Secondary T cell-T cell synaptic interactions drive the differentiation of protective CD8+ T cells.

Immunization results in the differentiation of CD8+ T cells, such that they acquire effector abilities and convert into a memory pool. Priming of T cells takes place via an immunological synapse formed with an antigen-presenting cell (APC). By disrupting synaptic stability at different times, we found that the differentiation of CD8+ T cells required cell interactions beyond those made with APCs. We identified a critical differentiation period that required interactions between primed T cells. We found that T cell-T cell synapses had a major role in the generation of protective CD8+ T cell memory. T cell-T cell synapses allowed T cells to polarize critical secretion of interferon-γ (IFN-γ) toward each other. Collective activation and homotypic clustering drove cytokine sharing and acted as regulatory stimuli for T cell differentiation.

The microRNA cluster miR-17∼92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.

Follicular helper T cells (TFH cells) are the prototypic helper T cell subset specialized to enable B cells to form germinal centers (GCs) and produce high-affinity antibodies. We found that expression of microRNAs (miRNAs) by T cells was essential for TFH cell differentiation. More specifically, we show that after immunization of mice with protein, the miRNA cluster miR-17∼92 was critical for robust differentiation and function of TFH cells in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17∼92 restrained the expression of genes 'inappropriate' to the TFH cell subset, including the direct miR-17∼92 target Rora. Removal of one Rora allele partially 'rescued' the inappropriate gene signature in miR-17∼92-deficient TFH cells. Our results identify the miR-17∼92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene-expression program.

ATF7ip Targets Transposable Elements for H3K9me3 Deposition to Modify CD8+ T Cell Effector and Memory Responses.

CD8+ T cells are critical for the immune response to pathogens and tumors, and CD8+ T cell memory protects against repeat infections. In this study, we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of CD8+ T cell immune responses. Mice with a T cell-specific deletion of ATF7ip have a CD8+ T cell-intrinsic enhancement of Il7r expression and Il2 expression leading to enhanced effector and memory responses. Chromatin immunoprecipitation sequencing studies identified ATF7ip as a repressor of Il7r and Il2 gene expression through the deposition of the repressive histone mark H3K9me3 at the Il7r gene and Il2-Il21 intergenic region. Interestingly, ATF7ip targeted transposable elements for H3K9me3 deposition at both the IL7r locus and the Il2-Il21 intergenic region, indicating that ATF7ip silencing of transposable elements is important for regulating CD8+ T cell function. These results demonstrate a new epigenetic pathway by which IL-7R and IL-2 production are constrained in CD8+ T cells, and this may open up new avenues for modulating their production.

Using the technology acceptance model to assess clinician perceptions and experiences with a rheumatoid arthritis outcomes dashboard: qualitative study.

BACKGROUND: Improving shared decision-making using a treat-to-target approach, including the use of clinical outcome measures, is important to providing high quality care for rheumatoid arthritis (RA). We developed an Electronic Health Record (EHR) integrated, patient-facing sidecar dashboard application that displays RA outcomes, medications, and lab results for use during clinical visits ("RA PRO dashboard"). The purpose of this study was to assess clinician perceptions and experiences using the dashboard in a university rheumatology clinic. METHODS: We conducted focus group (FG) discussions with clinicians who had access to the dashboard as part of a randomized, stepped-wedge pragmatic trial. FGs explored clinician perceptions towards the usability, acceptability, and usefulness of the dashboard. FG data were analyzed thematically using deductive and inductive techniques; generated themes were categorized into the domains of the Technology Acceptance Model (TAM). RESULTS: 3 FG discussions were conducted with a total of 13 clinicians. Overall, clinicians were enthusiastic about the dashboard and expressed the usefulness of visualizing RA outcome trajectories in a graphical format for motivating patients, enhancing patient understanding of their RA outcomes, and improving communication about medications. Major themes that emerged from the FG analysis as barriers to using the dashboard included inconsistent collection of RA outcomes leading to sparse data in the dashboard and concerns about explaining RA outcomes, especially to patients with fibromyalgia. Other challenges included time constraints and technical difficulties refreshing the dashboard to display real-time data. Methods for integrating the dashboard into the visit varied: some clinicians used the dashboard at the beginning of the visit as they documented RA outcomes; others used it at the end to justify changes to therapy; and a few shared it only with stable patients. CONCLUSIONS: The study provides valuable insights into clinicians' perceptions and experiences with the RA PRO dashboard. The dashboard showed promise in enhancing patient-clinician communication, shared decision-making, and overall acceptance among clinicians. Addressing challenges related to data collection, education, and tailoring dashboard use to specific patient populations will be crucial for maximizing its potential impact on RA care. Further research and ongoing improvements in dashboard design and implementation are warranted to ensure its successful integration into routine clinical practice.

Effect of Immunosuppression on the Immunogenicity of mRNA Vaccines to SARS-CoV-2 : A Prospective Cohort Study.

BACKGROUND: Patients with chronic inflammatory disease (CID) treated with immunosuppressive medications have increased risk for severe COVID-19. Although mRNA-based SARS-CoV-2 vaccination provides protection in immunocompetent persons, immunogenicity in immunosuppressed patients with CID is unclear. OBJECTIVE: To determine the immunogenicity of mRNA-based SARS-CoV-2 vaccines in patients with CID. DESIGN: Prospective observational cohort study. SETTING: Two U.S. CID referral centers. PARTICIPANTS: Volunteer sample of adults with confirmed CID eligible for early COVID-19 vaccination, including hospital employees of any age and patients older than 65 years. Immunocompetent participants were recruited separately from hospital employees. All participants received 2 doses of mRNA vaccine against SARS-CoV-2 between 10 December 2020 and 20 March 2021. Participants were assessed within 2 weeks before vaccination and 20 days after final vaccination. MEASUREMENTS: Anti-SARS-CoV-2 spike (S) IgG+ binding in all participants, and neutralizing antibody titers and circulating S-specific plasmablasts in a subset to assess humoral response after vaccination. RESULTS: Most of the 133 participants with CID (88.7%) and all 53 immunocompetent participants developed antibodies in response to mRNA-based SARS-CoV-2 vaccination, although some with CID developed numerically lower titers of anti-S IgG. Anti-S IgG antibody titers after vaccination were lower in participants with CID receiving glucocorticoids (n = 17) than in those not receiving them; the geometric mean of anti-S IgG antibodies was 357 (95% CI, 96 to 1324) for participants receiving prednisone versus 2190 (CI, 1598 to 3002) for those not receiving it. Anti-S IgG antibody titers were also lower in those receiving B-cell depletion therapy (BCDT) (n = 10). Measures of immunogenicity differed numerically between those who were and those who were not receiving antimetabolites (n = 48), tumor necrosis factor inhibitors (n = 39), and Janus kinase inhibitors (n = 11); however, 95% CIs were wide and overlapped. Neutralization titers seemed generally consistent with anti-S IgG results. Results were not adjusted for differences in baseline clinical factors, including other immunosuppressant therapies. LIMITATIONS: Small sample that lacked demographic diversity, and residual confounding. CONCLUSION: Compared with nonusers, patients with CID treated with glucocorticoids and BCDT seem to have lower SARS-CoV-2 vaccine-induced antibody responses. These preliminary findings require confirmation in a larger study. PRIMARY FUNDING SOURCE: The Leona M. and Harry B. Helmsley Charitable Trust, Marcus Program in Precision Medicine Innovation, National Center for Advancing Translational Sciences, and National Institute of Arthritis and Musculoskeletal and Skin Diseases.

Th1 cell induction in lymph nodes according to a red-blue chemokine map.

Lymph nodes are compartmentalized organs but whether this feature has a role in T cell differentiation has been unclear. In this issue, Groom et al. (2012) reveal that spatially separated expression of two CXCR3 ligands guides Th1 cell development.

MicroRNA-29 regulates T-box transcription factors and interferon-γ production in helper T cells.

MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.

Cutting edge: An in vivo reporter reveals active B cell receptor signaling in the germinal center.

Long-lasting Ab responses rely on the germinal center (GC), where B cells bearing high-affinity Ag receptors are selected from a randomly mutated pool to populate the memory and plasma cell compartments. Signaling downstream of the BCR is dampened in GC B cells, raising the possibility that Ag presentation and competition for T cell help, rather than Ag-dependent signaling per se, drive these critical selection events. In this study we use an in vivo reporter of BCR signaling, Nur77-eGFP, to demonstrate that although BCR signaling is reduced among GC B cells, a small population of cells exhibiting GC light zone phenotype (site of Ag and follicular helper T cell encounter) express much higher levels of GFP. We show that these cells exhibit somatic hypermutation, gene expression characteristic of signaling and selection, and undergo BCR signaling in vivo.

A sharp T-cell antigen receptor signaling threshold for T-cell proliferation.

T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77-eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery.

MicroRNA function in NK-cell biology.

The important role of microRNAs in directing immune responses has become increasingly clear. Here, we highlight discoveries uncovering the role of specific microRNAs in regulating the development and function of natural killer (NK) cells. Furthermore, we discuss the impact of NK cells on the entire immune system during global and specific microRNA ablation in the settings of inflammation, infection, and immune dysregulation.

Sex- and corticotropin-releasing factor receptor 2- dependent actions of urocortin 1 during inflammation.

We investigated whether corticotropin-releasing factor receptor 2 (CRF2) and its high-affinity agonist urocortin 1 (Ucn1) mediate sex-specific signaling and immune responses. Intrarectal trinitrobenzene sulfonic acid was used to induce experimental colitis in wild-type, CRF2 knockout (CRF2KO), and heterozygous (CRF2Ht) mice of both sexes. Changes in plasma extravasation, organ weight, survival, immune cell numbers, inflammatory cytokines, and the MAPK signaling pathway were assessed. Stored intestinal biopsies from patients with Crohn's disease (CD) and age- and sex-matched individuals without inflammatory bowel disease (IBD) were examined by immunofluorescence and confocal microscopy to characterize Ucn1 and CRF receptor expression. CRF2Ht mice of both sexes showed decreased survival during colitis compared with other genotypes. Ucn1 improved survival in male mice alone. Ucn1 restored colon length and spleen and adrenal weight and decreased colonic TNF-α, IL-6, and IL-1ß levels in male CRF2Ht mice alone. CRF2Ht mice of both sexes showed decreased phosphorylation of MAPK p38 and heat shock protein 27 (Hsp27) levels. Ucn1 restored p-Hsp27 levels in male CRF2Ht mice alone. Expression of the chaperone protein Hsp90 decreased during colitis, except in male CRF2Ht mice. Taken together, our data indicate that sex shows significant interaction with genotype and Ucn1 during colitis. Human duodenal and colonic biopsies revealed that sex-specific differences exist in levels of CRF receptors and Ucn1 expression in patients with CD compared with the matched non-IBD subjects. To conclude, Ucn1 mediates sex-specific immune and cellular signaling responses via CRF2, emphasizing the need for inclusion of females in preclinical studies.

B Cell-intrinsic TLR7 signaling is required for optimal B cell responses during chronic viral infection.

The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. TLRs were demonstrated to be critical for Ab responses to a variety of immunizations. In particular, recent evidence suggests that B cell-intrinsic TLR signaling is required for optimal responses to virus-like Ags, but the mechanisms by which TLR signaling impacts Ab responses during infection in vivo is unclear. In the current study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact Ab responses in mice during chronic viral infection. This effect was independent of T follicular helper cells and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone, GC B cells. These results suggest that B cell-intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of Ag-specific B cells in the GC.

IL-7 production in murine lymphatic endothelial cells and induction in the setting of peripheral lymphopenia.

IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.

Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection.

The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. In this study, we demonstrate a nonredundant functional requirement for both nucleic acid-sensing TLRs and RIG-I-like receptors in the control of a mouse model of chronic viral infection. Whereas the RIG-I-like receptor pathway was important for production of type I IFNs and optimal CD8(+) T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral Ab responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific Ab and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on Ab production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal Ab responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.

Expression of chemokine receptor CXCR3 on T cells affects the balance between effector and memory CD8 T-cell generation.

Generation of a robust immunological memory response is essential for protection on subsequent encounters with the same pathogen. The magnitude and quality of the memory CD8 T-cell population are shaped and influenced by the strength and duration of the initial antigenic stimulus as well as by inflammatory cytokines. Although chemokine receptors have been established to play a role in recruitment of effector CD8 T cells to sites of inflammation, their contribution to determination of T-cell fate and shaping of the long-lived memory T-cell population is not fully understood. Here, we investigated whether reduced access to antigen and inflammation through alterations in expression of inflammatory and homeostatic chemokine receptors has an impact on generation of effector and memory CD8 T cells. We found that in mice infected with lymphocytic choriomeningitis virus, colocalization of virus-specific CD8 T cells with antigen in spleen is dependent on expression of the inflammatory chemokine receptor, CXCR3. In addition, absence of CXCR3 expression on CD8 T cells leads to formation of fewer short-lived effector cells and more memory precursor cells. Furthermore, the memory CD8 T-cell population derived from CXCR3-deficient cells has fewer cells of the effector memory phenotype and exhibits a recall response of greater magnitude than that of WT cells. These data demonstrate that CD8 T-cell positioning relative to antigen and inflammatory cytokines in secondary lymphoid organs affects the balance of effector and memory T-cell formation and has both a quantitative and qualitative impact on the long-lived memory CD8 T-cell population.

Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate.

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.

Complement Factor I Gene Variant as a Treatable Cause of Recurrent Aseptic Neutrophilic Meningitis: A Case Report.

Mutations in the complement factor I (CFI) gene have previously been identified as causes of recurrent CNS inflammation. We present a case of a 26-year-old man with 18 episodes of recurrent meningitis, who had a variant in CFI(c.859G>A,p.Gly287Arg) not previously associated with neurologic manifestations. He achieved remission with canakinumab, a human monoclonal antibody targeted at interleukin-1 beta.

A simple point-of-care assay accurately detects anti-spike antibodies after SARS-CoV-2 vaccination.

Objective: Lateral flow assays (LFA) are sensitive for detecting antibodies to SARS-CoV-2 proteins within weeks after infection. This study tested samples from immunocompetent adults, and those receiving treatments for chronic inflammatory diseases (CID), before and after mRNA SARS-CoV-2 vaccination. Methods: We compared results obtained with the COVIBLOCK Covid-19 LFA to those obtained by anti-spike (S) ELISA. Results: The LFA detected anti-S antibodies in 29 of 29 (100%) of the immunocompetent and 110 of 126 (87.3%) of the CID participants after vaccination. Semiquantitative LFA scores were statistically significantly lower in samples from immunosuppressed participants, and were significantly correlated with anti-S antibody levels measured by ELISA. Conclusions: This simple LFA test is a practical alternative to laboratory-based assays for detecting anti-S antibodies after infection or vaccination. This type of test may be most useful for testing people in outpatient or resource-limited settings.

Sleep Disturbance and SARS-CoV-2 Vaccinations in Patients With Chronic Inflammatory Disease.

OBJECTIVE: Immunocompromised patients with chronic inflammatory disease (CID) may have experienced additional psychosocial burden during the COVID-19 pandemic due to their immunocompromised status. This study was undertaken to determine if vaccination would result in improved patient-reported outcomes longitudinally among individuals with CID undergoing SARS-CoV-2 vaccination regardless of baseline anxiety. METHODS: Data are from a cohort of individuals with CID from 2 sites who underwent SARS-CoV-2 vaccination. Participants completed 3 study visits before and after 2 messenger RNA vaccine doses in the initial vaccination series when clinical data were collected. Patient-reported outcomes were measured using the Patient-Reported Outcomes Measurement Information System 29-item Health Profile and expressed as T scores, with 2 groups stratified by high and low baseline anxiety. Mixed-effects models were used to examine longitudinal changes, adjusting for age, sex, and study site. RESULTS: A total of 72% of the cohort was female with a mean ± SD age of 48.1 ± 15.5 years. Overall, sleep disturbance improved following both doses of SARS-CoV-2 vaccinations, and anxiety decreased after the second dose. Physical function scores worsened but did not meet the minimally important difference threshold. When stratifying by baseline anxiety, improvement in anxiety, fatigue, and social participation were greater in the high anxiety group. Physical function worsened slightly in both groups, and sleep disturbance improved significantly in the high anxiety group. CONCLUSION: Sleep disturbance decreased in a significant and meaningful way in patients with CID upon vaccination. In patients with higher baseline anxiety, social participation increased, and anxiety, fatigue, and sleep disturbance decreased. Overall, results suggest that SARS-CoV-2 vaccination may improve mental health and well-being, particularly among those with greater anxiety.

CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs.

Naive lymphocytes continually enter and exit lymphoid organs in a recirculation process that is essential for immune surveillance. During immune responses, the egress process can be shut down transiently. When this occurs locally it increases lymphocyte numbers in the responding lymphoid organ; when it occurs systemically it can lead to immunosuppression as a result of the depletion of recirculating lymphocytes. Several mediators of the innate immune system are known to cause shutdown, including interferon alpha/beta (IFN-alpha/beta) and tumour necrosis factor, but the mechanism has been unclear. Here we show that treatment with the IFN-alpha/beta inducer polyinosine polycytidylic acid (hereafter 'poly(I:C)') inhibited egress by a mechanism that was partly lymphocyte-intrinsic. The transmembrane C-type lectin CD69 was rapidly induced and CD69-/- cells were poorly retained in lymphoid tissues after treatment with poly(I:C) or infection with lymphocytic choriomeningitis virus. Lymphocyte egress requires sphingosine 1-phosphate receptor-1 (S1P1), and IFN-alpha/beta was found to inhibit lymphocyte responsiveness to S1P. By contrast, CD69-/- cells retained S1P1 function after exposure to IFN-alpha/beta. In coexpression experiments, CD69 inhibited S1P1 chemotactic function and led to downmodulation of S1P1. In a reporter assay, S1P1 crosslinking led to co-crosslinking and activation of a CD69-CD3zeta chimaera. CD69 co-immunoprecipitated with S1P1 but not the related receptor, S1P3. These observations indicate that CD69 forms a complex with and negatively regulates S1P1 and that it functions downstream of IFN-alpha/beta, and possibly other activating stimuli, to promote lymphocyte retention in lymphoid organs.