed enhanced NF-κB-dependent Th1 and Th17 cell differentiation with exaggerated expression of markers associated with overactivation and premature immunosenescence/exhaustion on Th17 cells. The immunological phenotype was completely reversed in elderly Pdlim1^-/- mice with suppressed proinflammatory response and abrogated Th1/17 cell differentiation.

Conclusions

These data identify PDLIM1 as a novel regulator of Th17 differentiation causing inborn error of immunity in humans.

Background and Aims

Ras-related protein 1b (Rap1b) belongs to the Ras-superfamily of small GTPases. Its active form, GTP-Rap1, mediates integrin activation in platelets and lymphocytes thereby regulating adhesion and migration (Stefanini-2018; Shimonaka-2003).

Methods

Whole exome sequencing (WES), Next generation sequencing (NGS), RAP1B-overexpression, functional analysis.

Results

We investigated a boy presenting with neonatal thrombocytopenia, neutropenia, anaemia, low monocyte counts, absolute lymphopenia (balanced lymphocyte subsets, decreased naïve CD4+ and CD8+ T-cells) and profound hypogammaglobulinemia. WES revealed a heterozygous, unknown and predicted deleterious, de novo variant c.35G>A, p.G12E in RAP1B. G12 is located in the RAP1B active site. Interestingly, G12V leads to constitutively activated Rap1 (Scrima-2008).

The patient presented an abnormal increase in PBMC migration and integrin activation in resting platelets, as well as increased GTP-RAP1 expression and altered cell cycle in B-EBV cells. G12E and G12V-RAP1B overexpression in a megakaryocytic cell line recapitulated integrin overactivation pattern and abnormal increase in RAP1B-GTP activity (also demonstrated in HEK293T cells) supporting that G12E is a gain-of-function (GOF) mutation.

Interestingly, NGS identified RAP1B c.35G>A as a somatic mutation. Variant allele frequency (VAF) in patient’s peripheral blood cells decreased over time, while VAF in bone marrow cellular subsets remained stable, suggesting that RAP1B-G12E confers a negative advantage preferentially in the peripheral cell compartments.

Conclusions

We identified the first RAP1B-G12E mutation in a patient with combined immunodeficiency associated to severe platelet dysfunction. We show that this mutation is a de novo somatic GOF mutation suggesting that monoallelic RAP1B mutations should be considered as a novel cause of inborn errors of immunity associated to thrombocytopathy.

Background and Aims

Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations of Pik3cd, resulting in dysregulated immunity, recurrent respiratory infections and lymphoproliferation, yet underlying mechanisms behind these phenotypes remain unclear.

Methods

Using patient samples and a mouse model (Pik3cd^E1020K/+ mice), we have evaluated CD4 and CD8 T cell function both in culture and in vivo in response to infectious agents, evaluating cellular phenotypes, metabolism, gene expression and chromatin organization.

Results

We find that Pik3cd^E1020K/+ CD8⁺ T cells exhibited accelerated differentiation to short-lived effectors, associated with increased mTORC1 and c-Myc pathways, as well as altered metabolic, transcriptional and epigenetic circuits characterized by a pronounced IL-2/STAT5 signature associated with heightened IL-2 responses that prevented differentiation to memory-like cells in the presence of IL-15.. Conversely, Pik3cd^E1020K/+ CD8⁺ T cells failed to sustain expression of proteins critical for maintenance of long-lived memory cells, including TCF1, and mounted inadequate central memory responses in vivo with enhanced generation of recently-described long-lived effector populations. We now have examined responses to chronic infection using Clone 13 LCMV as a model pathogen, where we find that Pik3cd^E1020K/+ mice lose TCF1+ progenitor CD8⁺ T cells, leading to an altered balance of effector and exhausted cells associated with increased immunopathology. Similarly, we find altered differentiation of CD4 cells from Pik3cd^E1020K/+ mice, with increased inflammatory cytokine production and immunopathology in vivo.

Conclusions

Our data position PI3Kd as a central hub integrating multiple signaling nodes that promote an accelerated effector T cell program at the expense of central memory.

Background and Aims

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency, with heterogeneous clinical presentation. Our goal was to analyze CD8 T cell homeostasis in patients with infection only CVID, compared to those additionally affected by dysregulatory and autoimmune phenomena.

Methods

Flow and mass cytometry evaluation of peripheral blood of 40 patients with CVID and 17 healthy donors.

Results

CD8 T cells are skewed in patients with CVID, with loss of naïve and increase of effector memory stages, expansion of cell clusters with high functional exhaustion scores, and a highly activated population of cells with immunoregulatory features, producing IL-10. These findings correlate to clinically widely used B-cell-based EURO classification. Features of exhaustion, including loss of CD127, CD28 and expression of TIGIT and PD-1 in CD8 T cells are strongly associated with interstitial lung disease and autoimmune cytopenias, whereas CD8 T cell activation with elevated HLA-DR and CD38 expression predict non-infectious diarrhea.

Conclusions

We demonstrate features of advanced differentiation, exhaustion, activation and immunoregulatory capabilities within CD8 T cells of CVID patients. Assessment of CD8 T cell phenotype may allow risk-assessment of CVID patients, and provide new insights into CVID pathogenesis, including a better understanding of mechanisms underlying T cell exhaustion and regulation.

Background and Aims

Although previous studies implicated c-Jun N-terminal kinase 1 and 2 (JNK1/2) in helper CD4⁺ T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs (e.g. NF-κB) responsible for effector cell survival, proliferation, and differentiation. Beyond NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and JNK-dependent transcription in T cells.

Methods

We transfected CARD11 knockout (KO) Jurkat T cells with wild-type (WT) and DI CARD11 expression plasmids to assess effects on AgR-induced JNK signaling. RNA sequencing was performed on mitogen-activated WT and CARD11 KO T cells with or without JNK inhibition to define JNK-dependent transcriptomic changes.

Results

We found that AgR-dependent JNK1 and JNK2 phosphorylation is CARD11-dependent. Furthermore, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation and c-Jun accumulation with variable potency, mirroring the extent of impaired NF‑κB activation. Intriguingly, JNK inhibition in WT Jurkat and primary CD4⁺ T cells resulted in elevated expression of GATA3, the master transcriptional regulator of Th2 cell differentiation.

Conclusions

Our novel findings suggest that defective CARD11-dependent JNK signaling in CD4⁺ T cells may contribute to severe allergic disease manifestations noted in CADINS patients, unveiling a new potential therapeutic target.

Background and Aims

Partial T cell primary immunodeficiency disorders are a group of inborn errors of immunity (IEI) that have partial reduction of T cell number/function and are commonly associated with autoimmune and inflammatory complications. T cell receptor (TCR) signaling is initiated by Lymphocyte cell-specific protein-tyrosine kinase (LCK). A novel homozygous mutation in LCK (p.Pro440Ser, LCK P440S) was identified in two siblings who were born to consanguineous parents, presenting with T lymphopenia, recurrent viral and fungal infections, and gastrointestinal inflammatory complications.

Hypothesis: The P440S mutation impairs LCK expression/function such that T cell-mediated intestinal immunity is dysfunctional, leading to intestinal inflammation (colitis).

Aim 1: Determine effect of the P440S mutation on LCK expression/function in TCR signaling.

Aim 2: Determine etiology of colitis in mice harboring Lck P440S (lck mut).

Methods

We generated a cell line and a mouse model that harbor the mutant kinase, and performed downstream immunophenotypic and functional assessments.

Results

The P440S mutation results in shorter protein half-life, decreased protein expression, and defective TCR signaling responses. Similarly to the lck-/- mice, the lck Mut mice demonstrate impaired thymocyte development with T cell lymphopenia, and skewed memory phenotype of peripheral T cells. However, unlike the lck-/-, only the lck mut mice develop colitis, suggesting that the partial loss of Lck function drives colitis development.

Conclusions

Our findings explain the aberrant immunological presentation of the human P440S patients, advance our understanding of intestinal inflammation in the context of partial T cell defects, and establish a model system with which to develop therapies against intestinal inflammation in IEI.

Background and Aims

In patients with B-cell positive severe combined immunodeficiency (B+ SCID) due to mutations in IL2RG, B-cell function after HSCT depends on the engraftment of donor B lymphocytes. To study development and function of autologous and donor B-cells in patients with mixed chimerism after HLA-haploidentical HSCT, we analyzed blood samples of 12 long term surviving patients (1 to 34 years of follow up) with B+ SCID. All patients developed normal specific humoral immune function and are independent of Ig-substitution.

Methods

We used staining with allele-specific antibodies targeting HLA-molecules to characterize the maturation stages of donor- and autologous B-cells by flow-cytometry.

Results

We demonstrate a complete lack of switched memory B-cells (CD19+IgM-CD27+) in the autologous compartment but detected a percentage of up to 19% of CD27+ in CD19+ autologous cells, which were identified as IgM+CD27+ marginal-zone (MZ)-like B-cells. Class switched memory B-cells were strictly confined to donor derived B-cells and surprisingly a proportion of 0.9% of donor B-cells detected in peripheral blood was found sufficient to allow for normal B-cell function. Beyond that we found a negative correlation between the proportion of donor-cells and the percentage of switched memory B-cells in the donor derived B-cell compartment indicating a relative lack of naïve B-cells in patients with low B-cell-donorchimerism.

Conclusions

We identified unexpectedly low proportions (0,9-17%) of donor B-cells in the peripheral blood in 4/12 patients as sufficient for normal B-cell function, and further specified the phenotypic, transcriptional and epigenetic consequences of common-gamma-chain deficiency in autologous B-cells in the presence of normal T-cell function.