Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation.

BACKGROUND: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. METHODS: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. RESULTS: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. CONCLUSIONS: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.

Characterization of Ly108-H1 Signaling Reveals Ly108-3 Expression and Additional Strain-Specific Differences in Lupus Prone Mice.

Ly108 (SLAMF6) is a homophilic cell surface molecule that binds SLAM-associated protein (SAP), an intracellular adapter protein that modulates humoral immune responses. Furthermore, Ly108 is crucial for the development of natural killer T (NKT) cells and CTL cytotoxicity. Significant attention has been paid towards expression and function of Ly108 since multiple isoforms were identified, i.e., Ly108-1, Ly108-2, Ly108-3, and Ly108-H1, some of which are differentially expressed in several mouse strains. Surprisingly, Ly108-H1 appeared to protect against disease in a congenic mouse model of Lupus. Here, we use cell lines to further define Ly108-H1 function in comparison with other isoforms. We show that Ly108-H1 inhibits IL-2 production while having little effect upon cell death. With a refined method, we could detect phosphorylation of Ly108-H1 and show that SAP binding is retained. We propose that Ly108-H1 may regulate signaling at two levels by retaining the capability to bind its extracellular as well as intracellular ligands, possibly inhibiting downstream pathways. In addition, we detected Ly108-3 in primary cells and show that this isoform is also differentially expressed between mouse strains. The presence of additional binding motifs and a non-synonymous SNP in Ly108-3 further extends the diversity between murine strains. This work highlights the importance of isoform awareness, as inherent homology can present a challenge when interpreting mRNA and protein expression data, especially as alternatively splicing potentially affects function.

Generation of NK cells with chimeric-switch receptors to overcome PD1-mediated inhibition in cancer immunotherapy.

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.

IFNγ directly counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells potentially through the upregulation of multiple key survival factors.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.

A gain-of-function variant in the Wiskott-Aldrich syndrome gene is associated with a MYH9-related disease-like syndrome.

While loss-of-function variants in the WAS gene are associated with Wiskott-Aldrich syndrome and lead to microthrombocytopenia, gain-of-function variants of WAS are associated with X-linked neutropenia (XLN) and the absence of microthrombocytopenia. Only a few XLN families have been reported so far, and their platelet phenotype was not described in detail. To date, no renal involvement was described in XLN. In the present study, we report exome sequencing of individuals from 3 generations of a family with a dominant disease combining neutropenia, macrothrombocytopenia, and renal failure. We identified a heterozygous missense gain-of-function variant in the WAS gene (c.881T>C, p.I294T) that segregates with the disease and is already known to cause XLN. There was no pathogenic variant in MYH9, TUBB1, or ACTN1. This is the first report of a WAS gain-of-function variant associated with both the hematological phenotype of XLN (neutropenia, macrothrombocytopenia) and renal disease (proteinuria, renal failure) with glomerular tip lesion hyalinosis and actin condensations in effaced podocytes foot processes.

Overactive WASp in X-linked neutropenia leads to aberrant B-cell division and accelerated plasma cell generation.

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching.

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Constitutive activation of WASp leads to abnormal cytotoxic cells with increased granzyme B and degranulation response to target cells.

X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28-coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.

Wiskott-Aldrich syndrome gene mutations modulate cancer susceptibility in the p53± murine model.

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeleton in hematopoietic cells and mutated in two severe immunodeficiency diseases with high incidence of cancer. Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in WASp and most frequently associated with lymphoreticular tumors of poor prognosis. X-linked neuropenia (XLN) is caused by gain-of-function mutations in WASp and associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). To understand the role of WASp in tumorigenesis, we bred WASp+, WASp-, and WASp-XLN mice onto tumor susceptible p53+/- background and sub-lethally irradiated them to enhance tumor development. We followed the cohorts for 24 weeks and tumors were characterized by histology and flow cytometry to define the tumor incidence, onset, and cell origin. We found that p53+/-WASp+ mice developed malignancies, including solid tumors and T cell lymphomas with 71.4% of survival 24 weeks after irradiation. p53+/-WASp- mice showed lower survival rate and developed various early onset malignancies. Surprisingly, the p53+/-WASp-XLN mice developed malignancy mostly with late onset, which caused delayed mortality in this colony. This study provides evidence for that loss-of-function and gain-of-function mutations in WASp influence tumor incidence and onset.

Constitutive activation of WASp in X-linked neutropenia renders neutrophils hyperactive.

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.

Nuclear Wiskott-Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells.

BACKGROUND: The Wiskott-Aldrich syndrome protein (WASp) family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined. METHODS: We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq) in thymocytes and spleen CD4+ T cells. RESULTS: WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF)12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASpL272P and WASpI296T had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus. CONCLUSIONS: These data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development.

Slamf6 negatively regulates autoimmunity.

The nine SLAM family (Slamf) receptors are positive or negative regulators of adaptive and innate immune responses, and of several autoimmune diseases. Here we report that the transfer of Slamf6-/- B6 CD4+ T cells into co-isogenic bm12 mice causes SLE-like autoimmunity with elevated levels of autoantibodies. In addition, significantly higher percentages of Tfh cells and IFN-γ-producing CD4+ cells, as well as GC B cells were observed. Interestingly, the expression of the Slamf6-H1 isoform in Slamf6-/- CD4+ T cells did not induce this lupus-like phenotype. By contrast, Slamf1-/- or Slamf5-/- CD4+ T cells caused the same pathology as WT CD4+ T cells. As the transfer of Slamf [1+6]-/- or Slamf [1+5+6]-/- CD4+ T cells induced WT levels of autoantibodies, the presence of Slamf1 was requisite for the induction of increased levels of autoantibodies by Slamf6-/- CD4+ T cells. We conclude that Slamf6 functions as an inhibitory receptor that controls autoimmune responses.

Deletion of Wiskott-Aldrich syndrome protein triggers Rac2 activity and increased cross-presentation by dendritic cells.

Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8(+) T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFNγ-producing CD8(+) T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8(+) T cells at the expense of CD4(+) T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8(+) T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells.

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies.

Humoral immunodeficiency caused by mutations in the Wiskott-Aldrich syndrome protein (WASp) is associated with failure to respond to common pathogens and high frequency of autoimmunity. Here we addressed the question how deficiency in WASp and the homologous protein N-WASp skews the immune response towards autoreactivity. Mice devoid of WASp or both WASp and N-WASp in B cells formed germinal center to increased load of apoptotic cells as a source of autoantigens. However, the germinal centers showed abolished polarity and B cells retained longer and proliferated less in the germinal centers. While WASp-deficient mice had high titers of autoreactive IgG, B cells devoid of both WASp and N-WASp produced mainly IgM autoantibodies with broad reactivity to autoantigens. Moreover, B cells lacking both WASp and N-WASp induced somatic hypermutation at reduced frequency. Despite this, IgG1-expressing B cells devoid of WASp and N-WASp acquired a specific high affinity mutation, implying an increased BCR signaling threshold for selection in germinal centers. Our data provides evidence for that N-WASp expression alone drives WASp-deficient B cells towards autoimmunity.

The cell surface receptor Slamf6 modulates innate immune responses during Citrobacter rodentium-induced colitis.

The homophilic cell surface receptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6(-/-) C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram(-) bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag(-/-) mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6(-/-) Rag(-/-) mice were markedly reduced as compared with those of Rag(-/-) mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6(-/-) Rag(-/-) mice than in Rag(-/-) animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag(-/-) mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram(-) bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag(-/-) mice.

Congenital defects in neutrophil dynamics.

Neutrophil granulocytes are key effector cells of the vertebrate immune system. They represent 50-70% of the leukocytes in the human blood and their loss by disease or drug side effect causes devastating bacterial infections. Their high turnover rate, their fine-tuned killing machinery, and their arsenal of toxic vesicles leave them particularly vulnerable to various genetic deficiencies. The aim of this review is to highlight those congenital immunodeficiencies which impede the dynamics of neutrophils, such as migration, cytoskeletal rearrangements, vesicular trafficking, and secretion.

SAP modulates B cell functions in a genetic background-dependent manner.

Mutations affecting the SLAM-associated protein (SAP) are responsible for the X-linked lympho-proliferative syndrome (XLP), a severe primary immunodeficiency syndrome with disease manifestations that include fatal mononucleosis, B cell lymphoma and dysgammaglobulinemia. It is well accepted that insufficient help by SAP-/- CD4+ T cells, in particular during the germinal center reaction, is a component of dysgammaglobulinemia in XLP patients and SAP-/- animals. It is however not well understood whether in XLP patients and SAP-/- mice B cell functions are affected, even though B cells themselves do not express SAP. Here we report that B cell intrinsic responses to haptenated protein antigens are impaired in SAP-/- mice and in Rag-/- mice into which B cells derived from SAP-/- mice together with wt CD4+ T cells had been transferred. This impaired B cells functions are in part depending on the genetic background of the SAP-/- mouse, which affects B cell homeostasis. Surprisingly, stimulation with an agonistic anti-CD40 causes strong in vivo and in vitro B cell responses in SAP-/- mice. Taken together, the data demonstrate that genetic factors play an important role in the SAP-related B cell functions. The finding that anti-CD40 can in part restore impaired B cell responses in SAP-/- mice, suggests potentially novel therapeutic interventions in subsets of XLP patients.