Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production

Self Cite

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256

The best-understood mechanisms for achieving antibody self/nonself discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgM low IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgM low IgD+ B cells form twice as many GC progeny as naïve IgM hi IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.self-tolerance | affinity maturation | clonal selection | autoimmunity

Section: Methodsmentioning

confidence: 99%

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity

Sabouri

Schofield

Horikawa

et al. 2014

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214

256

“…In murine DCs, the DHR2 domain has been implicated in regulating the Rho GTPase CDC42 (cell division control protein 42 homolog), which in turn maintains cell polarity of mature DCs during migration (9,10). Furthermore, mice harboring inactivating mutations in Dock8 lack marginal zone B-cell development, long-term antibody production following immunization, and memory CD8 + T-cell responses to viral infections (11,12). In humans, inactivating mutations in Dock8 were recently identified as the primary genetic cause underlying autosomal recessive hyper-IgE syndrome (13).…”

mentioning

confidence: 99%

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration

Krishnaswamy¹,

Singh²,

Gowthaman³

et al. 2015

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.D endritic cells (DCs) are crucial for the initiation of an adaptive immune response. Upon acquiring antigens in the periphery, DCs undergo a maturation process that includes antigen processing, cytokine production, and up-regulation of costimulatory molecules. A mature DC must then migrate from peripheral tissues to draining lymph nodes (LNs) to fulfill its role as an antigen-presenting cell that primes naïve T cells (1). Although the signals that induce this maturation process are now well-established (1), relatively little is understood about DC migration aside from the primary chemotactic cue provided by CCR7 that guides DCs to the LN (2, 3).We recently described a genetically modified NLRP10 (NLR family, pyrin domain containing 10) knockout strain in which this migration step was disrupted while leaving the remainder of the DC maturation program, including CCR7 expression, intact (4).NLRP10 is the only NOD-like receptor (NLR) without a leucine-rich repeat domain, the putative pathogen-associated molecular pattern (PAMP)-binding domain. It has been proposed to both positively and negatively regulate other NLRs, such as NOD1 and NLRP3, respectively (5, 6). Although we found that NLRP3 inflammasome activation was unaltered in the absence of NLRP10, we discovered that Nlrp10 −/− mice could ...

“…Flow cytometry staining with tetramers enabled enumeration of CD8 + T cells specific for two immunodominant epitopes of the virus, GP [33][34][35][36][37][38][39][40][41] and NP [396][397][398][399][400][401][402][403][404] , and CD4 + T cells specific for the immunodominant GP 66-77 virus epitope. Compared with wild-type controls, there were 10-fold fewer antigen-specific CD8 + and CD4 + T cells in LCMV-infected homozygotes for either the exon 2 deleted or the C166X null allele, although there was still a detectable effector T-cell response in both strains (Fig.…”

Section: Etaa1 Mutant Mice Have Defective Effector T-cell Responses Amentioning

confidence: 99%

“…To track CD8 + T cells bearing a defined T-cell receptor (TCR) for virus antigen, we crossed the Etaa1 ΔEx2 allele to congenically marked (CD45.1 + ) P14 TCR transgenic C57BL/6 mice, which express a TCR specific for LCMV GP [33][34][35][36][37][38][39][40][41] antigen. Naïve CD8 + T cells expressing the P14 TCR developed and accumulated normally despite ETAA1 deficiency.…”

Section: Etaa1-deficient T Cells Proliferate Normally In Vitro and Inmentioning

confidence: 99%

“…6C). To test in vitro proliferative capacity following antigen-specific stimulation, we stimulated Etaa1 +/+ or Etaa1-ΔEx2/ΔEx2 P14 CD8 + T cells with GP [33][34][35][36][37][38][39][40][41] peptide and IL-2 in vitro and analyzed them daily for 5 d. ETAA1-deficient P14 cells acquired the Ki67 cell cycle marker and increased in number comparably to their wild-type counterparts, and also exhibited a comparable percentage of viable cells at each time point (Fig. 6D), demonstrating that ETAA1 is dispensable for clonal expansion in vitro.…”

Section: Etaa1-deficient T Cells Proliferate Normally In Vitro and Inmentioning

confidence: 99%

See 1 more Smart Citation

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

Miosge

Sontani

Chuah

et al. 2017

Self Cite

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8 + and CD4 + T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.T cell | DNA damage | replication stress | Etaa1 | immunity