CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

Sex Matters: Physiological Abundance of Immuno-Regulatory CD71+ Erythroid Cells Impair Immunity in Females.

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.

Galectin-9 and VISTA Expression Define Terminally Exhausted T Cells in HIV-1 Infection.

We report significant upregulation of Galectin-9 (Gal-9) and VISTA on both CD4+ and CD8+ T cells in HIV-infected human patients. Gal-9 and VISTA expression was associated with impaired T cells effector functions. Although Gal-9 was coexpressed with other coinhibitory receptors such as TIGIT, CD160, CD39, and VISTA, it was simultaneously coexpressed with PD-1. Coexpression of Gal-9 with PD-1 was associated with a more terminally exhausted T cell phenotype in HIV-1 patients. This was marked by higher expression of EOMES, blimp1, and Glut1 in Gal-9+ versus Gal-9- T cells, which is consistent with an exhausted T cell phenotype. Gal-9+ T cells exhibited the phenotype characteristics of effector T cells (CD45RA+, CD45RO-/lo, CD62L-, CD27lo) with higher T-bet expression. A positive correlation between the plasma viral load with the plasma Gal-9 levels in treatment-naive HIV patients and an inverse correlation between CD4 count with the frequency of CD4+Gal-9+ T cells were observed. Increased percentages of Gal-9+ T cells was evident in HIV-treated patients. Enhanced expression of Gal-9 on T cells following PMA stimulation via protein kinase C suggests persistent TCR stimulation as a potential contributing factor in Gal-9 upregulation in HIV patients. This was supported by the constant degranulation of Gal-9+ T cells. Moreover, CD44 clustering by Gal-9 may influence cytoskeleton rearrangement and coclustering of CD3, which likely impact initiation of signal transduction via TCR. Our preliminary data also confirm upregulation of Gal-9 on T cells in hepatitis B virus and HPV infections. These results demonstrate a novel role for Gal-9 and VISTA in HIV pathogenesis.

The Profile of Human Milk Metabolome, Cytokines, and Antibodies in Inflammatory Bowel Diseases Versus Healthy Mothers, and Potential Impact on the Newborn.

BACKGROUND AND AIMS: For women with inflammatory bowel disease [IBD], it is not very well known how IBD or IBD treatment affects their breast milk components. We aimed to investigate whether breast milk composition differs in healthy control [HC] versus IBD mothers in terms of antibodies, cytokines, and metabolite,s to identify potential impact of IBD breast milk on neonatal immune system. METHODS: Breast milk specimens from HC [n = 17] and IBD [n = 31 for Crohn's disease [CD]; and n = 41 for ulcerative colitis [UC]; were collected at 3 and 6 months postpartum [PP3] and [PP6], respectively. Faecal samples were also collected. Cytokines and immunoglobulins [IgA/IgG/IgE] were analysed by multiplex Meso Scale Discovery [MSD] and commercial kits. Moreover, breast milk metabolites were analysed by 1H nuclear magnetic resonance [NMR]. RESULTS: We found that breast milk from IBD mothers showed significantly lower levels of IgA, sugar metabolite [lactose], and 2-aminobutyrate. In contrast, we observed that breast milk from mothers with IBD had increased levels of pro-inflammatory cytokines and higher energy metabolites [lactate and succinate] than milk from healthy mothers. In addition, we noticed that the type of treatment [5-aminosalicylic acid versus biologics] influenced the milk cytokines and metabolites profile. CONCLUSIONS: The reduction in immunoprotective components of IBD breast milk such as sIgA and lactose theoretically may modulate the potential protective effects of breastfeeding. On the other hand, presence of higher levels of pro-inflammatory cytokines, lactate, and succinate may predispose the offspring to an inflammatory condition or impact on the gut microbiome. Better understanding of the role of succinate in infants and its potential effects on microbiome or mucosal immunity merits further investigations.

Inulin-type fructans improve active ulcerative colitis associated with microbiota changes and increased short-chain fatty acids levels.

The intestinal microbiota is involved in ulcerative colitis (UC) pathogenesis. Prebiotics are hypothesized to improve health through alterations to gut microbiota composition and/or activity. Our aim was therefore to determine if inulin-type fructans induce clinical benefits in UC, and identify if benefits are linked to compositional and/or functional shifts of the luminal (fecal) and mucosal (biopsy) bacterial communities. Patients (n = 25) with mild/moderately active UC received 7.5 g (n = 12) or 15 g (n = 13) daily oral oligofructose-enriched inulin (Orafti®Synergy1) for 9 weeks. Total Mayo score, endoscopic activity and fecal calprotectin were assessed. Fecal and mucosal bacterial communities were characterized by 16S rRNA tag sequencing, and short chain fatty acids (SCFA) production were measured in fecal samples. Fructans significantly reduced colitis in the high-dose group, with 77% of patients showing a clinical response versus 33% in the low-dose group (P = 0.04). Fructans increased colonic butyrate production in the 15 g/d dose, and fecal butyrate levels were negatively correlated with Mayo score (r = -0.50; P = 0.036). The high fructan dose led to an increased Bifidobacteriaceae and Lachnospiraceae abundance but these shifts were not correlated with improved disease scores. In summary, this pilot study revealed that 15 g/d dose inulin type fructans in UC produced functional but not compositional shifts of the gut microbiota, suggesting that prebiotic-induced alterations of gut microbiota metabolism are more important than compositional changes for the benefits in UC. The findings warrant future well-powered controlled studies for the use of ß-fructans as adjunct therapy in patients with active UC.

Lower Abundance and Impaired Function of CD71+ Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy.

BACKGROUND AND AIMS: CD71+ erythroid cells are enriched during pregnancy with immuno suppressive properties. We investigated the frequency and functionality of CD71+ erythroid cells in peripheral blood, cord blood, and placenta of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. We aimed to determine their role in IBD pathogenesis during pregnancy. METHODS: Peripheral blood was collected at preconception, the first, second and third trimesters, and postpartum. Cord blood and placental tissues were collected at the time of birth. Cells from different specimens were subjected to immune-phenotyping and functional assays. CD71+ erythroid cells were purified for quantitative polymerase chain reaction [qPCR] analysis. Using an allogeneic mouse model of pregnancy, the effects of CD71+ erythroid cells depletion on intestinal homeostasis and dysbiosis was studied. RESULTS: IBD patients had lower CD71+ erythroid cells during pregnancy compared with HCs. Placenta and cord blood CD71+ erythroid cells from IBD patients exhibited impaired functionality and expressed lower inhibitory molecules including VISTA, TGF-ß, and reactive oxygen species [ROS]. Lower CD71+ erythroid cells were correlated with reduced regulatory T cells and increased immune-activation in IBD patients. Depletion of CD71+ erythroid cells in an allogeneic pregnancy model resulted in upregulation of TLRs, IL-6, and CXCL-1, and enhanced production of TNF-α, in intestinal tissues. In contrast, TGF-ß gene expression was reduced. Excessive inflammatory response in the gut [e.g. TNF-α] affects intestinal integrity and CD71+ erythroid cells impact on the gut's bacterial composition. CONCLUSIONS: Reduced frequency and/or impaired functionality of CD71+ erythroid cells during pregnancy may predispose IBD patients to a more pro-inflammatory milieu in their gastrointestinal tract, characterised by lower Tregs, higher IL-6, and TNF-α, and dysbiosis.

CD71+ Erythroid Suppressor Cells Promote Fetomaternal Tolerance through Arginase-2 and PDL-1.

Survival of the allogeneic pregnancy depends on the maintenance of immune tolerance to paternal alloantigens at the fetomaternal interface. Multiple localized mechanisms contribute to the fetal evasion from the mother's immune rejection as the fetus is exposed to a wide range of stimulatory substances such as maternal alloantigens, microbes and amniotic fluids. In this article, we demonstrate that CD71+ erythroid cells are expanded at the fetomaternal interface and in the periphery during pregnancy in both humans and mice. These cells exhibit immunosuppressive properties, and their abundance is associated with a Th2 skewed immune response, as their depletion results in a proinflammatory immune response at the fetomaternal interface. In addition to their function in suppressing proinflammatory responses in vitro, maternal CD71+ erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-α and IFN-γ production through arginase-2 activity and PD-1/programmed death ligand-1 (PDL-1) interactions. Their depletion leads to the failure of gestation due to the immunological rejection of the fetus. Similarly, fetal liver CD71+ erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71+ erythroid cells on both sides (mother/fetus) is crucial for fetomaternal tolerance. Thus, our results reveal a previously unappreciated role for CD71+ erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immunoregulatory responses during pregnancy.

Changes in the functional characteristics of tumor and normal cells after treatment with extracts of white dead-nettle.

Lamium album L. is a perennial herb widely used in folk medicine. It possesses a wide spectrum of therapeutic activities (anti-inflammatory, astringent, antiseptic, antibiotic, antispasmodic, antioxidant and anti-proliferative). Preservation of medicinal plant could be done by in vitro propagation to avoid depletion from their natural habitat. It is important to know whether extracts from L. album plants grown in vitro possess similar properties as extracts from plants grown in vivo. For these reasons, it is important to examine changes in the composition of secondary metabolites during in vitro cultivation of the plant and how they affect the biological activity. We used A549 human cancer cell line and normal kidney epithelial cells MDCKII (Madin-Darby canine kidney cells II) as controls in assessing the anti-cancer effect of plant extracts. To elucidate changes in some key functional characteristics, adhesion test, MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER), immunofluorescence staining and trypan blue exclusion test were performed. Methanol and chloroform extracts of in vivo and in vitro propagated plants affected differently cancerous and non-cancerous cells. The most pronounced differences were observed in the morphological analysis and in the cell adhesive properties. We also detected suppressed epithelial transmembrane electrical resistance of MDCK II cells, by treatment with plant extracts, compared to non-treated MDCK II cells. A549 cells did not polarize under the same conditions. Altered organization of actin filaments in both cell types were noticed suggesting that extracts from L. album L. change TER and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II cells.