Expression of the Plasma Cell Transcriptional Regulator Blimp-1 by Dark Zone Germinal Center B Cells During Periods of Proliferation

Long-lived plasma cells (PCs) develop in germinal centers (GCs) by the differentiation of affinity matured B cells. Antibody affinity maturation involves iterative rounds of somatic hypermutation in dark zones (DZs) and selection in light zones (LZs), however the details of where, when and how PC commitment occurs are not well-understood. Fate bifurcation at the time of selection is one possibility, with the very highest affinity GC B cells differentiating as an alternative to DZ re-entry. However, how this model fits with a need to also retain these clones in the response is not clear. Here, we show that subsets of bona fide DZ cells express the plasma cell master regulator Blimp-1 at low levels during periods of proliferation. Ex vivo culture experiments demonstrate that these cells are not yet committed to plasma cell differentiation but that they may be sensitized to go down that route. Contrary to models in which T cells directly select GC B cells to begin expressing Blimp-1, we found that expression of this transcriptional regulator occurred even when follicular helper T cells were ablated. We speculate that Blimp-1 may be induced during proliferation in the DZ, and that as such single selected cells might give rise to both GC and post-GC progeny

Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication

Hepcidin-mediated hypoferremia disrupts immune responses to vaccination and infection

Background: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. Methods: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. Findings: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. Conclusions: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Germinal centers output clonally diverse plasma cell populations expressing high- and low-affinity antibodies

Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses.</p

Phenotypic and Morphological Properties of Germinal Center Dark Zone Cxcl12-Expressing Reticular Cells.

The germinal center (GC) is divided into a dark zone (DZ) and a light zone (LZ). GC B cells must cycle between these zones to achieve efficient Ab affinity maturation. Follicular dendritic cells (FDCs) are well characterized for their role in supporting B cell Ag encounter in primary follicles and in the GC LZ. However, the properties of stromal cells supporting B cells in the DZ are relatively unexplored. Recent work identified a novel stromal population of Cxcl12-expressing reticular cells (CRCs) in murine GC DZs. In this article, we report that CRCs have diverse morphologies, appearing in open and closed networks, with variable distribution in lymphoid tissue GCs. CRCs are also present in splenic and peripheral lymph node primary follicles. Real-time two-photon microscopy of Peyers patch GCs demonstrates B cells moving in close association with CRC processes. CRCs are gp38(+) with low to undetectable expression of FDC markers, but CRC-like cells in the DZ are lineage marked, along with FDCs and fibroblastic reticular cells, by CD21-Cre- and Ccl19-Cre-directed fluorescent reporters. In contrast to FDCs, CRCs do not demonstrate dependence on lymphotoxin or TNF for chemokine expression or network morphology. CRC distribution in the DZ does require CXCR4 signaling, which is necessary for GC B cells to access the DZ and likely to interact with CRC processes. Our findings establish CRCs as a major stromal cell type in the GC DZ and suggest that CRCs support critical activities of GC B cells in the DZ niche through Cxcl12 expression and direct cell-cell interactions