T follicular helper (Tfh) cells are critical for germinal centers (GC), the specialized microenvironment where long-lived humoral immunity is generated in response to vaccination or infection. Within the GC, B cells engage with Tfh cells and elicit their help in the form of cytokines and cell-surface co-stimulator molecules. Tfh helper activity must be rapidly available in response to B cell engagement, yet the mechanisms controlling this helper activity remain poorly characterized. Post-transcriptional regulation of mRNA decay and translation offers one way to rapidly and temporally tune Tfh cell activity. ZFP36L1, a member of the ZFP family of RNA-binding proteins, is a candidate modulator of Tfh cell helper activity, as it controls cytokine production and responses in other T cell lineages, modulating their differentiation and function. We sought to determine if ZFP36L1 is also important for Tfh cell biology. In this study, we show expression of ZFP36L1 by Tfh cells. We selectively delete ZFP36L1 from Tfh cells and analyze the effect this has on the GCs. Surprisingly, we find that the GC response and affinity maturation are resilient to deletion of ZFP36L1 from Tfh cells.

The germinal center (GC) reaction drives the production of high-affinity antibodies by iterative cycles of B cell somatic hypermutation, selection, and proliferation. How GC B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen sensing and protect GC B cells from replication stress by controlling a cell cycle-related posttranscriptional regulon. They safeguard the successful completion of mitosis by balancing CDK1 and p21-mediated regulation of cell-cycle progression. In their absence, GC B cells are prone to arrest in the G2-M phase and die by apoptosis, resulting in curtailed GC responses. DNA replication forks stalled at active replication initiation zones, causing replication stress and increased activity of the ATR-CHK1 DNA damage response. Thus, RNA binding proteins guide posttranscriptional gene regulation and maintain a functional G2-M checkpoint in GC B cells.

Acquisition of cytotoxic activity in CD4+ T cells (TCTX) can promote potent anti-tumor activity thus holding promise as a therapeutic approach. However, how this activity is regulated remains poorly understood. Here, we demonstrate that tumor-infiltrating CD4+ TCTX activity is restrained by a post-transcriptional regulatory checkpoint. In untreated tumors, CD4+ TCTX exist in a poised state, characterized by abundant Gzmb mRNA but limited Granzyme B (GzmB) protein. Differentiation into poised TCTX is regulated by the Blimp-1-Bcl6 axis and requires type-I interferon signaling. Treatment with anti-CTLA-4 or anti-LAG-3 plus anti-PD-1 removed the block to GzmB protein production by repressing expression of the post-transcriptional regulator Zfp36l1 . Constitutive Zfp36l1 expression abrogated the effects of anti-CTLA-4 while deletion of Zfp36l1 and its paralog Zfp36 triggered GzmB protein production and promoted tumor control. These data identify ZFP36/ZFP36L1 as a key post-transcriptional regulatory checkpoint of CD4+ TCTX activity and a potential immunotherapy target in cancer. ### Competing Interest Statement M.T. has a funded collaboration with AZ on a topic unrelated to this study. The remaining authors declare no competing interests Cancer Research UK, C416/A18088, CANCTA-2022/100001, C36463/A22246, C36463/A20764, C33499/A20265 Medical Research Council, MR/W002337/1, MR/T028270/1 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BBS/E/B/000C0407

RNA binding proteins (RBP) of the ZFP36 family limit the differentiation and effector functions of CD4 and CD8 T cells, but little is known of their expression or function in regulatory T (Treg) cells. By using Treg cell-restricted deletion of Zfp36 family members we identify the role of Zfp36l1 and Zfp36l2 in Treg cells to maintain immune homeostasis. Mice with Treg cells deficient in these RBP display an inflammatory phenotype with an expansion in the numbers of type-2 conventional dendritic cells, T effector cells, T follicular helper and germinal center B cells and elevated serum cytokines and immunoglobulins. In the absence of Zfp36l1 and Zfp36l2, the pool of cycling CTLA-4 in naïve Treg cells is reduced, Treg cells are less sensitive to IL-2 and IL-7 but are more sensitive to IFNγ. In mice lacking both RBP in Treg cells, the deletion of a single allele of Ifng is sufficient to ameliorate the pathology. Our results indicate that ZFP36L1 and ZFP36L2 regulate the availability of IFNγ and are required for the maintenance of Treg cell stability. Thus, ZFP36L1 and ZFP36L2 regulate multiple pathways that enable Treg cells to enforce immune homeostasis.

How germinal centre (GC) B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA-binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen-sensing and protect GC B cells from replication stress by controlling a cell cycle-related RNA post-transcriptional regulon. ZFP36L1 and ZFP36L2 safeguard faithful completion of mitosis by restraining the expression of CDK1 and cyclin B1, whilst controlling their activity through regulation of a p21-mediated negative feedback loop. In the absence of ZFP36L1 and ZFP36L2, GC B cells arrest in G2-M and die by apoptosis, resulting in curtailed GC responses. This is associated with stalling of the DNA replication fork at active replication initiation zones, which causes replication stress and increased activity of the ATR/CHK1 DNA damage response. Our findings reveal that gene regulation by RNA-binding proteins is essential for a functional G2-M checkpoint to operate in GC B cells. ### Competing Interest Statement The authors have declared no competing interest.

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal disorder of dogs wherein the esophagus is dilated and swallowing activity is reduced, causing regurgitation of ingesta. Affected individuals experience weight loss and malnourishment and are at risk for aspiration pneumonia, intussusception, and euthanasia. Great Danes have among the highest incidences of CIM across dog breeds, suggesting a genetic predisposition. We generated low-pass sequencing data for 83 Great Danes and used variant calls to impute missing whole genome single-nucleotide variants (SNVs) for each individual based on haplotypes phased from 624 high-coverage dog genomes, including 21 Great Danes. We validated the utility of our imputed data set for genome-wide association studies (GWASs) by mapping loci known to underlie coat phenotypes with simple and complex inheritance patterns. We conducted a GWAS for CIM with 2,010,300 SNVs, identifying a novel locus on canine chromosome 1 ( P -val = 2.76 × 10 −10 ). Associated SNVs are intergenic or intronic and are found in two clusters across a 1.7-Mb region. Inspection of coding regions in high-coverage genomes from affected Great Danes did not reveal candidate causal variants, suggesting that regulatory variants underlie CIM. Further studies are necessary to assess the role of these non-coding variants.

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

CD8 + T cell differentiation into effector cells is initiated early after antigen encounter by signals from the T cell antigen receptor and costimulatory molecules. The molecular mechanisms that establish the timing and rate of differentiation however are not defined. Here we show that the RNA binding proteins (RBP) ZFP36 and ZFP36L1 limit the rate of differentiation of activated naïve CD8 + T cells and the potency of the resulting cytotoxic lymphocytes. The RBP function in an early and short temporal window to enforce dependency on costimulation via CD28 for full T cell activation and effector differentiation by directly binding mRNA of NF-κB , Irf8 and Notch1 transcription factors and cytokines, including Il2 . Their absence in T cells, or the adoptive transfer of small numbers of CD8 + T cells lacking the RBP, promotes resilience to influenza A virus infection without immunopathology. These findings highlight ZFP36 and ZFP36L1 as nodes for the integration of the early T cell activation signals controlling the speed and quality of the CD8 + T cell response.

Introduction: Postpartum hemorrhage (PPH) is the leading cause of maternal morbidity in the UK. Visual estimation of blood loss is unreliable yet remains common practice. As part of a national quality improvement project to improve care during PPH, standardized, quantitative measurement of blood loss (QBL) for all deliveries was introduced into a tertiary obstetric unit in Cardiff, Wales. Methods: Retrospective analysis of 875 consecutive maternities between December 2017 and February 2018 was undertaken. Of these, 372 mothers had both pre- and post-partum hemoglobin (Hb) were recorded. Regression analyses were performed to investigate the relationship between change in Hb adjusted for red cell transfusion and QBL. Results: The correlation coefficient between QBL and adjusted change in Hb for all deliveries (n = 372) was 0.57. This corresponded to an estimated fall of adjusted change in Hb of 15.3 g/L (95% CI: 13.1, 17.6) per 1000 mL blood loss. Discussion: QBL has been shown to be reliable across all maternity settings, with reproducible results in theater and delivery rooms (on the obstetric unit and alongside midwifery-led unit). QBL is moderately correlated with adjusted change in Hb for all volumes of bleeding and gives clinicians more accurate knowledge of blood loss than visual estimation. This low-cost, low-fidelity intervention can influence the timely escalation of clinical care and therefore patient outcome.

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal (GI) motility disorder of dogs in which reduced peristaltic activity and dilation of the esophagus prevent the normal transport of food into the stomach. Affected puppies regurgitate meals and water, fail to thrive, and experience complications such as aspiration pneumonia that may necessitate euthanasia. The German shepherd dog (GSD) has the highest disease incidence, indicative of a genetic predisposition. Here, we discover that male GSDs are twice as likely to be affected as females and show that the sex bias is independent of body size. We propose that female endogenous factors ( e . g ., estrogen) are protective via their role in promoting relaxation of the sphincter between the esophagus and stomach, facilitating food passage. A genome-wide association study for CIM revealed an association on canine chromosome 12 ( P -val = 3.12x10 -13 ), with the lead SNPs located upstream or within Melanin-Concentrating Hormone Receptor 2 ( MCHR2 ), a compelling positional candidate gene having a role in appetite, weight, and GI motility. Within the first intron of MCHR2 , we identified a 33 bp variable number tandem repeat (VNTR) containing a consensus binding sequence for the T-box family of transcription factors. Across dogs and wolves, the major allele includes two copies of the repeat, whereas the predominant alleles in GSDs have one or three copies. The single-copy allele is strongly associated with CIM ( P -val = 1.32x10 -17 ), with homozygosity for this allele posing the most significant risk. Our findings suggest that the number of T-box protein binding motifs may correlate with MCHR2 expression and that an imbalance of melanin-concentrating hormone plays a role in CIM. We describe herein the first genetic factors identified in CIM: sex and a major locus on chromosome 12, which together predict disease state in the GSD with greater than 75% accuracy.

Introduction: Antenatal anaemia contributes to significant perinatal morbidity. [1] Allen LH. Anaemia and iron deficiency: effects on pregnancy outcome. Am J Clinical Nutrition. 2000; Vol 71; 1280s-4s. Google Scholar In March 2020 a revised antenatal anaemia pathway was implemented in anticipation of reduced blood stocks due to COVID-19. Changes included alternate day oral iron for all patients from booking, measurement of ferritin at booking and 28 weeks and a revised intravenous (IV) iron pathway. Baseline data were compared with re-audit data following pathway adoption to assess the impact on anaemia, ferritin levels and blood transfusion.

The germinal centre (GC) is required for the generation of high affinity antibodies and immunological memory. Here we show that the RNA binding protein HuR has an essential function in GC B cells to sustain the GC response. In its absence, the GC reaction and production of high-affinity antibody is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively. The expression and splicing patterns of hundreds of genes are altered in the absence of HuR. Among these genes, HuR is required for the expression of Myc and a Myc-dependent transcriptional program that controls GC B cell proliferation and Ig somatic hypermutation. Additionally, HuR regulates the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle, and it modulates a gene signature associated with DNA deamination protecting GC B cells from DNA damage and cell death.

Cell migration relies on coordinated activity of chemotactic and guidance receptors. Here, we report a specific role for the RNA-binding protein ZFP36L1 in limiting the abundance of molecules involved in the homing of antibody-secreting cells (ASCs) to the bone marrow (BM). In the absence of ZFP36L1, ASCs build up in the spleen and the liver and show diminished accumulation in the BM. ZFP36L1 facilitates migration by directly regulating G protein–coupled receptor kinase 2 (GRK2) and the integrin chains α4 and β1 in splenic ASCs. Expression of CXCR4 and of the integrins α4 and β1 is differentially regulated on ASCs produced at the early and late stages of the immune response. Consequently, deletion of the Zfp36l1 gene has a stronger effect on BM accumulation of high-affinity ASCs formed late in the response. Thus, ZFP36L1 is an integral part of the regulatory network controlling gene expression during ASC homing.