BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

Reduced IFNL1 and/or IFNL2, but not IFNL3 is associated with worse outcome in patients with COVID-19.

The recent pandemic was caused by the emergence of a new human pathogen, SARS-CoV-2. While the rapid development of many vaccines provided an end to the immediate crisis, there remains an urgent need to understand more about this new virus and what constitutes a beneficial immune response in terms of successful resolution of infection. Indeed, this is key for development of vaccines that provide long lasting protective immunity. The interferon lambda (IFNL) family of cytokines are produced early in response to infection and are generally considered anti-viral and beneficial. However, data regarding production of IFNL cytokines in COVID-19 patients is highly variable, and generally from underpowered studies. In this study, we measured all three IFNL1, IFNL2 and IFNL3 cytokines in plasma from a well characterised, large COVID-19 cohort (n=399) that included good representation from patients with a more indolent disease progression, and hence a beneficial immune response. While all three cytokines were produced, they differed in both the frequency of expression in patients, and the levels produced. IFNL3 was produced in almost all patients but neither protein level nor IFNL3/IFNL4 SNPs were associated with clinical outcome. In contrast, both IFNL1 and IFNL2 levels were significantly lower, or absent, in plasma of patients that had a more severe disease outcome. These data are consistent with the concept that early IFNL1 and IFNL2 cytokine production is protective against SARS-CoV-2 infection.

Redefining WILD syndrome: a primary lymphatic dysplasia with congenital multisegmental lymphoedema, cutaneous lymphovascular malformation, CD4 lymphopaenia and warts.

BACKGROUND: Primary lymphoedema (PL) syndromes are increasingly recognised as presentations of complex genetic disease, with at least 20 identified causative genes. Recognition of clinical patterns is key to diagnosis, research and therapeutics. The defining criteria for one such clinical syndrome, 'WILD syndrome' (Warts, Immunodeficiency, Lymphoedema and anogenital Dysplasia), have previously depended on a single case report. METHODS AND RESULTS: We present 21 patients (including the first described case) with similar clinical and immunological phenotypes. All had PL affecting multiple segments, with systemic involvement (intestinal lymphangiectasia/pleural or pericardial effusions) in 70% (n=14/20). Most (n=20, 95%) had a distinctive cutaneous lymphovascular malformation on the upper anterior chest wall. Some (n=10, 48%) also had hyperpigmented lesions resembling epidermal naevi (but probably lymphatic in origin). Warts were common (n=17, 81%) and often refractory. In contrast to the previous case report, anogenital dysplasia was uncommon-only found in two further cases (total n=3, 14%). Low CD4 counts and CD4:CD8 ratios typified the syndrome (17 of 19, 89%), but monocyte counts were universally normal, unlike GATA2 deficiency. CONCLUSION: WILD syndrome is a previously unrecognised, underdiagnosed generalised PL syndrome. Based on this case series, we redefine WILD as 'Warts, Immunodeficiency, andLymphatic Dysplasia' and suggest specific diagnostic criteria. The essential criterion is congenital multisegmental PL in a 'mosaic' distribution. The major diagnostic features are recurrent warts, cutaneous lymphovascular malformations, systemic involvement (lymphatic dysplasia), genital swelling and CD4 lymphopaenia with normal monocyte counts. The absence of family history suggests a sporadic condition, and the random distribution of swelling implicates mosaic postzygotic mutation as the cause.

Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients.

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

'We want it all': ART preferences assessed by Desirability of Outcome Ranking.

OBJECTIVES: Understanding how people living with HIV (PLWH) view antiretroviral therapy (ART) prescribing choices is fundamental to patient-centred care. We used the Desirability of Outcome Ranking (DOOR) approach to explore patient ART preferences. METHODS: Seventy-four PLWH entered the study, 20 into the 'pilot study', and 54 in the 'comparative study'. Participants ranked five different hypothetical patient stories by desirability. Each story comprised five narrative lines, each line addressing one treatment characteristic drawn from one of five pre-selected domains (treatment failure, treatment difficulty, adverse effects, long-term complications, life events). Narrative lines could be favourable or adverse. In the pilot study the number of adverse domains varied from one to five. Comparative study stories were fixed at two adverse versus three favourable domains, to test the relative ranking of different domains. RESULTS: The pilot study identified a relationship between the number of adverse domains and rank (R2  = 0.54; p < 0.0001, Friedman test), however pairwise differences in ranking were not significant beyond three adverse domains. In the comparative study, all domains were ranked equally across the cohort (p = 0.88; Friedman test). In pre-defined demographic subgroup analyses, women ranked the 'treatment failure' domain significantly less desirable than men (p = 0.0014, Mann-Whitney test). CONCLUSIONS: People living with HIV appear to care equally about all aspects of ART. The observation that male and female PLWH have different treatment priorities merits further investigation in larger studies. Interindividual differences highlight the importance of individualized shared decision-making and treatment personalization. DOOR may have a role as a pre-treatment assessment tool as well as a research technique.

IgG Seroconversion and Pathophysiology in Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29-May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%-8.5% of persons did not seroconvert 3-6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection.

Human TSCM cell dynamics in vivo are compatible with long-lived immunological memory and stemness.

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell-like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell-like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory.

Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day.

The dynamic co-evolution of memory and regulatory CD4+ T cells in the periphery.

Whereas memory T cells are required to maintain immunity, regulatory T cells have to keep the immune system in check to prevent excessive inflammation and/or autoimmunity. Both cell types must be present during the lifetime of the organism. However, it is not clear whether both subsets are regulated in tandem or independently of each other, especially because thymic involution severely restricts the production of T-cell populations during ageing. In this Opinion article, we discuss recent evidence in both mice and humans that supports the hypothesis that some CD4(+)CD25(+)FOXP3(+)regulatory T cells can differentiate from rapidly proliferating memory T cells in the periphery.

Murine IL-4Δ2 splice variant down-regulates IL-4 activities independently of IL-4Rα binding and STAT-6 phosphorylation.

IL-4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL-4 and its splice variant IL-4Δ2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL-4Δ2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL-4Δ2 form which is suggestive of its possible biological role in this species. Recombinant murine IL-4Δ2 inhibited IL-4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL-4 mediated inhibition of IFN-γ induced nitric oxide release by macrophages, (ii) inhibited IL-4 mediated induction of T cell proliferation, and (iii) prevented IL-4 stimulation of IgE synthesis by B cells. However, IL-4Δ2 did not compete with IL-4 for IL-4Rα binding and did not interfere with the downstream STAT-6 phosphorylation in T cells, suggesting an alternative mechanism for its antagonism of specific IL4-driven effects. These findings suggest that the mouse is a suitable experimental model for studies of the biology of IL-4 and its alternative splice variant.

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.

Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation.

Protection versus pathology in aviremic and high viral load HIV-2 infection-the pivotal role of immune activation and T-cell kinetics.

BACKGROUND: Many human immunodeficiency virus (HIV)-2-infected individuals remain aviremic and behave as long-term non-progressors but some progress to AIDS. We hypothesized that immune activation and T-cell turnover would be critical determinants of non-progressor/progressor status. METHODS: We studied 37 subjects in The Gambia, West Africa: 10 HIV-negative controls, 10 HIV-2-infected subjects with low viral loads (HIV-2-LV), 7 HIV-2-infected subjects with high viral loads (HIV-2-HV), and 10 with HIV-1 infection. We measured in vivo T-cell turnover using deuterium-glucose labeling, and correlated results with T-cell phenotype (by flow cytometry) and T-cell receptor excision circle (TREC) abundance. RESULTS: Immune activation (HLA-DR/CD38 coexpression) differed between groups with a significant trend: controls

High production rates sustain in vivo levels of PD-1high simian immunodeficiency virus-specific CD8 T cells in the face of rapid clearance.

Programmed Death 1 (PD-1) expression by human/simian immunodeficiency virus (HIV/SIV)-specific CD8 T cells has been associated with defective cytokine production and reduced in vitro proliferation capacity. However, the cellular mechanisms that sustain PD-1(high) virus-specific CD8 T cell responses during chronic infection are unknown. Here, we show that the PD-1(high) phenotype is associated with accelerated in vivo CD8 T cell turnover in SIV-infected rhesus macaques, especially within the SIV-specific CD8 T cell pool. Mathematical modeling of 5-bromo-2' deoxyuridine (BrdU) labeling dynamics demonstrated a significantly increased generation rate of PD-1(high) compared to PD-1(low) CD8 T cells in all memory compartments. Simultaneous analysis of Ki67 and BrdU kinetics revealed a complex in vivo turnover profile whereby only a small fraction of PD-1(high) cells, but virtually all PD-1(low) cells, returned to rest after activation. Similar kinetics operated in both chronic and acute SIV infection. Our data suggest that the persistence of PD-1(high) SIV-specific CD8 T cells in chronic infection is maintained in vivo by a mechanism involving high production coupled with a high disappearance rate.

Comparing HIV-1 and HIV-2 infection: Lessons for viral immunopathogenesis.

HIV-1 and HIV-2 share many similarities including their basic gene arrangement, modes of transmission, intracellular replication pathways and clinical consequences: both result in AIDS. However, HIV-2 is characterised by lower transmissibility and reduced likelihood of progression to AIDS. The underlying mechanistic differences between these two infections illuminate broader issues of retroviral pathogenesis, which remain incompletely understood. Comparisons between these two infections from epidemiological, clinical, virologic and immunologic viewpoints provide a basis for hypothesis generation and testing in this 'natural experiment' in viral pathogenesis. In terms of epidemiology, HIV-2 remains largely confined to West Africa, whereas HIV-1 extends worldwide. Clinically, HIV-2 infected individuals seem to dichotomise, most remaining long-term non-progressors, whereas most HIV-1 infected individuals progress. When clinical progression occurs, both diseases demonstrate very similar pathological processes, although progression in HIV-2 occurs at higher CD4 counts. Plasma viral loads are consistently lower in HIV-2, as are average levels of immune activation. Significant differences exist between the two infections in all components of the immune system. For example, cellular responses to HIV-2 tend to be more polyfunctional and produce more IL-2; humoral responses appear broader with lower magnitude intratype neutralisation responses; innate responses appear more robust, possibly through differential effects of tripartite motif protein isoform 5 alpha. Overall, the immune response to HIV-2 appears more protective against disease progression suggesting that pivotal immune factors limit viral pathology. If such immune responses could be replicated or induced in HIV-1 infected patients, they might extend survival and reduce requirements for antiretroviral therapy.

Chromoblastomycosis Treated With Posaconazole and Adjunctive Imiquimod: Lending Innate Immunity a Helping Hand.

Chromoblastomycosis (CBM) is a difficult-to-treat, chronic fungal infection of the skin and subcutaneous tissue. The evidence base for treatment is scarce, with no standardized therapeutic approach. Chronicity of CBM infection is postulated to be due in part to a failure of host cell-mediated immunity to generate a proinflammatory response sufficient for fungal clearance. We present a case of a chronic chromoblastomycosis lesion of the hand present for nearly 4 decades, previously refractory to itraconazole monotherapy, that was successfully treated with a combination of posaconazole and adjunctive immunotherapy with topical imiquimod, a Toll-like receptor 7 agonist. Serial biopsies and images demonstrate the clinical and histopathological improvement of the lesion. Randomized trials of antifungal therapy with adjunctive imiquimod are warranted to determine whether a combination of antifungal and host-directed therapy improves outcomes for this neglected tropical mycosis.

Profound and selective lymphopaenia in primary lymphatic anomaly patients demonstrates the significance of lymphatic-lymphocyte interactions.

Introduction: The lymphatic system has a pivotal role in immune homeostasis. To better understand this, we investigated the impact of Primary Lymphatic Anomalies (PLA) on lymphocyte numbers and phenotype. Methods: The study comprised (i) a retrospective cohort: 177 PLA subjects from the National Primary Lymphatic Anomaly Register with clinical and laboratory data, and (ii) a prospective cohort: 28 patients with PLA and 20 healthy controls. Patients were subdivided using established phenotypic diagnostic categories and grouped into simplex (localised tissue involvement only) and systemic (involvement of central lymphatics). Further grouping variables included genital involvement and the likelihood of co-existent intestinal lymphangiectasia. Haematology laboratory parameters were analysed in both cohorts. In the prospective cohort, prospective blood samples were analysed by flow cytometry for markers of proliferation, differentiation, activation, skin-homing, and for regulatory (CD4+Foxp3+) T cells (Treg). Results: In patients with PLA, lymphopaenia was frequent (22% of subjects), affected primarily the CD4+ T cell subset, and was more severe in subjects with systemic versus simplex patterns of disease (36% vs 9% for lymphopaenia; 70% vs 33% for CD4+ cells). B cells, NK cells and monocytes were better conserved (except in GATA2 deficiency characterised by monocytopaenia). Genital oedema and likelihood of concomitant intestinal lymphangiectasia independently predicted CD4+ T cell depletion. Analysing CD4+ and CD8+ T cells by differentiation markers revealed disproportionate depletion of naïve cells, with a skewing towards a more differentiated effector profile. Systemic PLA conditions were associated with: increased expression of Ki67, indicative of recent cell division, in naïve CD4+, but not CD8+ T cells; increased levels of activation in CD4+, but not CD8+ T cells; and an increased proportion of Treg. Skin-homing marker (CCR10, CLA and CCR4) expression was reduced in some patients with simplex phenotypes. Discussion: Patients with PLA who have dysfunctional lymphatics have a selective reduction in circulating lymphocytes which preferentially depletes naïve CD4+ T cells. The presence of systemic disease, genital oedema, and intestinal lymphangiectasia independently predict CD4 lymphopaenia. The association of this depletion with immune activation and increased circulating Tregs suggests lymphatic-lymphocyte interactions and local inflammatory changes are pivotal in driving immunopathology.

KIR-HLA interactions extend human CD8+ T cell lifespan in vivo.

BACKGROUNDThere is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell-mediated control of chronic viral infection and that these results are consistent with an increase in the CD8+ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo.METHODSWe used stable isotope labeling with deuterated water to quantify memory CD8+ T cell survival in healthy individuals and patients with chronic viral infections.RESULTSWe showed that an individual's iKIR-ligand genotype was a significant determinant of CD8+ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8+ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8+ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8+ and CD4+ T cell immune aging phenotype.CONCLUSIONSTogether, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival.FUNDINGWellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.

Increased monocyte distribution width in COVID-19 and sepsis arises from a complex interplay of altered monocyte cellular size and subset frequency.

INTRODUCTION: Monocyte distribution width (MDW), a parameter generated alongside full blood counts (FBC) in new-generation haematology analysers, has been proposed as a diagnostic test for severe infection/sepsis. It represents the standard deviation (SD) of the monocyte mean volume (MMV). METHODS: This study aimed to compare monocyte volumetric parameters retrieved by the UniCel DxH 900 haematology analyser (MMV and MDW) against corresponding parameters from the same sample measured using flow cytometry (forward scatter [FSC] mean and SD) in combination with phenotypic characterization of monocyte subtypes. We analysed blood samples from healthy individuals (n = 11) and patients with conditions associated with elevated MDW: sepsis (n = 26) and COVID-19 (n = 15). RESULTS: Between-instrument comparisons of monocyte volume parameters (MMV vs. FSC-mean) showed relatively good levels of correlation, but comparisons across volume variability parameters (MDW vs. FSC-SD) were poor. Stratification on sample type revealed this lack of correlation only within the sepsis group. Flow cytometry analysis revealed that in healthy controls intermediate monocytes are the largest and non-classical the smallest cells. In each disease state, however, each monocyte subset undergoes different changes in volume and frequency that together determine the overall configuration of the monocyte population. Increased MDW was associated with reduced classical monocyte frequency and increased intermediate monocyte size. In COVID-19, the range of monocyte sizes (smallest to largest) reduced, whereas in sepsis it increased. CONCLUSION: Increased MDW in COVID-19 and sepsis has no single flow cytometric phenotypic correlate. It represents-within a single value-the delicate equipoise between monocyte subset frequency and size.

Identification of proliferative and non-proliferative subpopulations of leukemic cells in CLL.

Pathogenesis in chronic lymphocytic leukemia (CLL) is strongly linked to the potential for leukemic cells to migrate to and proliferate within lymph-nodes. Previous in vivo studies suggest that all leukemic cells participate in cycles of migration and proliferation. In vitro studies, however, have shown heterogeneous migration patterns.To investigate tumor subpopulation kinetics, we performed in vivo isotope-labeling studies in ten patients with IgVH-mutated CLL (M-CLL). Using deuterium-labeled glucose, we investigated proliferation in sub-populations defined by CXCR4/CD5 and surface (sIgM) expression. Mathematical modeling was performed to test the likelihood that leukemic cells exist as distinct sub-populations or as a single population with the same proliferative capacity. Further labeling studies in two patients with M-CLL commencing idelalisib investigated the effect of B-cell receptor (BCR) antagonists on sub-population kinetics.Modeling revealed that data were more consistent with a model comprising distinct sub-populations (p = 0.008) with contrasting, characteristic kinetics. Following idelalisib therapy, similar labeling suppression across all sub-populations suggested that the most proliferative subset is the most sensitive to treatment. As the quiescent sub-population precedes treatment, selection likely explains the persistence of such residual non-proliferating populations during BCR-antagonist therapy. These findings have clinical implications for discontinuation of long-term BCR-antagonist treatment in selected patients.