Immunoregulatory CD71⁺ Erythroid Cells (CECs) Expand in Multiple Myeloma and Impair Control of L. Monocytogenes Infection

Introduction

Although the clinical management of multiple myeloma (MM) patients has significantly improved over recent years, MM remains a largely incurable disease. One of the most common complication of MM, which correlates with the worse clinical outcome, is anemia. Moreover, MM patients are prone to develop life-threatening infections mainly due to the disease-related impairment of the immune system function. Recently, CD71⁺ erythroid progenitor and precursor cells (CECs) arose as a relevant immunomodulatory population. CECs have been shown to create an immunosuppressive microenvironment via multiple mechanisms including arginase 2 (ARG2), PDL-1, and TGF-beta. It was recently observed in our laboratory that mice bearing late-stage tumors are characterized by the presence of an increased percentage of CECs in their blood and spleens. Moreover, the tumor-associated CECs show elevated levels of arginases. The abovementioned facts may suggest an important role of CECs and their related mechanisms in compromising the immune response in MM.

Methods

Vk*MYC MM model

Recipient C57BL/6 (wild type or ARG2 KO) mice were injected via tail vein with 1 × 10⁶ Vk*MYC cells. M-spike levels were evaluated with serum protein electrophoresis. Five weeks later they were challenged with L. monocytogenes.

L.monocytogenes infection and bacterial burden evaluation

Mice were injected intraperitoneally with 1 x 10³L. monocytogenes cells (strain EGDe). Bacterial burden was analyzed 72h and 7 days post infection. Briefly, spleens and livers of sacrificed mice were isolated, weighed, and homogenized. The homogenates were serially diluted and plated on culture plates. Bacterial burden was calculated using the number of colonies and the dilution factor.

Flow cytometry analysis

Flow cytometry analyses (immunophenotyping of spleen and bone marrow (BM) cells as well as cytokine beads arrays, Legenplex, BioLegend) were performed using BD Fortessa X-20 flow cytometer operated by BD FACSDiva Software ver. 8.0. The acquired data were re-analyzed using FlowJo™ ver. 10 software.

Arginase inhibitor

OAT-1746 arginase inhibitor (Molecure SA) was administered intraperitoneally at doses of 5 or 20 mg/kg twice daily, starting from the second or the fifth day before the bacterial challenge, respectively. The treatment was continued until the end of the experiment.

Results

In Vk*MYC-bearing mice we observed an accumulation of CECs in the blood and spleen in time, correlating with the development of anemia. At the late stage of the disease MM-associated CECs showed less differentiated phenotype and increased expression of arginase-1 (ARG1) and ARG2. MM-associated CECs inhibited T cells activation and proliferation in an ARG and reactive oxygen species-dependent manner. Interestingly, MM development was delayed in ARG2 KO vs WT mice. In the BM aspirates of MM patients we observed increased percentage of CECs expressing high levels of both ARG1 and ARG2 comparing with healthy controls. Moreover, MM impaired control of L. monocytogenes infection as we observed an elevated bacterial burden in the livers and spleens of Vk*MYC-bearing vs control mice. Infected MM-bearing mice had fewer NK cells and activated CD4⁺ and CD8⁺ T cells in the spleen than infected control mice. Furthermore, MM altered the dynamics of cytokines involved in the control of intracellular infection. 24h post Listeria challenge we observed their significant decrease followed by an increase at 72h comparing to infected non-MM mice. These results indicate that MM-bearing mice have delayed initial immune response leading to increased bacterial burden followed by excessive cytokines response resulting in sepsis-like condition leading to increased mice mortality. Finally, ARG inhibitor decreased L. monocytogenes burden in MM-bearing mice. Altogether, our results show that accumulation of CECs during MM progression leads to impaired bacterial infection control, at least partially dependent on ARG2 activity.

Funding: National Science Centre in Poland [grants No 2017/25/B/NZ6/01139 and 2019/35/B/NZ6/00540]; Polish Ministry of Education and Science [grant No 013/RID/2018/19 (Regional Initiative for Excellence, project budget 12,000,000 PLN)].

Juszczynski:RYVU Therapeutics: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees.