Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive myeloid cells developing from myeloid progenitors, which are particularly enriched in pathological conditions such as cancer, infections, inflammation and sepsis [1‐3]. These pathological conditions determine an increase in circulating colony-stimulating factors (CSFs) as well as chemokines that stimulate the myelopoiesis and, consequently, the generation of immature MDSCs. Different subsets of human MDSCs have been documented in several types of tumors, and it appears that all MDSC phenotypes can be allocated to one of the three main subsets, each of them containing more than one cell population. Monocytic MDSCs (M-MDSCs) share the morphology of monocytes and are characterized by the expression of CD14 and lack of CD15, polymorphonuclear MDSCs (PMN-MDSCs) are instead defined by the opposite expression of the myeloid markers (CD15⁺/CD14⁻), while more immature MDSCs (early-stage, e-MDSCs) lack the expression of both markers [4].

These cells are released into the bloodstream and recruited to the affected tissues, where they proliferate and are activated by inflammatory factors, and suppress acute inflammatory reactions by inhibiting the functions of distinct components of innate and adaptive immunity [5]. In tumors and inflammatory disorders T cells represent the main target of MDSC-induced immune tolerance [1, 6]. Indeed, MDSCs are involved in tumor angiogenesis, drug resistance and tumor progression and could represent a potential therapeutic target both in cancer and in chronic inflammatory diseases [3, 7].

During aging several changes take place such as the dysregulation of the immune, central and peripheral nervous, endocrine and metabolic system. In particular, aging is associated with a decline of functional capacity of both the adaptive and innate immune systems, in a process defined as immunoscenescence. Although the adaptive immune response has been more extensively investigated, some works document the significant impact of ageing on the innate compartment as well [6, 8, 9]. In fact, neutrophils, dendritic cells and monocytes in aged individuals show a reduced functional activity [10‐13]. In addition, during aging an increase of regulatory T cells, a loss of T helper CD4⁺ and cytotoxic CD8⁺ T cells and an alteration of their functional capacities is observed, probably due to lower production of lymphoid cells by the bone marrow and to thymic involution, which reduces the release of naïve T cells. This is accompanied by a number of events including a reduced proliferative ability upon stimuli, as well as telomeres erosion, accumulation of memory T cells from chronic infections and replicative senescence upon persistent antigen exposure [14‐16]. In the same context MDSCs accumulate and exacerbate the process by impairing T cell proliferation and function, and producing large amounts of pro-inflammatory cytokines [5].

Aging is also associated with a chronic, low-grade inflammation state called inflammaging [17]. It appears that in the microenvironment of the bone marrow (BM) inflammaging affects haematopoietic stem cells, with a possible rebound on the myelopoiesis and lymphopoiesis process [18]. In particular, one of the hallmarks of the alterations in the BM during aging is the increased myelopoiesis, associated with a concomitant decrease in lymphopoiesis.

Our group demonstrated that GM-CSF, G-CSF, and IL-6 allow the in vitro generation of MDSCs from precursors present in human bone marrow aspirates of healthy donors, and named such cells BM-derived MDSCs (BM-MDSCs). Of note, these cells share the phenotype and the suppressive function of MDSCs isolated from cancer patients [19]. BM-MDSCs are a heterogeneous collection of immature myeloid cells, but only the most immature subset from BM-MDSCs, with morphology and phenotype of promyelocytes (immature-BM-MDSC, i-BM-MDSC) is entirely responsible for the suppression mediated by BM-MDSCs [19].

Most of the works that advance that MDSC levels increase during aging have been obtained in mouse models in which these suppressive populations have been evaluated in the BM, spleen and circulation [20, 21], while only a few works have documented it also in humans [22‐24].

It is not clear if the increase of myelopoiesis, which occurs in aging, is associated with an increase of MDSCs generation in the BM of humans, and consequently with an increase of these cells in the blood circulation. In addition, there are no data showing whether aging impacts on the immunosuppressive ability of MDSCs.

Aim of this study is to compare the induction of MDSCs from the BM of young and old individuals by using an optimized method to generate in 4 days MDSCs from precursor cells through cytokines treatment. Such cells are equivalent to MDSCs present in the blood of cancer patients and gives us the possibility to evaluate not only the expansion ability of the precursors, but also the immunosuppressive ability of the induced myeloid suppressor cells.

In this study a total of 20 pediatric and 24 elderly donors were enrolled. Demographic data are reported in Table 1, showing a median age of 6 years for pediatric donors, and of 79 years for elderly individuals. BM aspirates from both pediatric and elderly donors with normal cytologic characteristics were freshly characterized for the myeloid differentiation by flow cytometry using by combining the expression of the markers CD11b and CD16. To induce the expansion of BM-MDSCs from myeloid cell precursors, we cultured BM cells for 4 days with the combination of the cytokines G-CSF and GM-CSF, and assessed the maturation of the myelomonocytic precursors by flow cytometry, as previously reported [19, 25]. A representative example of the immunophenotype of a BM before and after cell culture of a pediatric and an adult individual is shown in Fig. 1a, while the cumulative result of several independent experiments is shown in Fig. 1b. Three different myeloid subsets were analyzed: the most immature and immune suppressive fraction corresponding to CD11b^low/−/CD16⁻ (immature-BM-MDSCs, i-BM-MDSCs), and the two more differentiated but immature and non-suppressive CD11b⁺/CD16⁻ and CD11⁺/CD16⁺ subsets [19] (Fig. 1 a-b).

When we compared the ex vivo distribution of the three myeloid cell subsets between pediatric and elderly donors, we found a similar distribution of the myeloid differentiating cells (Fig. 1a, left panels; Fig. 1b), suggesting that there is no significant change in their distribution with aging, despite a reported shift toward myelopoiesis [26]. Instead, after 4 days of cell culture in the presence of myelomonocytic cytokines we observed a significant reduction in the percentage of suppressive i-BM-MDSCs in the group of elderly with respect to the pediatric group (median 12.6% vs 8.1%, p = 0.0001 by pairwise comparison) (Fig. 1c). Moreover, compared to the freshly isolated BM cells, in which polymorphonuclear (PMN) cells corresponding to CD11b⁺/CD16^high are clearly present, after cell culture with the cytokines only a very low presence of these mature cells can be detected, although significantly higher in pediatric donors (median 8.4%, n = 20) compared to elderly donors (median 2.4%, n = 19) (Fig. 1a left panels; Fig. 1c).

To assess whether i-BM-MDSCs isolated from the two groups of donors displayed immune suppression on T cells, after 4 days of cell culture two fractions of BM-MDSCs were isolated, corresponding to the more immature CD11b⁻ and the more differentiated CD11b⁺ cell fraction. A functional assay was set up with allogenic peripheral blood mononuclear cells (PBMCs) activated with anti-CD3 and anti-CD28 for 4 days in the presence of the two myeloid cell fractions. Previous work from our laboratory already demonstrated that the CD11b⁻ fraction corresponds to the suppressive i-BM-MDSC, while CD11b⁺ cells are devoid of significant immune suppressive activity [19]. Evaluation of the proliferation of T cells was assessed by CellTrace profile (Fig. 2), as previously described [27]. In line with previous results, only the most immature CD11b⁻ cell fraction showed the ability to suppress the proliferation of T cells in both groups of donors (Fig. 2 a-b) [19] and, importantly, no significant differences were found comparing the suppression ability of CD11b⁻BM-MDSCs isolated from pediatric and elderly donors, thus suggesting that the suppressive ability of MDSCs is maintained with aging. Of note, the immune suppressive capacity is acquired only after cytokines induction, as demonstrated by functional tests performed on ex vivo sorted immature CD11b⁻ cells from the BM of both the pediatric [19] and elderly (data not shown) donors, that failed to suppress T cell proliferation.

We previously demonstrated that MDSCs can be induced in vitro by using a combination of cytokines and that CD11b⁻/CD16⁻ cells phenotypically and functionally resembled to myeloid suppressor cells present in the blood of cancer patients [19, 25]. In the present work, we extend these data and observe that a reduced percentage of immune suppressive myeloid cells can be generated with G-CSF and GM-CSF combination from the BM of elderly donors, suggesting a less effective ability to induce this mechanism. Of note, the reduced frequency of BM-MDSCs in elderly donors does not translate in a worsened MDSCs function, since this population maintains the same immunosuppressive capacity of pediatric donors, as demonstrated by the elevated suppression observed in a functional assay. Literature data report that the aging process is associated with a significant increase in MDSCs [23], and accompanied by an augmented myeloid-to-lymphoid ratio [26], although it was never clarified the mechanism of this age-related process. Actually, conflicting results exist concerning the cellular complexity of the bone marrow niche during aging [28], and our understanding of MDSC generation during aging remains limited [22]. However, our findings suggest that in vitro induction of MDSCs from the BM is reduced with aging and, this may be due to an impaired ability of myeloid precursors to proliferate, as observed in T cells [14], or could be due to an impaired response to cytokine stimuli [29]. Therefore, the increased peripheral MDSC levels observed in cancer patients might be dependent on cancer-associated mechanisms, rather than on the aging process itself.