RAC2 and primary human immune deficiencies Lougaris, Vassilios; Baronio, Manuela; Gazzurelli, Luisa ...
Journal of leukocyte biology,
August 2020, Letnik:
108, Številka:
2
Journal Article
Recenzirano
RAC2 is a GTPase that is exclusively expressed in hematopoietic cells. Animal models have suggested important roles for RAC2 in the biology of different cell types, such as neutrophils and ...lymphocytes. Primary immunodeficiencies represent “experimentum naturae” and offer priceless insight on the function of the human immune system. Mutations in RAC2 have been identified in a small number of patients giving rise to different forms of primary immunodeficiencies ranging from granulocyte defects caused by dominant negative mutations to combined immunodeficiency due to dominant activating mutations. This review will focus on the clinical and immunologic phenotype of patients with germline mutations in RAC2.
Germline Rac2 mutations result in variable clinical and immunological presentation in patients with primary immune deficiencies
The underlying genetic mechanisms have been elucidated in the last few years in less than 10% to 15% of the cases and involve mutations in CD19, MS4A1 (CD20), CR2 (CD21), ICOS, TNFRSF13C, TNFRSF13B, ...PLCG2 (phospholipase Cg2), CD81, LRBA, and PRKCD (protein kinase Cd).1-3 Recently, germline heterozygous mutations in NFKB2 were identified in 10 patients to be associated with early-onset CVID with autoimmunity in most cases,4,5 profound B-cell deficiency,6 or a CVID-like phenotype.7 All affected patients had hypogammaglobulinemia with variable association of the following clinical and immunologic features: central adrenal insufficiency (ACTH insufficiency), alopecia totalis or areata, trachyonychia, variable natural killer (NK) cell numbers, and defects in peripheral T and B cells. Appendix Lymphocyte subsets Index patient (%) Normal range for age (%) Proliferation (cpm) Index patient Healthy control T cells (CD3+) 89.8 60.5-79.8 CD3+CD4+ 63.8 30.3-48.3 Naive (CD45RA+CCR7+) 85.8 34.3-74.6 RTE (CD45RA+CCR7+CD31+) 66.5 21.1-63.5 Central memory (CD45RA-CCR7+) 8.5 13.0-43.5 Effector memory (CD45RA-CCR7-) 4.4 8.5-28.1 Terminally differentiated (CD45RA+CCR7-) 1.2 0.7-6.6 CD3+CD8+ 22.3 13.8-37.5 Naive (CD45RA+CCR7+) 92.0 26.7-72.9 Central memory (CD45RA-CCR7+) 1.8 1.2-11.6 Effector memory (CD45RA-CCR7-) 4.0 6.0-53.6 Terminally differentiated (CD45RA+CCR7-) 2.3 3.9-72.0 TCR γ/δ 1.7 0.5-21.5 B cells (CD19+) 4.8 5.7-19.7 RBE (CD38hiCD21dim/loCD27-) 31.4 15.0-35.3 Naive (CD38dim/loCD21hiCD27-) 60.5 33.8-79.6 CD19hiCD21lo 1.9 1.1-10 Switched memory (IgD-CD27+) 0.4 2.7-20.6 IgM memory (IgD+CD27+) 2.0 3.5-24.1 Terminally differentiated (CD38hiCD27hiCD21lo) 0.17 0.16-8.70 Plasma cells (CD38hiCD20-CD138+) 0.00 0.04-3.20 NK cells (CD3-CD16+CD56+) 4.5 4.6-27.8 CD3 227,000 77,000 CD3 + IL2 200,000 152,000 PHA 116,000 120,000 PMA + Ionomycin 418,000 285,000 Background 4,000 7,000 Table I Immunologic profile from the index patient mutated in NFKB2 PMA, Phorbol 12-myristate 13-acetate; RBE, recent bone marrow emigrants; RTE, recent thymic emigrants; TCR, T-cell receptor.
RAC2D57N also disrupts the polymerization of filamentous actin, thereby reducing myeloid cell and lymphocyte chemotaxis and adhesion.2,3 Although conditional deletion of either RAC1 or RAC2 in ...lymphocytes is sufficient for intact T-cell and B-cell development in mice, reduced numbers of CD4+ T cells and B cells have been found in one patient with RAC2D57N and in another kindred lacking RAC2 protein expression due to a homozygous RAC2W56X mutation.4,5 Somatic activating variants in RAC2 have been associated with melanoma,1 but there are no published reports of germline gain-of-function mutations in any RAC family members. The mutation was confirmed by Sanger sequencing (Fig 1, B) and had a deleterious Combined Annotation Dependent Depletion Phred-like score of 32.7 The mutated P34 residue resides within the highly conserved Switch 1 domain important for interactions with guanine exchange factors and downstream effectors (Fig 1, C).1 Immunoblotting revealed similar levels of RAC2 in patients and controls (Fig E1, B). Because the patients' primary cells express both wild-type (WT) and RAC2P34H, we expressed Flag-tagged WT or mutant RAC2 in HEK293T cells and confirmed that WT and mutant forms of RAC2 had comparable expression (Fig 1, D). Filamentous actin accumulation is known to reduce mitochondrial membrane potential, increase reactive oxidant species, and trigger proapoptotic pathways, all of which increased susceptibility to apoptosis.8 In addition, the conserved Switch 1 domain in the Rac guanosine triphosphatases binds to the p100β catalytic subunit and activates phosphoinositide-3-kinase (PI3K).9 PI3K overactivation, due to germline gain-of-function mutations in PIK3CD or PIK3R1, results in a combined immunodeficiency with certain features shared by our patients, including reduced percentages of naive T cells, the accumulation of senescent CD8+CD57+ lymphocytes, and increased T- and B-cell apoptosis.10 The lymphopenia in patients with PI3K overactivation has been attributed to TCR-mediated apoptotic signaling as well as metabolic shifts that favor the expansion of memory and senescent T cells over naive cells.10 However, because patients with activated phosphoinositide 3-kinase δ syndrome have additional features, including increased IgM levels, lymphoproliferative disease, and autoimmunity, additional patients and studies are needed to investigate the PI3K-AKT axis in patients with gain-of-function mutations in RAC2. Variable Patient 1 Patient 2 Patient 3 Age at the time of testing (y) 31 7 2 Hemogram (normal range) Hemoglobin (g/dL) 15.6 (10.9-15) 12.6 (10.5-13.8) 11.8 (11-12.8) WBCs (103 cells/μL) 3.670 (5.8-9.3) 1.640 (5.4-9.7) 2.960 (5.9-10.4) Neutrophils (103 cells/μL) 2.822 (3.3-6.3) 0.730 (2.5-5.9) 1.557 (2.5-6) Lymphocytes (103 cells/μL) 0.499 (1.14-2.28) 0.350 (1.28-2.76) 0.400 (1.33-4.77) Monocytes (103 cells/μL) 0.184 (0.29-0.7) 0.172 (0.19-0.81) 0.266 (0.19-0.94) Platelets (103 cells/μL) 153 (174-333) 214 (187-367) 216 (208-413) Lymphocyte subsets CD3+ (103 cells/μL) 359 (1000-2600) 248 (1400-3700) 252 (2100-6200) CD3+CD4+ (103 cells/μL) 72 (530-1500) 140 (700-2200) 98 (1300-3400) CD45RA+CD31+ recent thymic emigrants, % CD4+ 0.0 (9.8-43) 32 (45-63) 29 (57-76) CD45RA+CCR7+CD31– naive, % CD4+ 0.7 (21-61) 36.4 (57.1-84.8) 32.7 (65-84) CD45RA–CCR7– effector memory, % CD4+ 95.8 (7.6-25) 44.1 (3.3-15.2) 35.7 (2.9-9.8) CD45RA–CCR7+ central memory, % CD4+ 3.3 (26-62) 16.9 (11.2-26.7) 29.8 (10-22.3) CD3+CD8+ (103 cells/μL) 224 (330-1100) 55 (490-1300) 65 (620-2000) CD45RA+CCR7+CD31– naive, % CD8+ 0.4 (11-66) 4.6 (28-80) 14.7 (39-89) CD45RA–CCR7– effector memory, % CD8+ 60.9 (16-54) 56.2 (6.2-29.3) 36.4 (3.4-28) CD45RA–CCR7+ central memory, % CD8+ 0.8 (3.7-32) 1.2 (1.2-4.5) 6.3 (1-5.7) CD45RA+CCR7− TEMRA, % CD8+ 38.1 (5.6-43) 38 (9.1-49.1) 42.7 (4.8-30) CD57+, % CD8+∗ 67.9 (2-38) 50 (2-20) 42.8 (2-16) CD19+ (103 cells/μL) 12 (110-570) 40 (390-1400) 19 (720-2600) CD27−IgD+IgM+ naive, % CD19+ 60.8 (48-97) 30.2 (47-77) 25.0 (54-88) CD27+IgD+ unswitched memory, % CD19+ 27.4 (7-23) 8.8 (5.2-20.4) 11.9 (2.7-19) CD27+IgD– switched memory, % CD19+ 9.4 (8.8-27) 21.7 (10.9-30.4) 19.6 (3.3-7.4) CD38hiIgMhitransitional, % CD19+ 0.8 (2.2-13) 35.6 (7.2-23) 37.8 (10-30) CD3–CD56+ (103 cells/μL) 101 (70-480) 56 (130-0.720) 116 (160-920) Immunoglobulins IgG (mg/dL) 804† (639-1349) 353 (463-1236) 239 (345-1213) IgM (mg/dL) 83 (56-352) 14 (43-196) 12 (14-106) IgA (mg/dL) 226 (70-312) 17 (25-154) 13 (43-173) Tetanus vaccine titer n.d. before IVIG started UD UD Hepatitis vaccine titer n.d. before IVIG started UD UD Proliferation‡ Anti-CD3 (% CD4+ divided) 65.3 (84.2-93.8) 89.05 (84.2-93.8) 24.5 (84.2-93.8) Phytohemmaglutinin (% CD4+ divided) 6.04 (62.9-85) 60 (62.9-85) 63.85 (62.9-85) Anti-CD3 (% CD8+ divided) 54.5 (87-92.7) 64.45 (87-92.7) 34.3 (87-92.7) Phytohemmaglutinin (% CD8+ divided) 3.81 (83.1-93.5) 21.6 (83.1-93.5) 20.75 (83.1-93.5) Table I Immune phenotyping of the patients' lymphocytes Chromosome Position Reference Patient Consequence Gene name CADD score 1 3328670 A C Non-synonymous PRDM16 19.7 1 22310701 C A Non-synonymous CELA3B 19.63 1 27023871 G T Non-synonymous ARID1A 21.1 1 27679943 C T Non-synonymous SYTL1 34 1 27695833 C T Non-synonymous FCN3 23.3 1 27695833 C T Upstream MAP3K6 23.3 2 220420994 G A Non-synonymous OBSL1 32 3 100373932 G A Non-synonymous GPR128 18.75 5 40681232 G T Non-synonymous PTGER4 33 5 79354543 G A Non-synonymous THBS4 23.9 5 102887994 A G Non-synonymous NUDT12 24.2 5 112884695 G A Non-synonymous YTHDC2 22.9 5 137713429 C A Non-synonymous KDM3B 17.27 5 147796767 G A Non-synonymous FBXO38 27.8 5 150227995 T A Non-synonymous IRGM 22.7 9 1052014 G T Non-synonymous DMRT2 33 9 121976315 T A Non-synonymous BRINP1 16.83 9 130826079 C G Upstream SLC25A25 22.1 9 130826079 C G Non-synonymous NAIF1 22.1 9 140057403 G C Non-synonymous GRIN1 24 10 14870199 A G Non-synonymous CDNF 24.3 10 71966072 G A Non-synonymous PPA1 22.9 12 10962022 C T Non-synonymous TAS2R9 15.52 12 10962022 C T Upstream TAS2R8 15.52 12 29786258 T C Non-synonymous TMTC1 29.3 12 56030724 T G Non-synonymous OR10P1 22.1 12 57578143 C T Non-synonymous LRP1 22.4 14 21780617 A G Non-synonymous RPGRIP1 27.3 14 47600951 A T Non-synonymous MDGA2 22.5 14 72196852 C G Non-synonymous SIPA1L1 22.6 17 26684394 T G Upstream TMEM199 15.59 19 1592525 A C Non-synonymous MBD3 28.3 19 1592543 G C Non-synonymous MBD3 23.3 19 6452423 C T Non-synonymous SLC25A23 19.64 19 44662108 A G Non-synonymous ZNF234 25 19 50832152 T C Upstream NR1H2 16.25 19 50832152 T C Non-synonymous KCNC3 16.25 22 24953707 C T Upstream GUCD1 22.8 22 24953707 C T Non-synonymous SNRPD3 22.8 22 37637633 G T Non-synonymous RAC2 32 Table E1 Variants shared by all 3 patients, which are rare (<30 alleles in gnomAD) and deleterious (CADD PHRED-like score >15)
To the Editor: The nuclear factor kappa B (NF-kB) signaling pathway plays an important role in immune cell biology.1 Both the classical (nuclear factor kappa B, subunit 1 NF-kB1; p105/p50) and the ...alternative (nuclear factor kappa B, subunit 2 NF-kB2; p100/p52) NF-kB pathways have been largely studied mainly in animal models.1 Regarding B cells, the role of NF-kB1 was underlined in a murine nf-kb1 knockout model in which peripheral B cells showed defective maturation, defective isotype switching, and impaired humoral immune responses.1-3 A similar, although more pronounced, immunologic phenotype was observed in the nf-kb2 knockout mice, with defective secondary lymphoid organ development and impaired B-cell development both in early (bone marrow) and in late (periphery) stages with defective humoral responses both to T-dependent and to T-independent antigens.1 The role of NF-kB2 in human B-cell development was recently defined in patients carrying monoallelic mutations in NF-kB2, leading to common variable immunodeficiency (CVID)-like disease with autoimmunity and defects in late stages of peripheral B-cell maturation.4,5 Monoallelic mutations in NF-kB1 leading to p50 haploinsufficiency were recently described in a limited number of patients with CVID6; however, data regarding the effect of monoallelic NF-kB1 mutations on B-cell development are scarce. Appendix Features Patient 1 Patient 2low * Sex Male Female Age at diagnosis (y) 7 30 Current age (y) 40 55 IgG (mg/dL)dagger 160 181 IgA (mg/dL)dagger 14 6 IgM (mg/dL)dagger 14 48 Recall response to vaccinations Tetanus toxoid Absent NA Hepatitis B Absent NA Lymphocyte subsetsdagger CD3 (%) 83.984.3 Â CD4 (%) 39.758 Â CD8 (%) 53.523.2 Â CD19 (%) 3.210.5 Â CD56 (%) 10.64.7 Â Proliferative response to mitogens Anti-CD3, anti-CD3 + IL-2, PMA + I, PHA Normal NA Respiratory infections Pneumonia, sinusitis Pneumonia, recurrent sinusitis Bronchiectasis -- -- Autoimmunity Autoimmune thyroiditis, autoimmune enteropathy --
Ciliogenesis proteins orchestrate vesicular trafficking pathways that regulate immune synapse (IS) assembly in the non-ciliated T-cells. We hypothesized that ciliogenesis-related genes might be ...disease candidates for common variable immunodeficiency with impaired T-cell function (T-CVID). We identified a heterozygous, predicted pathogenic variant in the ciliogenesis protein CCDC28B present with increased frequency in a large CVID cohort. We show that CCDC28B participates in IS assembly by regulating polarized T-cell antigen receptor (TCR) recycling. This involves the CCDC28B-dependent, FAM21-mediated recruitment of the actin regulator WASH to retromer at early endosomes to promote actin polymerization. The CVID-associated CCDC28B
variant failed to interact with FAM21, leading to impaired synaptic TCR recycling. CVID T cells carrying the ccdc28b 211 C > T allele displayed IS defects mapping to this pathway that were corrected by overexpression of the wild-type allele. These results identify a new disease gene in T-CVID and pinpoint CCDC28B as a new player in IS assembly.
Background Bruton tyrosine kinase (BTK) plays an essential role in various biologic functions of different cell types. Mutations in BTK lead to X-linked agammaglobulinemia (XLA) in humans. BTK was ...recently linked to the innate immune system, in particular, the Toll-like receptor (TLR) pathway; however, the TLR9 pathway has never been studied in dendritic cells (DCs) of patients with XLA. Objective The aim of this study was to investigate the role of BTK in human DC activation upon TLR9 stimulation. Methods DCs of patients with XLA and healthy donors were stimulated via TLR4/9 and evaluated for cell activation and cytokine production. Results We showed that BTK plays an essential role in DC responses to unmethylated CpG oligodeoxynucleotide: although responses to lipopolysaccaride/TLR4 induce normal DC activation in terms of upregulation of specific markers (CD86, CD83, CD80, HLA-DR), the CpG/TLR9 pathway is completely impaired in patients with XLA. Furthermore, cytokine production upon TLR9 activation in patients with XLA is radically impaired in terms of IL-6, IL-12, TNF-α, and IL-10 production. Interestingly, BTK mediated STAT1/3 upregulation in a TLR9-dependent manner. The important role of BTK in human DC activation was confirmed after incubation of healthy DCs with ibrutinib, the specific BTK inhibitor, which resulted in impairment of TLR9 responses as seen in patients with XLA. Conclusion Analysis of these data suggests that BTK regulates human DC responses upon TLR9 engagement in terms of activation, cytokine production, and STAT1/3 upregulation. These findings may be of important significance for better understanding and managing different clinical conditions, such as agammaglobulinemia and lymphoid malignancies.
Mutations affecting the non-canonical pathway of NF-κB were recently identified to underlie a form of common variable immunodeficiency strongly associated with autoimmunity. Although intrinsic B-cell ...abnormalities explain most of the humoral defects of this disease, detailed data on the impact of NFKB2 on follicular helper (Tfh) and regulatory (Tregs) T cells are scarce. Here, we show that Tfh, CXCR5+, and CXCR5− Treg cell subsets were significantly reduced in patients heterozygous for a truncating mutation of NFKB2. Plasma CXCL13 levels were reduced, underlining an important role for NFKB2 in regulating the germinal center (GC) response. Proinflammatory IFNγ, IL-17 and IL-10 cytokine production by CD4 T cells was lower in the mutated patients, but the production of IL-4 and IL-21 was not altered. Taken together, our findings show that NFKB2 influences the quality and efficiency of human GC reaction, by affecting not only the B cells but also GC-relevant T cell subsets.
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•Circulating Tfh, CXCR5+ and CXCR5− Treg cells are significantly reduced in patients with a dominant negative mutation in NFKB2.•Plasma CXCL13 levels are much lower in mutated patients as compared to healthy controls.•CD4 IFNγ and IL-17 production is reduced, but IL-21 and IL-4 expression is unaltered in patients.
Rate of survival without any neurological consequence after cardiac arrest is driven not only by early recognition but also by high-quality cardiopulmonary resuscitation. Because the effectiveness of ...the manual cardiopulmonary resuscitation is usually impaired by rescuers’ fatigue, devices have been devised to improve it by appliances or ergonomic solutions. However, some devices are thought to replace the manual resuscitation altogether, either mimicking its action or generating hemodynamic effects with working principles which are entirely different. This article reviews such devices, both manual and automatic. They are mainly classified by actuation method, applied force, working space, and positioning time. Most of the trials and meta-analyses have not demonstrated that chest compressions given with automatic devices are more effective than those given manually. However, advances in clinical research and technology, with an improved understanding of the organizational implications of their use, are constantly improving the effectiveness of such devices.
Venous leg ulcers are one of the most common nonhealing conditions and represent an important clinical problem. The application of pulsed radiofrequency electromagnetic fields (PRF-EMFs), already ...applied for pain, inflammation, and new tissue formation, can represent a promising approach for venous leg ulcer amelioration. This study aims to evaluate the effect of PRF-EMF exposure on the inflammatory, antioxidant, cell proliferation, and wound healing characteristics of human primary dermal fibroblasts collected from venous leg ulcer patients. The cells' proliferative and migratory abilities were evaluated by means of a BrdU assay and scratch assay, respectively. The inflammatory response was investigated through TNFα, TGFβ, COX2, IL6, and IL1β gene expression analysis and PGE2 and IL1β production, while the antioxidant activity was tested by measuring GSH, GSSG, tGSH, and GR levels. This study emphasizes the ability of PRF-EMFs to modulate the TGFβ, COX2, IL6, IL1β, and TNFα gene expression in exposed ulcers. Moreover, it confirms the improvement of the proliferative index and wound healing ability presented by PRF-EMFs. In conclusion, exposure to PRF-EMFs can represent a strategy to help tissue repair, regulating mediators involved in the wound healing process.
Purpose
Jacobsen syndrome (JS) is a rare form of genetic disorder that was recently classified as a syndromic immunodeficiency. Available detailed immunological data from JS patients are limited.
...Methods
Clinical and immunological presentation of twelve pediatric patients with JS by means of revision of clinical records, flow cytometry, real-time PCR, and lymphocyte functional testing were collected.
Results
Recurrent infections were registered in 6/12 patients (50%), while bleeding episodes in 2/12 (16.7%). White blood cell and absolute lymphocyte counts were reduced in 8/12 (66.7%) and 7/12 (58.3%) patients, respectively. Absolute numbers of CD3
+
and CD4
+
T cells were reduced in 8/12 (66.7%) and 7/12 (58.3%), respectively. Of note, recent thymic emigrants (RTE) were reduced in all tested patients (9/9), with T-cell receptor excision circle analysis (TRECs) showing a similar trend in 8/9 patients; naïve CD4
+
T cells were low only in 5/11 patients (45.4%). Interestingly, B-cell counts, IgM memory B cells, and IgM serum levels were reduced in 10/12 (83.3%) patients. Natural killer (NK) cell counts were mostly normal but the percentages of CD16
+
CD56
low/−
cells were expanded in 7/7 patients tested. The observed immunological alterations did not correlate with patients’ age. Finally, responses to proliferative stimuli were normal at presentation for all patients, although they may deteriorate over time.
Conclusions
Our data suggest that patients affected with JS may display important numeric and maturational alterations in the T-, B-, and NK-cell compartments. These findings suggest that JS patients should be regularly monitored from an immunological point of view.
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