key: cord-0298556-vxy5e975
authors: Grzywa, Tomasz M.; Sosnowska, Anna; Rydzynska, Zuzanna; Lazniewski, Michal; Plewczynski, Dariusz; Klicka, Klaudia; Malecka-Gieldowska, Milena; Rodziewicz-Lurzynska, Anna; Ciepiela, Olga; Justyniarska, Magdalena; Pomper, Paulina; Grzybowski, Marcin M.; Blaszczyk, Roman; Wegrzynowicz, Michal; Tomaszewska, Agnieszka; Basak, Grzegorz; Golab, Jakub; Nowis, Dominika
title: Potent but transient immunosuppression of T-cells is a general feature of CD71+ erythroid cells
date: 2021-10-04
journal: bioRxiv
DOI: 10.1101/2021.01.18.427109
sha: f5ec8e02c62016e9ab51fc704a770dd383f85263
doc_id: 298556
cord_uid: vxy5e975

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation.

Accordingly, we found that both cytoplasmic and nuclear ROS levels were higher in 142 anemia-induced CECs as compared with mature RBCs (Fig. 3a, Supplementary Fig.   143 5a,b). ROS reached the highest values in the CECs at the earliest stages of their 144 maturation ( Supplementary Fig. 5c,d) i.e. in CD45 + CECs ( Supplementary Fig. 5e,f) . 145 Interestingly, in contrast to human CECs 13 , ROS levels in murine CECs, including 146 CD45 + CECs, were significantly lower than in the cells of non-erythroid lineages such 147 as myeloid cells or T-cells (Fig. 3b) . 148 Murine CECs expressed ARG2, a mitochondrial arginase isoform (Fig. 3c,d) , but had Arg1 tm1Lky /J mice that express eYFP under Arg1 promoter (Fig. 3f,g) . Similar to ROS, 152 the levels of ARG1 and ARG2 were the highest in early-stage CECs and 153 consequently decreased during maturation (Supplementary Fig. 6a-g) . Intriguingly, 154 while the level of ARG2 (Fig. 3c) , as well as the percentage of ARG2 + CECs, were 155 similar in all groups (Fig. 3d) , the fraction of ARG1 + cells was substantially higher in 156 HA-PHZ mice as determined by intracellular staining (Fig. 3e ). This finding seems 157 counterintuitive considering that ARG-dependent degradation of ʟ-arginine leads to 158 T-cell suppression 18, 19 , and we did not observe the suppression of T-cells in HA-PHZ 159 mice in vivo (Fig. 2b) . Moreover, CECs from HA-PHZ mice exerted the weakest Supplementary Fig. 7b ), but no increase 165 in ARG2 protein levels was observed (Fig. 3d) . Surprisingly, despite robust 166 7 upregulation of ARG1 levels, total arginase activity in both CECs isolated from HA-167 PHZ mice and CECs-conditioned medium was lower even than that in CECs from 168 NHA mice (Fig. 3i,j) . Moreover, CECs cultured ex vivo in the presence of PHZ 169 strongly upregulated ARG1 expression (Fig. 3k,l) . Then, we sought to confirm 170 whether PHZ is responsible for the attenuation of CECs immunoregulatory 171 properties. Indeed, we found that CECs isolated from NHA lose their suppressive 172 effects on T-cells proliferation in the presence of PHZ ( Supplementary Fig. 8a,b) . slightly reduced by concomitant incubation with N-acetylcysteine (ROSi) (Fig. 4d) . 203 Moreover, ROS scavengers did not prevent ARG1 induction by PHZ in vivo 204 ( Supplementary Fig. 10a ,b) nor in vitro ( Supplementary Fig. 10c,d) . Thus, we 205 demonstrated that PHZ targets ARG leading to the diminishment of CECs 206 immunoregulatory properties, however, the exact mechanism that would explain 207 PHZ-mediated inhibition of ARG activity remains elusive. restored the proliferation of T-cells that was inhibited by co-culture with CECs 217 isolated from NHA mice (Fig. 5b) , similar to CECs isolated from neonates 218 ( Supplementary Fig. 11 ). Likewise, CECs-conditioned medium had a suppressive 219 effect on T-cell proliferation, and supplementation with either of ʟ-arginine or ARGi 220 restored T-cell proliferation to percentages akin to the control group (Fig. 5c ).

To confirm that early-stage CECs that have the highest ROS levels (Supplementary T-cells (Fig. 5f ). Further studies revealed that the expansion of CECs in anemic mice 231 leads to the substantial increase of the total arginase activity (Fig. 5g ). This effect 232 was caused by an increased ARG2 but not ARG1 levels in the spleen ( Fig. 5h-j) .

Even though the concentration of ʟ-arginine was only slightly decreased in the serum 234 of anemic mice ( Supplementary Fig. 12) , their splenic CD4 + T-cells and CD8 + T-cells 235 had decreased levels of CD3ζ (Fig. 5k,l) , a marker of ʟ-arginine T-cell starvation 25,26 .

Accordingly, ex vivo stimulation of T-cells with anti-CD3/CD28 beads in the presence 237 of CECs resulted in a decrease in CD3ζ, which was prevented by ARGi and 238 completely restored by the combination of ARGi and ROSi (Fig. 5m,n) . Noteworthy, 239 the decrease in CD3ζ was not observed in the lymph nodes of anemic mice, where 240 CECs are a relatively rare population ( Supplementary Fig. 13a-c) . Altogether, these 241 10 results show that CECs suppress T-cells response in anemic mice via both arginase 242 and ROS and their local accumulation in the spleen impairs T-cell immunity.

To further study the role of ARG2 in the modulation of immune response by CECs, 244 we assessed the suppressive effects of CECs isolated from anemic mice lacking 245 functional Arg2 gene (Arg2 -/-, Arg2 tm1Weo/ J mice 27 ). Arg2 -/mice had effective stress 246 erythropoiesis ( Supplementary Fig. 14a ). Despite a slightly increased percentage of 247 ARG1 + CECs compared to wild-type mice (Arg2 +/+ ) ( Supplementary Fig. 14b ), no 248 significant changes in total ARG1 levels were observed in these cells (Supplementary 249 Fig. 14c ). In contrast to wild-type mice, expansion of CECs in the spleen of anemic Table 1, Supplementary Table 2 ). The percentages of CECs (CD71 + CD235a + ) in the 260 peripheral blood were substantially increased in anemic patients compared to non-261 anemic control individuals (Fig. 7a,b) . The number of CECs in the blood (Fig. 7c) 262 reversely correlated with hemoglobin concentration (Fig. 7d) and was the highest in 263 patients with moderate and severe anemia (Fig. 7e) .

In anemic patients, CECs constituted a substantial fraction of peripheral blood 265 mononuclear cells (PBMCs) (Fig. 7f,g) and were predominantly at the latest stages of 266 differentiation with a very small percentage of CD45 + CECs (Supplementary Fig.   267 15a,b). We found that the production of IFN-γ in response to CD3/CD28 stimulation 268 was suppressed in T-cells from anemic individuals when compared to non-anemic 269 controls (Fig. 7h, i) . However, T-cells proliferation ( Supplementary Fig. 15c,d) 289 Further, we investigated the immunoregulatory properties of model human Supplementary Fig. 20a,b) . Moreover, isolated PBMC-derived CECs (Fig.   312 10a) potently suppressed both CD4 + and CD8 + human T-cell proliferation ( Fig.   313 10b,c).

Next, we aimed to study the possible changes in immunoregulatory properties of Supplementary Fig. 22c ), and proliferation ( Supplementary Fig. 23a,b) . Loss of the 332 suppressive properties corresponded with a decrease in CD71 (Fig. 10h) , an 333 increase in CD235a (Fig. 10i ) as well as a decrease in CD49d (Fig. 10k) Spleens were isolated from mice and mechanically dispersed by pressing gently 596 through a 70 μm nylon cell strainer using a rubberized 1 ml syringe piston. Murine 597 bone marrow was isolated from the femur by the centrifugation method. Briefly, 598 femurs were dissected, followed by the removal of any muscle or connective tissue.

The condyles and epiphysis were removed and a cleared bone was placed in once again with the same dose of OVA-ALUM. NHA mice were divided into three 684 groups. NHA before mice were phlebotomized before first immunization, NHA boost 685 mice were phlebotomized before second OVA immunization, and NHA both were 686 phlebotomized before first and second immunization (see Supplementary Fig. 3c) . Rahman barostat. During simulation short-range nonbonded interactions were cut off 848 at 1.4nm, with long-range electrostatics calculated using the particle mesh Ewald 849 (PME) algorithm. Bonds were constrained using the lincs algorithm. Simulations were 850 carried out with Gromacs 67 using the gromos54a7 force field, modified to include 851 parameters for Mn 2+ ion adopted from 68 . Spc model was used for water molecules. 

Mediate <em>trans</em>-Infection, and Harbor Infective Viral 1021

Erythroid precursors and progenitors suppress adaptive 1023 immunity and get invaded by SARS-CoV-2

Erythropoietic precursors in mice with phenylhydrazine-1026 induced anemia

Erythropoietic precursors in mice under erythropoietic 1029 stimulation and suppression

Resolving the distinct stages in erythroid differentiation based 1031 on dynamic changes in membrane protein expression during erythropoiesis

CD71+ Erythroid Cells in Human Neonates Exhibit 1035

Immunosuppressive Properties and Compromise Immune Response Against 1036

Systemic Infection in Neonatal Mice

Arginase I production in the tumor microenvironment by 1039 mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-1040 cell responses

Local suppression of T cell responses by arginase-induced 1043

L-arginine depletion in nonhealing leishmaniasis

Discovery of (R)-2-amino-6-borono-2

yl)ethyl)hexanoic acid and congeners as highly potent inhibitors of human 1047

arginases I and II for treatment of myocardial reperfusion injury

Mechanism of induction of haemolytic 1050 anaemia by phenylhydrazine

Small extracellular vesicles containing 1053 arginase-1 suppress T-cell responses and promote tumor growth in ovarian 1054 carcinoma

Inhibition of arginase modulates T-cell response in the 1057 tumor microenvironment of lung carcinoma

A Novel Oral Arginase 1/2 Inhibitor Enhances the Antitumor 1060 Effect of PD-1

Regulation of T cell receptor CD3zeta chain expression 1064 by L-arginine

Small extracellular vesicles containing 1067 arginase-1 suppress T-cell responses and promote tumor growth in ovarian

Generation of a mouse model for arginase II deficiency by targeted disruption 1072 of the arginase II gene

Isolation and functional characterization of human erythroblasts at 1075 distinct stages: implications for understanding of normal and disordered 1076 erythropoiesis in vivo

Advances in 1079 understanding erythropoiesis: evolving perspectives

Time-course of butyric 1082 acid-induced differentiation in human K562 leukemic cell line: rapid increase in 1083 γ-globin, porphobilinogen deaminase and NF-E2 mRNA levels

From Erythroblasts to Mature Red 1086

Myeloid Cell-Derived Arginase in Cancer Immune 1089

Arginase-producing myeloid suppressor cells in renal cell 1091 carcinoma patients: a mechanism of tumor evasion

Granulocytic myeloid derived suppressor cells expand in 1094 human pregnancy and modulate T-cell responses

Oxidative stress by 1097 tumor-derived macrophages suppresses the expression of CD3 zeta chain of 1098

T-cell receptor complex and antigen-specific T-cell responses

The 1101 prevalence of anemia and its association with 90-day mortality in hospitalized 1102 community-acquired pneumonia

Multiple myeloma hinders erythropoiesis and causes anaemia 1105 owing to high levels of CCL3 in the bone marrow microenvironment

Preoperative anaemia and postoperative outcomes in 1108 non-cardiac surgery: a retrospective cohort study

Perioperative anemia: an independent risk factor for infection, mortality, and 1112 resource utilization in surgery

The immunoregulatory function of peripheral blood 1115 CD71+ erythroid cells in systemic-onset juvenile idiopathic arthritis

Immune system development varies according to age, location, 1118 and anemia in African children

Normal and pathological erythropoiesis in adults: from gene 1121 regulation to targeted treatment concepts

Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells 1124

Radiotherapy and immunotherapy converge on elimination of 1127 tumor-promoting erythroid progenitor cells through adaptive immunity

Putative regulators for the continuum of erythroid 1131 differentiation revealed by single-cell transcriptome of human BM and UCB 1132 cells

Erythroid Suppressor Cells Compromise Neonatal 1135

Immune Response against <em>Bordetella pertussis</em>. The Journal of 1136

Inflammation and Cancer: Triggers

Lower Abundance and Impaired Function of CD71+

Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy

Chronic IFN-γ production in mice induces anemia by 1146 reducing erythrocyte life span and inhibiting erythropoiesis through an IRF-1147

1 axis

Impact of interferon-γ on 1150 hematopoiesis

Rats offer a superior model of human stress erythropoiesis

During EPO or anemia challenge, erythroid progenitor cells 1156 transit through a selectively expandable proerythroblast pool

Structure and function of the 1159 immune system in the spleen

Tumor Immune 1162

Evasion Induced by Dysregulation of Erythroid Progenitor Cells Development

Assessment of 1168 peripheral blood lymphocyte subsets in children with iron deficiency anemia

Revisiting WHO haemoglobin thresholds to define anaemia 1172 in clinical medicine and public health

Chitin induces accumulation in tissue of innate immune 1175 cells associated with allergy

T cell receptor antagonist peptides induce positive 1178 selection

Practical murine hematopathology: a comparative 1180 review and implications for research

MUSCLE: multiple sequence alignment with high accuracy and 1182 high throughput

Comparative Protein Structure Modeling Using 1185

MolProbity: More and better reference data for improved 1188 all-atom structure validation

Development and 1191 validation of a genetic algorithm for flexible docking

Surflex: fully automatic flexible molecular docking using a molecular 1194 similarity-based search engine

GROMACS 4.5: a high-throughput and highly parallel open 1197 source molecular simulation toolkit

X-Ray and molecular dynamics studies of 1200 concanavalin-A glucoside and mannoside complexes Relating structure to 1201 thermodynamics of binding

An Automated Force Field Topology Builder (ATB) and 1204 Repository: Version 1.0

Large-scale in vitro production of red blood cells from 1207 human peripheral blood mononuclear cells