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1.
Cancer Immunol Immunother ; 72(5): 1153-1167, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-36355079

RESUMEN

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.


Asunto(s)
Antígeno B7-H1 , Mieloma Múltiple , Humanos , Antígeno B7-H1/metabolismo , Ligandos , Células Asesinas Naturales , Citocinas/metabolismo , Receptores de Células Asesinas Naturales/metabolismo , Inmunoterapia/métodos , Microambiente Tumoral
2.
J Allergy Clin Immunol ; 149(3): 1069-1084, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34384840

RESUMEN

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.


Asunto(s)
Linfocitos B , Neutropenia , Proteína del Síndrome de Wiskott-Aldrich , Animales , Linfocitos B/citología , División Celular , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Inmunoglobulina A , Ratones , Neutropenia/genética , Células Plasmáticas/patología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo
3.
Int J Mol Sci ; 24(5)2023 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-36902453

RESUMEN

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.


Asunto(s)
Antígenos Ly , Transducción de Señal , Animales , Ratones , Antígenos Ly/genética , Línea Celular , Fosforilación , Isoformas de Proteínas/genética
4.
Nat Immunol ; 11(10): 920-7, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20818396

RESUMEN

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.


Asunto(s)
Antígenos CD/metabolismo , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Macrófagos/inmunología , Fagosomas/inmunología , Receptores de Superficie Celular/metabolismo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Antígenos CD/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Bacterianas/genética , Beclina-1 , Células Cultivadas , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Macrófagos/microbiología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Chaperonas Moleculares/genética , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Fagocitosis , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Porinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Proteína de Clasificación Vacuolar VPS15
5.
Clin Immunol ; 173: 19-26, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27368806

RESUMEN

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.


Asunto(s)
Autoanticuerpos/inmunología , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/trasplante , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Interferón gamma/inmunología , Lupus Eritematoso Sistémico/inmunología , Ratones Transgénicos , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología
6.
Int Immunol ; 27(9): 447-57, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25957267

RESUMEN

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.


Asunto(s)
Antígenos CD/inmunología , Citrobacter rodentium/inmunología , Colitis/inmunología , Inmunidad Innata/inmunología , Receptores de Superficie Celular/inmunología , Animales , Colitis/microbiología , Colon/inmunología , Colon/microbiología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Células Th17/inmunología , Interleucina-22
7.
J Autoimmun ; 62: 81-92, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26143192

RESUMEN

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.


Asunto(s)
Autoanticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Eliminación de Gen , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunoglobulina M/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genética , Animales , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/inmunología , Formación de Anticuerpos , Antígenos CD19/genética , Apoptosis/genética , Apoptosis/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Linfocitos B/citología , Trasplante de Médula Ósea , Diferenciación Celular , Haptenos , Hemocianinas/inmunología , Inmunoglobulina M/sangre , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Quimera por Trasplante
8.
Blood ; 119(17): 3966-74, 2012 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-22411869

RESUMEN

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.


Asunto(s)
Linfocitos B/citología , Linfocitos B/fisiología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Proteína del Síndrome de Wiskott-Aldrich/fisiología , Animales , Western Blotting , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiotaxis , Ficoll/análogos & derivados , Ficoll/farmacología , Citometría de Flujo , Hematopoyesis/fisiología , Inmunización , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Ratones , Ratones Noqueados , Trinitrobencenos/farmacología
9.
Blood ; 120(1): 122-9, 2012 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-22613797

RESUMEN

One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell-dependent B cell responses were absent in SAP(-/-).B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP(-/-).BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAP(fl/fl).tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell-mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.


Asunto(s)
Linfocitos B/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Células Asesinas Naturales/inmunología , Trastornos Linfoproliferativos/inmunología , Animales , Antineoplásicos Hormonales/farmacología , Linfocitos B/citología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Femenino , Galactosilceramidas/metabolismo , Galactosilceramidas/farmacología , Expresión Génica/inmunología , Centro Germinal/inmunología , Haptenos/inmunología , Haptenos/metabolismo , Células Asesinas Naturales/citología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Tamoxifeno/farmacología
10.
Blood ; 119(12): 2819-28, 2012 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-22302739

RESUMEN

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.


Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Recuento de Células , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genética
11.
FASEB J ; 27(8): 3123-31, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23629864

RESUMEN

The costimulatory receptor Slamf6 partially controls lupus-related autoimmunity in congenic Sle1b mice; for instance, the presence of the protein isoform Slamf6-H1 in Sle1b.Slamf6-H1 mice mitigates disease. Here, we report that young Sle1b mice, but not Sle1b.Slamf6-H1 or B6 mice, contain a memory T-helper cell subset identified by ]mt]2-fold increase in expression of 17 genes, chief among which is Spp1, encoding the cytokine osteopontin (OPN). These T follicular helper (TFH) cells, including OPN(+) TFH cells, expand concomitantly with severity of the disease. By contrast, Sle1b.Slamf6-H1 or Sle1b.SAP(-)/(-) mice do not develop autoantibodies and the number of T(FH) cells is 5 times lower than in age-matched Sle1b mice. By comparing Sle1b and Sle1b.OPN(-)/(-) mice, we find that the lack of OPN expression impedes early autoantibody production. Furthermore, on the adoptive transfer of Sle1b.OPN(-)/(-) CD4(+) T cells into bm12 recipients autoantibody production and germinal center formation is reduced compared to recipients of Sle1b.OPN(+/+) CD4(+) T cells. We propose a model in which OPN provides a survival signal for a precursor T(FH) cell subset, which is a key factor in autoimmunity.


Asunto(s)
Antígenos CD/inmunología , Autoinmunidad/inmunología , Osteopontina/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Autoinmunidad/genética , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Femenino , Citometría de Flujo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones , Ratones Noqueados , Osteopontina/genética , Osteopontina/metabolismo , Receptor de Muerte Celular Programada 1 , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Receptores CXCR5/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Transcriptoma/inmunología
12.
J Immunol ; 188(8): 3567-71, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22422882

RESUMEN

The contribution of individual molecular aberrations to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease that affects multiple organs, is often difficult to evaluate because of the presence of abundant confounding factors. To assess the effect of increased expression of the phosphatase protein phosphatase 2A (PP2A) in T cells, as recorded in SLE patients, we generated a transgenic mouse that overexpresses the PP2Ac subunit in T cells. The transgenic mouse displays a heightened susceptibility to immune-mediated glomerulonephritis in the absence of other immune defects. CD4(+) T cells produce increased amounts of IL-17 while the number of neutrophils in the peripheral blood is increased. IL-17 neutralization abrogated the development of glomerulonephritis. We conclude that increased PP2Ac expression participates in SLE pathogenesis by promoting inflammation through unchecked IL-17 production and facilitating the development of end-organ damage.


Asunto(s)
Expresión Génica/inmunología , Glomerulonefritis/inmunología , Interleucina-17/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteína Fosfatasa 2/inmunología , Animales , Anticuerpos Neutralizantes/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Glomerulonefritis/inducido químicamente , Humanos , Interleucina-17/biosíntesis , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo
13.
J Immunol ; 187(1): 21-5, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21622868

RESUMEN

Several genes within a syntenic region of human and mouse chromosome 1 are associated with predisposition to systemic lupus erythematosus. Analyses of lupus-prone congenic mice have pointed to an important role for the signaling lymphocyte activation molecule family (slamf)6 surface receptor in lupus pathogenesis. In this article, we demonstrate that a second member of the Slamf gene family, Slamf4 (Cd244), contributes to lupus-related autoimmunity. B6.Slamf4(-/-) mice spontaneously develop activated CD4 T cells and B cells and increased numbers of T follicular helper cells and a proportion develop autoantibodies to nuclear Ags. B6.Slamf4(-/-) mice also exhibit markedly increased autoantibody production in the B6.C-H-2bm12/KhEg → B6 transfer model of lupus. Although slamf4 function is best characterized in NK cells, the enhanced humoral autoimmunity of B6.Slamf4(-/-) mice is NK cell independent, as judged by depletion studies. Taken together, our findings reveal that slamf4 has an NK cell-independent negative regulatory role in the pathogenesis of lupus a normally non-autoimmune prone genetic background.


Asunto(s)
Antígenos CD/fisiología , Autoanticuerpos/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Receptores Inmunológicos/fisiología , Animales , Antígenos CD/genética , Cromatina/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Humanos , Tolerancia Inmunológica/genética , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Familia de Moléculas Señalizadoras de la Activación Linfocitaria
14.
Proc Natl Acad Sci U S A ; 107(7): 3024-9, 2010 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-20133615

RESUMEN

DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4(R278H/R278H) (Lig4(R/R)) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4(R/R) mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans.


Asunto(s)
Anomalías Múltiples/genética , Formación de Anticuerpos/genética , ADN Ligasas/genética , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , Mutación Missense/genética , Inmunodeficiencia Combinada Grave/genética , Animales , Apoptosis/inmunología , Southern Blotting , Niño , ADN Ligasa (ATP) , ADN Ligasas/inmunología , Citometría de Flujo , Humanos , Inmunoglobulinas/sangre , Inmunofenotipificación , Ratones , Mutación Missense/inmunología , Síndrome
15.
Biomolecules ; 13(4)2023 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-37189377

RESUMEN

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.


Asunto(s)
Anticuerpos Antiproteína Citrulinada , Artritis Reumatoide , Ratones , Animales , Anticuerpos Antiproteína Citrulinada/metabolismo , Neutrófilos/metabolismo , Ácidos Aminosalicílicos , Artritis Reumatoide/metabolismo , Células Clonales
16.
Int Immunol ; 23(2): 149-58, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21278219

RESUMEN

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.


Asunto(s)
Antígenos CD/genética , Antígenos CD/inmunología , Autoanticuerpos/inmunología , Glomerulonefritis/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Animales , Glomerulonefritis/genética , Humanos , Inmunohistoquímica , Endogamia , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones de la Cepa 129 , Ratones Congénicos , Ratones Noqueados , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria
17.
Oncoimmunology ; 11(1): 2109861, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35979386

RESUMEN

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.


Asunto(s)
Interferón gamma , Leucemia Mielógena Crónica BCR-ABL Positiva , Antígenos CD34/metabolismo , Antígenos CD34/farmacología , Apoptosis , Línea Celular Tumoral , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre/metabolismo , Regulación hacia Arriba
18.
Blood Adv ; 6(18): 5279-5284, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-35404999

RESUMEN

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.


Asunto(s)
Neutropenia , Insuficiencia Renal , Síndrome de Wiskott-Aldrich , Actinas/genética , Mutación con Ganancia de Función , Pérdida Auditiva Sensorineural , Humanos , Mutación , Cadenas Pesadas de Miosina/genética , Neutropenia/genética , Trombocitopenia/congénito , Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/genética
19.
Cell Death Dis ; 12(10): 875, 2021 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-34564697

RESUMEN

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.


Asunto(s)
Antineoplásicos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiofenos/farmacología , Antígenos CD34/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Clonales , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Mesilato de Imatinib/administración & dosificación , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína bcl-X/metabolismo
20.
JCI Insight ; 6(6)2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33621210

RESUMEN

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.


Asunto(s)
Degranulación de la Célula , Granzimas/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Animales , Estudios de Casos y Controles , Ratones , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T Citotóxicos/inmunología , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patología
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