Normal numbers of stem cell memory T cells despite strongly reduced naive T cells support intact memory T cell compartment in Ataxia Telangiectasia

Ataxia Telangiectasia (AT) is a rare inherited disorder characterized by progressive cerebellar ataxia, chromosomal instability, cancer susceptibility and immunodeficiency. AT is caused by mutations in the ATM gene, which is involved in multiple processes linked to DNA double strand break repair. Immunologically, ATM mutations lead to hampered V(D)J recombination and consequently reduced numbers of naive B and T cells. In addition, class switch recombination is disturbed resulting in antibody deficiency causing common, mostly sinopulmonary, bacterial infections. Yet, AT patients in general have no clinical T cell associated infections and numbers of memory T cells are usually normal. In this study we investigated the naive and memory T cell compartment in five patients with classical AT and compared them with five healthy controls using a 24-color antibody panel and spectral flow cytometry. Multidimensional analysis of CD4 and CD8 TCR alpha beta(+) cells revealed that early naive T cell populations, i.e. CD4(+)CD31(+) recent thymic emigrants and CD8(+)CCR7(++)CD45RA(++) T cells, were strongly reduced in AT patients. However, we identified normal numbers of stem cell memory T cells expressing CD95, which are antigen-experienced T cells that can persist for decades because of their self-renewal capacity. We hypothesize that the presence of stem cell memory T cells explains why AT patients have an intact memory T cell compartment. In line with this novel finding, memory T cells of AT patients were normal in number and expressed chemokine receptors, activating and inhibitory receptors in comparable percentages as controls. Comparing memory T cell phenotypes by Boolean gating revealed similar diversity indices in AT compared to controls. We conclude that AT patients have a fully developed memory T cell compartment despite strongly reduced naive T cells. This could be explained by the presence of normal numbers of stem cell memory T cells in the naive T cell compartment, which support the maintenance of the memory T cells. The identification of stem cell memory T cells via our spectral flow cytometric approach is highly relevant for better understanding of T cell immunity in AT. Moreover, it provides possibilities for further research on this recently identified T cell population in other inborn errors of immunity.Transplantation and immunomodulatio

Recurrent respiratory tract infections (RRTI) in the elderly: A late onset mild immunodeficiency?

Transplantation and immunomodulatio

T and NK cells in IL2RG-deficient patient 50 years after hematopoietic stem cell transplantation

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56(dim)CD27(+) NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR V beta antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR V beta repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27(-) and/or CD28(-) memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57(+)KLRG1(+)CX3CR1(+) phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.Transplantation and immunomodulatio

NK cell-dependent antibody-mediated immunotherapy is improved in vitro and in vivo when combined with agonists for toll-like receptor 2 in head and neck cancer models

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.Transplantation and immunomodulatio

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome

Absent B cells, agammaglobulinemia, and hypertrophic cardiomyopathy in folliculin-interacting protein 1 deficiency

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondria! numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1(-/-) animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of I immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.Molecular Technology and Informatics for Personalised Medicine and Healt

Comment to: Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia

Transplantation and immunomodulatio

CD56(dim)CD16(-) NK cell phenotype can be induced by cryopreservation

Transplantation and immunomodulatio

A Comprehensive Workflow for Applying Single-Cell Clustering and Pseudotime Analysis to Flow Cytometry Data

The introduction of single-cell platforms inspired the development of high-dimensional single-cell analysis tools to comprehensively characterize the underlying cellular heterogeneity. Flow cytometry data are traditionally analyzed by (subjective) gating of sub-populations on two-dimensional plots. However, the increasing number of parameters measured by conventional and spectral flow cytometry reinforces the need to apply many of the recently developed tools for single-cell analysis on flow cytometry data, as well. However, the myriads of analysis options offered by the continuously released novel packages can be overwhelming to the immunologist with limited computational background. In this article, we explain the main concepts of such analyses and provide a detailed workflow to illustrate their implications and additional prerequisites when applied on flow cytometry data. Moreover, we provide readily applicable R code covering transformation, normalization, dimensionality reduction, clustering, and pseudotime analysis that can serve as a template for future analyses. We demonstrate the merit of our workflow by reanalyzing a public human dataset. Compared with standard gating, the results of our workflow provide new insights in cellular subsets, alternative classifications, and hypothetical trajectories. Taken together, we present a well-documented workflow, which utilizes existing high-dimensional single-cell analysis tools to reveal cellular heterogeneity and intercellular relationships in flow cytometry data.Transplantation and immunomodulatio

Mesenchymal stromal cells isolated from children with systemic juvenile idiopathic arthritis suppress innate and adaptive immune responses

Transplantation and immunomodulatio