Subpopulations of M-MDSCs from mice infected
by an immunode
ﬁciency-causing retrovirus and their differential
suppression of T- vs B-cell responses
Megan A. O
, Whitney W. Fua
, Kathy A. Greena
, William R. Greena,b,n aDepartment of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
bNorris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
a r t i c l e i n f o
Article history: Received 5 June 2015 Returned to author for revisions 27 July 2015
Accepted 31 July 2015 Available online 27 August 2015 Keywords:
Myeloid derived suppressor cells Retrovirus
a b s t r a c t
Monocytic (CD11bþLy6G7 /LoLy6Cþ) myeloid derived suppressor cells (M-MDSCs) expand following murine retroviral LP-BM5 infection and suppress ex vivo polyclonal T-cell and B-cell responses. M-MDSCs 3 weeks post LP-BM5 infection have decreased suppression of T-cell, but not B-cell, responses and alterations in the degree of iNOS/NO dependence of suppression. M-MDSCs from LP-BM5 infected mice were sorted into four quadrant populations (Ly6C/CD11b density): all quadrants suppressed B-cell responses, but only M-MDSCs expressing the highest levels of Ly6C and CD11b (Q2) signiﬁcantly suppressed T-cell responses. Further subdivision of this Q2 population revealed the Ly6Cþ /HiM-MDSC subpopulation as the most suppressive, inhibiting T- and B-cell responses in a full, or partially, iNOS/NO-dependent manner, respectively. In contrast, the lower/moderate levels of suppression by the Ly6Cþ /Lo and Ly6Cþ /MidM-MDSC Q2 subpopulations, whether versus T- and/or B-cells, displayed little/no iNOS dependency for suppression. These results highlight differential phenotypic and functional immuno-suppressive M-MDSC subsets in a retroviral immunodeﬁciency model.
Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Myeloid derived suppressor cells (MDSCs) are a heterogeneous cell population, which dampens T-cell mediated immune responses against several types of cancer, including melanoma, lung, mammary, and colon carcinomas (Marigo et al., 2008; Sica and Bronte, 2007; Solito et al., 2014). More recently, MDSCs have been identiﬁed as altering immune responses to viral infections, including cytomegalo-virus (CMV), hepatitis C cytomegalo-virus (HCV), human immunodeﬁciency virus (HIV), simian immunodeﬁciency virus (SIV), and murine LP-BM5 retrovirus (Daley-Bauer et al., 2012; Garg and Spector, 2014; Green et al., 2013; Qin et al., 2013; Sui et al., 2014; Tacke et al., 2012). In murine models, MDSCs are classically deﬁned as CD11bþGR-1þ, with
further subdivision into two subsets: CD11bþLy6G7/LoLy6Cþ /Hi monocytic-MDSCs (M-MDSCs) and CD11bþLy6Gþ /HiLy6C7/Lo granu-locytic MDSCs (G-MDSCs) (aka polymorphonuclear (PMN)-MDSCs) (Sica and Bronte, 2007; Gabrilovich and Nagaraj, 2009; Condamine and Gabrilovich, 2011). MDSCs display varying co-expression of other surface markers including, but not limited to, TLR-4, F4/80, Fc
γRIII/II (CD16/32), IL-4R
α(CD124), and/or CD115 (Marigo et al., 2008; Sica and
Bronte, 2007; Green et al., 2013; Gabrilovich and Nagaraj, 2009). Murine MDSCs may also express different combinations of chemokine receptors, especially CCR2, CXCR4, CXCR2, and/or CX3CR1, which are
important for egress of MDSCs out of the bone marrow and/or migration to tumor sites or sites of infection (Brandau et al., 2011; Hart et al., 2014; Highﬁll et al., 2014; Huang et al., 2006; Lesokhin et al., 2012; Obermajer et al., 2011; Sawanobori et al., 2008; Shi and Pamer, 2011; Zhu et al., 2014; Zhao et al., 2015). MDSCs suppress T-cell responses by secretion of nitric oxide (NO), arginase-1 (Arg-1), reactive oxygen species (ROS), and other mechanisms. Arguably, M-MDSCs express higher levels of NO and lower levels of ROS, while G-MDSCs produce higher levels of ROS, along with lower levels of NO, with both subsets capable of producing some Arg-1 (Gabrilovich and Nagaraj, 2009; Goh et al., 2013).
In recent years, the earliest descriptions of MDSCs in retroviral infections were focused in the HIV, SIV, and LP-BM5 murine systems (Garg and Spector, 2014; Green et al., 2013; Qin et al., 2013; Sui et al., 2014; Vollbrecht et al., 2012). In recent studies, HIV-infected patients had increased MDSC frequency, which correlated with exacerbated HIV disease (increased viral load and decreased CD4 T cell counts) (Qin et al., 2013; Vollbrecht et al., 2012; Bowers et al., 2014). MDSC frequency decreased after initiation of highly active antiretroviral therapy (HAART), consis-tent with an active role of MDSCs during HIV infection (Qin et al., 2013; Vollbrecht et al., 2012). Ex vivo enrichment from the Contents lists available atScienceDirect
0042-6822 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
nCorresponding author at: Department of Microbiology and Immunology, Geisel
School of Medicine at Dartmouth, Lebanon, NH, USA.
peripheral blood mononuclear cells (PBMCs) of HIV-infected patients revealed two MDSC phenotypes: granulocytic-like MDSCs (Vollbrecht et al., 2012); and monocytic-like MDSCs (Qin et al., 2013), both capable of suppressing in vitro T-cell responses. It remains unclear which mechanism(s) are used by these MDSCs to suppress immune responses ex vivo and which, if any, differential cell-surface or mechanistic MDSC phenotype(s) are contributing to in vivo retrovirus-induced immunodeﬁciency. Additional studies are warranted in systems, such as murine retroviral models, which allow well-controlled experimentation to examine MDSCs in retroviral systems.
LP-BM5 retrovirus-induced murine AIDS (MAIDS) causes pro-found and progressive immunodeﬁciency in mice, resulting in early activational events, such as splenomegaly, lymphadenopathy, and hypergammaglobulinemia (Aziz et al., 1989; Cerny et al., 1990; Klinman and Morse, 1989; Morse et al., 1989; Mosier et al., 1985), similar to human HIV/AIDS. LP-BM5-induced disease also features severe deﬁciencies of the responsiveness of T and B cells, an increased incidence of B-cell lymphomas, and higher susceptibility to opportunistic infections (Cerny et al., 1990; Mosier et al., 1985). Recently, following LP-BM5 infection, our lab identiﬁed an expanded CD11bþ suppressive M-MDSC population, which was further characterized as Ly6G7/LoLy6Cþ (Green et al., 2013). A proportion of these M-MDSCs co-express Fc
γRIII/II, F4/80, and/or TLR4 (Green et al., 2013). M-MDSCs from LP-BM5 infected mice suppress T-cell responses in an iNOS-dependent, indoleamine 2,3-dioxygenase (IDO)-independent, and arginase-independent manner (Green et al., 2013; O’Connor and Green, 2013a). In addition, and
distinctively, these M-MDSCs suppress B-cell responsiveness, in part due to an iNOS-dependent mechanism (Green et al., 2013).
The goal of this study was to further deﬁne the cell-surface phenotype and function of M-MDSCs from LP-BM5 infected mice, utilizing sorted M-MDSC populations and subpopulations. We show differential suppression of T- and/or B-cell responses by distinct M-MDSC subsets, which in some cases suppress via different mechanisms. These studies provide a further under-standing of these murine LP-BM5-derived M-MDSCs and may have broad implications, especially for understanding how MDSCs contribute to immunodeﬁciency and retroviral pathogenesis dur-ing SIV/HIV retroviral infections.
M-MDSCs derived earlier in the course of LP-BM5 infection exhibit decreased suppression of T-cell responses
Our lab has previously described retrovirus infection associated monocytic MDSCs (M-MDSCs) capable of suppressing polyclonal T-, and a novelﬁnding, B-cell responsiveness, following LP-BM5 infection of B6 mice (Green et al., 2013), but it remains unknown when these cells obtain their suppressive capabilities. To give insight into the functional development of these M-MDSCs we compared M-MDSCs from LP-BM5 infected mice 5 weeks post infection (wpi), the typical timepoint of M-MDSC assessment, versus an earlier 3 wpi timepoint.
Fig. 1. M-MDSCs derived earlier in the course of LP-BM5 infection exhibit decreased suppression of T-cell responses. M-MDSCs were isolated from LP-BM5 infected mice 3 or 5 wpi, co-cultured with naïve responder cells stimulated with LPS (B-Cell) (A) or anti-CD3/CD28 (T-cell) (C) in a standard suppression assay, as previously described (Green et al., 2013). Histograms (A–E) represent the means of triplicate samples with standard deviations of a representative experiment, of a total of at least three independent experiments. The percent suppression of B-cell (A) or T-cell (C) responsiveness by M-MDSCs is depicted for both standard co-cultures and co-cultures with the addition of the iNOS inhibitor, L-NIL. The effect on suppression (blockade) by L-NIL addition on B-cell (B) versus T-cell (D) responsiveness is compared. (E) Supernates derived from parallel suppression assays in which responder cells were stimulated with anti-CD3/CD28, in the absence or presence of M-MDSCs, were assessed for nitric oxide (NO) using the Griess reagent for nitrite.npo0.05,nnpo0.01.
M-MDSCs were standardly isolated from LP-BM5 infected C57BL/6 (B6) mice, by Ly6G-depletion, followed by CD11b-enrichment (see Materials and methods section), from pooled splenocytes 3 or 5 wpi, and used in a suppression assay, as previously described (Green et al., 2013). Brieﬂy, M-MDSCs were co-cultured with naïve responder cells stimulated with polyclonal T- (anti-CD3/CD28) or B-cell (LPS) activators for 3 days. Suppres-sion was calculated from changes in proliferation of responder cells in the absence or presence of M-MDSCs, as measured by [3H]
thymidine incorporation. M-MDSCs from 3 wpi mice displayed nearly equivalent suppression of B-cell responses (Fig. 1A), as compared to M-MDSCs from 5 wpi mice. Suppression of B-cell responses was signiﬁcantly blocked (up to 40%) when cultures were treated with L-NIL, an iNOS-inhibitor (Fig. 1A and B), as we previously described with another iNOS inhibitor (Green et al., 2013), with no signiﬁcant difference of L-NIL deﬁned iNOS/NO dependency in cultures containing M-MDSCs from 3 versus 5 wpi mice.
In contrast, M-MDSCs from 3 wpi mice had a signiﬁcant impair-ment, but not complete abrogation, of the suppression T-cell responses, as compared to M-MDSCs from 5 wpi mice (Fig. 1C). Addition of the iNOS inhibitor completely blocked the suppression of T-cell responses by M-MDSCs from 5 wpi mice, but surprisingly, did not block the lower level of suppression of T-cell responses by M-MDSCs from 3 wpi mice (Fig. 1C and D). These data indicated that 3 wpi M-MDSCs suppressed T-cell responses in an iNOS-independent manner, in contrast to the iNOS-dependent manner of 5 wpi M-MDSCs. As measured by Griess reagent, NO produced in suppression assays containing M-MDSCs from 3 wpi mice, was signiﬁcantly lower than cultures containing M-MDSCs from 5 wpi mice (Fig. 1E). There-fore, the amount of NO produced by M-MDSCs, in vitro, from 3 wpi mice may not be sufﬁcient for an iNOS-dependent mechanism of suppression of T-cell responses. These collective data indicated that suppression of T- versus B-cell responses, and/or suppression via an iNOS/NO dependent mechanism, by M-MDSCs may proceed at different kinetic rates post infection, suggesting that subpopulations within these heterogeneous cells may display differential suppres-sion of T- and B-cell responses.
M-MDSC populations, as deﬁned by ﬂow cytometric quadrant analysis of CD11b vs Ly6C expression, differentially suppress B- and T-cell responsiveness
M-MDSCs from LP-BM5 infected mice display substantial hetero-geneity with regard to Ly6C expression (Green et al., 2013), and this heterogeneity may be associated with the differential suppression observed inFig. 1. M-MDSCs isolated from 5 wpi mice were sorted (seeMaterials and methods section) into indicated quadrant popula-tions, delineated by isotype controls, present at the following mean proportions: Ly6C7/Lo CD11bþ (Quadrant 1, Q1: 8.2%71.7%); Ly6CþCD11bþ (Quadrant 2, Q2: 61.0%76.2%); Ly6C7/LoCD11b7/Lo
(Quadrant 3, Q3: 18.2%73.4%); and Ly6CþCD11b7/Lo (Quadrant 4, Q4: 12.6%72.4%) cells (Fig. 2A). When sorted M-MDSCs were tested in suppression assays, as described above, at equivalent R:S ratios, cells from all four quadrants suppressed B-cell responsiveness (Fig. 2B), with no signiﬁcant difference in suppression between quadrant populations. In contrast, only sorted M-MDSCs in Q2 signiﬁcantly inhibited T-cell responsiveness (Fig. 2C), with the other M-MDSC quadrants (Q1, Q3, and Q4) contributing to little/no (depending on the overall activity levels of the individual experi-ments) suppression of the T-cell response. The possibility of FoxP3þT regulatory (Tregs) cells contributing to the observed suppression of B-and/or T-cell responses was very unlikely due to their infrequency (o0.5%) in enriched M-MDSC preparations. Regarding suppression of T cells in particular, the prototypic target of Treg cells, two additional ﬁndings argue against this possibility: 1) the essentially complete
iNOS/NO dependency of the mechanism of suppression by unsorted MDSCs and 2) the somewhat preferential distributions of the few contaminating CD4þFoxP3þ Tregs in quadrants which had little/no suppression of T-cell responses (Q1, Q3, and Q4). To provide insight into the total suppressive capacity of the M-MDSC quadrants, suppression was adjusted for the proportionality of cells (Fig. 2A) and Q2 cells were clearly the major contributor of the suppression of both B-, and especially T-cell, responsiveness (Fig. 2D).
For suppression of B-cell responses, Ly6Cþ /HiM-MDSCs constitute the single-most suppressive subpopulation, whereas suppressive function cannot be detected for the Ly6Cþ /Midsubpopulation
As cells in Q2 contributed substantially to the suppression of B-cell responses and T-B-cell responses (Fig. 2D), and because of the varied Ly6C density of Q2 (Fig. 2A), we further sub-divided these cells into three independent subpopulations, present at the fol-lowing average proportions: Ly6Cþ /Lo (6.4%70.7%), Ly6Cþ /Mid
(30.6%77.3%), and Ly6Cþ /Hi (20.6%71.6%) M-MDSCs (Fig. 3).
Having only one remaining sorting gate available, cells from the remaining three quadrants (Q1, Q3, and Q4), which display varying (lower) levels of both Ly6C and/or CD11b expression, and all which displayed suppression of B-cell responses (Fig. 2B), were sorted into a single population: Q1,3,4 (39.0%76.2%) (Fig. 3).
The suppression of polyclonal B-cell responses, utilizing the sorting scheme of M-MDSC Q2 subpopulations as depicted in Fig. 3, was ﬁrst examined. M-MDSCs from the pooled Q1,3,4 populations and the Ly6Cþ /Lo Q2 subpopulation had low/inter-mediate, but signiﬁcant, levels of suppression of B-cell respon-siveness, compared to sorted Ly6Cþ /Mid M-MDSCs (Fig. 4A). Unexpectedly, suppression of B-cell responsiveness by the Ly6Cþ /Midsubpopulation was consistently undetectable (Fig. 4A). In contrast, the Ly6Cþ /HiM-MDSC subset displayed potent inhibi-tory activity, signiﬁcantly suppressing B-cell responses at higher levels compared to any other subpopulation (Fig. 4A). Although the frequency of the Ly6Cþ /Hisubpopulation was a minority, about 20% of all M-MDSCs (Fig. 3) and about 35% of Q2 M-MDSCs (Interpretation from Fig. 3), it was a major contributor to the suppression of B-cell responsiveness (Fig. 4B).
To assess potential differential use of the iNOS/NO mechanism to suppress polyclonal B-cell responsiveness, M-MDSC subpopula-tions (except the Ly6Cþ /Lo M-MDSCs due to limited cell yield) were treated with the iNOS inhibitor L-NIL in standard suppres-sion assays (Fig. 4C). As a control, as previously described (Green et al., 2013), iNOS inhibition lead to signiﬁcant, but partial blockade of the suppression of B-cell responses by unsorted M-MDSCs (Fig. 4C and D). There was also signiﬁcant partial blockade (up to one third) of suppression by Ly6Cþ /Hicells (Fig. 4C and D). In sharp contrast, little/no iNOS-dependent suppression was con-sistently observed for the pooled Q1,3,4 M-MDSC population (Fig. 4C and D). Ly6Cþ /Midcells were unable to suppress B-cell responses (Fig. 4A), and therefore there was no“suppression” to be blocked. Nonetheless as an internal control, addition of L-NIL to a co-culture of responder cells and this M-MDSC subpopulation had no effect on responder cell proliferation (Fig. 4C and D). Collec-tively, these data indicated that Q2 M-MDSC subpopulations had differential suppressive activities, and exhibited varying levels of iNOS-dependency for suppression of B-cell responses.
The Ly6Cþ /HiM-MDSC subpopulation contributes to the majority of the suppression of T-cell responsiveness
In direct comparison to unsorted M-MDSCs, which exhibited strong levels of suppressive activity (80% inhibition), the suppression of T-cell responses by these same M-MDSC subpopulations (Fig. 3) was examined (Fig. 5). The pooled Q1,3,4 M-MDSC populations had no/low
levels of suppression of T-cell responses (Fig. 5A), and corroborated the results observed from the individual quadrant analyses (Fig. 2C). On an equivalent R:S ratio basis, the Ly6Cþ /Lo and Ly6Cþ /Mid M-MDSC subpopulations provided low/intermediate levels of suppression of T-cell responses (Fig. 5A), with no signiﬁcant difference in suppression between these two cell subsets overﬁve independent experiments. However, given the substantially higher distribution of the Ly6Cþ /Mid subpopulation (50% of Q2 M-MDSCs; calculation fromFig. 3), the total suppressive activity of the Ly6Cþ /Midsubpopulation was greater than that of the Ly6Cþ /Lo subpopulation (Fig. 5B). In contrast, the Ly6Cþ /Hicells were signiﬁcantly more suppressive of T-cell respon-siveness, compared to any other single M-MDSC subset, as demon-strated using multiple R:S ratios within a given experiment (Fig. 5A and B). Therefore, even when again taking into account the minority frequency (35% of Q2 M-MDSCs; calculation fromFig. 3) of this subpopulation, Ly6Cþ /Hicells were the single-most suppressive M-MDSC subpopulation of both T- (Fig. 5) and B-cell (Fig. 4) responses.
As previously described (Green et al., 2013), essentially all the suppression of T-cell responses by unsorted M-MDSCs was blocked
by the addition of L-NIL (Fig. 5C and D). Similarly, the suppression of T-cell responses by the highly active Ly6Cþ /HiM-MDSC subpopulation was also almost completely blocked by L-NIL (Fig. 5C and D) and indicated a very strong iNOS-dependence for suppression of T-cell responses. In contrast, the suppression of T-cell responses by the pooled Q1,3,4 and Ly6Cþ /Mid M-MDSCs was essentially iNOS-independent (Fig. 5C and D). The infrequency of FoxP3þTregs within all M-MDSC subpopulations studied, as described above, was incon-sistent with any substantial contribution to suppression. Thus, although all three M-MDSC Q2 subpopulations could suppress T-cell responses, to varying degrees, the Ly6Cþ /HiM-MDSCs subpopulation was by far more suppressive and its inhibitory activity almost completely iNOS-mediated.
The highly suppressive Ly6Cþ /HiM-MDSC subpopulation displays increased F4/80 expression
M-MDSCs from LP-BM5 infected mice co-express established MDSC markers, including TLR-4, F4/80, Fc
γRIII/II (Green et al., 2013),
Fig. 2. M-MDSC populations, as deﬁned by ﬂow cytometric quadrant analysis of CD11b vs Ly6C expression, differentially suppress B- and T-cell responsiveness. M-MDSCs were enriched from LP-BM5 infected mice, 5-6 wpi. (A) A representativeﬂow cytometry dot plot along with the quadrant gating scheme and frequencies of quadrant populations, is depicted: Ly6C7 /LoCD11bþ (Quadrant 1, Q1), Ly6CþCD11bþ(Quadrant 2, Q2); Ly6C7 /LoCD11b7 /Lo(Quadrant 3, Q3), and Ly6CþCD11b7 /Lo
(Quadrant 4, Q4) cells. Histogram (A) depicts the means and standard deviations for seven independent experiments. The percent suppression of B-cell (LPS) (panel B) and T-cell (ConA) (panel C) responsiveness by sorted M-MDSC quadrant populations, versus unsorted (U) M-MDSCs, is given. Histograms (B and C) represent means of triplicate samples with standard deviations, and are representative of four independent experiments, with similar patterns of results. No signiﬁcance differences in the suppression of B-cell responses were found between the quadrant subpopulations (B). In C, po0.01 for: § versus Q1; ǂ versus Q3; # versus Q4. (D) Overall contribution to suppression by each sorted M-MDSC quadrant population, as adjusted for the frequency of enriched M-MDSCs distributed into that quadrant (from A). Pie charts represent the means of four independent experiments.
α(unpublished data). Identiﬁcation of an additional MDSC marker(s) to further phenotype the described ﬂow-sorted M-MDSC subpopulations, could aid in understanding the functional underpinnings of their differential suppression of T versus B cell responsiveness. To fairly compare several MDSC surface markers over multiple experiments, we looked at co-expression of MDSC markers on the M-MDSCs subpopulations, relative to the expression levels on the unsorted M-MDSCs as a whole. Co-expression of TLR-4, Fc
γRIII/II, or IL-4R
αwas similar between all Q2 M-MDSC subpopulations (Fig. 6A), which displayed their
deﬁning differential level of Ly6C expression. In contrast, F4/80 co-expression was signiﬁcantly higher on the Ly6Cþ /Hisubpopulation,
as compared to the Ly6Cþ /Lo and Ly6Cþ /Mid subpopulations, with no signiﬁcant difference between these latter subpopulations (Fig. 6A).
In Fig. 1 we demonstrated decreased suppression of T-cell responses by M-MDSCs from 3 wpi versus standard 5 wpi mice, and wondered if M-MDSCs from these two timepoints were phenotypically distinct. First, the relative percentage of the Q2 M-MDSC subpopulations (Ly6Cþ /Lo, Ly6Cþ /Mid, and Ly6Cþ /Hi) (Fig. 6B),
Fig. 3. M-MDSC Q2 subpopulation gating scheme. M-MDSCs were enriched from LP-BM5 infected mice. A representativeﬂow cytometry dot plot, along with the Q2 subpopulation gating scheme, is depicted to deﬁne, based on Ly6C expression, Q2 subpopulations: Ly6Cþ /Lo, Ly6Cþ /Mid, and Ly6Cþ /HiM-MDSCs. Quadrants Q1, Q3, and Q4
were sorted into a common pool for testing as a single combined population (Q134). The frequencies of M-MDSC subpopulations are indicated by histogram means with standard deviations for eight independent experiments.
Fig. 4. For suppression of B-cell responses, Ly6Cþ /HiM-MDSCs constitute the single-most suppressive subpopulation, whereas suppressive function cannot be
detected for the Ly6Cþ /Midsubpopulation. Unsorted (U) or sorted M-MDSC subpopulations (gating scheme inFig. 3) were co-cultured with naïve responder cells
stimulated with LPS in a standard suppression assay. All histograms (A, C, D) represent the means of triplicate samples, with indicated standard deviations and are from a representative experiment, with similar patterns of results, of a total of four (A) and three (C and D) independent experiments. (A) Suppression of B-cell responses by unsorted and sorted M-MDSC subpopulations is given. po0.01: § versus Q134 population; ǂ versus the Ly6Cþ /Lo; or# versus Ly6Cþ /Midsubpopulations. (B) Overall
suppressive contribution of each Q2 subpopulation, adjusted for the frequency of M-MDSCs distributed into quadrant 2 (fromFig. 3). The pie chart depicts the means of four independent experiments. (C and D) Suppression assays were standard or included the addition of the iNOS inhibitor, L-NIL. The amount of suppression (C) and L-NIL blockade (D) are given. No detectible suppression indicated (ND), and not applicable (NA).nnpo0.01, (ns) no statistical difference.
and the overall expression of Ly6C (data not shown), were similar between 3 wpi and 5 wpi M-MDSCs. In addition, the entire expres-sion of TLR4 and Fc
γRIII/II of MDSCs and expression within M-MDSC subpopulations was also similar between M-M-MDSCs from 3 and 5 wpi mice (data not shown). F4/80 expression of the entire M-MDSC population was similar between 3 wpi and 5 wpi M-MDSCs (data not shown), but again as seen inFig. 6A, increased Ly6C expression was strongly associated with increased F4/80 expression also on 3 wpi M-MDSCs (Fig. 6C).
Unfortunately, the functions of Ly6C and F4/80, especially in the context of MDSC development and function, remain poorly deﬁned (Gordon et al., 2011; Lee et al., 2013; Lin et al., 2005, 2010), therefore other phenotypic marker(s) associated with a
functional role of MDSCs were needed to further deﬁne M-MDSC subpopulations from LP-BM5 infected mice
Chemokine receptor expression further delineates M-MDSCs from LP-BM5 infected mice
To gain insight into the function and/or development of the M-MDSCs, we examined the phenotypic expression of chemokine receptors (CCR2, CX3CR1, CXCR2, and CXCR4) reported to be variably
present on MDSC populations in other systems and involved in MDSC egress into circulation and/or recruitment to tumor sites and sites of infection (Hart et al., 2014; Lesokhin et al., 2012; Zhu et al., 2014; Zhao et al., 2015; Gallina et al., 2006; Talmadge and Gabrilovich, 2013;
Fig. 5. The Ly6Cþ /HiM-MDSC subpopulation contributes to the majority of the suppression of T-cell responsiveness. Unsorted (U) or sorted M-MDSC subpopulations
(gating scheme inFig. 3) were co-cultured with naïve responder cells stimulated with anti-CD3/CD28, at multiple responder:suppressor (R:S) ratios (indicated below each graph). The dashed lines separate the standard R:S 3:1 from M-MDSC titration to yield R:S 6:1 and 9:1 ratios. All histograms (A, C, D) represent the means of triplicate samples, with indicated standard deviations, and are from a representative of a total ofﬁve (A) or four (C and D) independent experiments. (A) Suppression of T-cell responses by unsorted and sorted M-MDSC subpopulations. po0.01: § versus Q134 population; ǂ versus the Ly6Cþ /Lo; or# versus Ly6Cþ /Midsubpopulation. (B) Overall
contribution to suppression by Q2 subpopulations, adjusted for proportionality of cells (fromFig. 3). The pie chart represents the mean ofﬁve independent experiments. (C) Suppression assays were standard or included the addition of the iNOS inhibitor, L-NIL. The amount of suppression (C) and L-NIL blockade (D) are given. No detectible L-NIL blockade indicated (ND).nnpo0.01, (ns) no statistical difference.
Fig. 6. The highly suppressive Ly6Cþ /HiM-MDSC subpopulation displays increased F4/80 expression. (A) The meanﬂuorescent intensity (MFI) of TLR4, FcγRIII/II, IL-4Rα, and F4/80 expression by M-MDSC subpopulations (gating scheme fromFig. 3) were normalized to the expression by all enriched M-MDSCs. Histograms represent the relative MFI means of at leastﬁve independent experiments with standard deviations. po0.01: ǂ versus Ly6Cþ /Lo;#versus Ly6Cþ /Mid. (B) Representativeﬂow cytometry dot plots
and average frequencies of M-MDSC subpopulations from 3 wpi or 5 wpi mice. No signiﬁcant differences between 3 wpi and 5 wpi M-MDSC subpopulations were observed. (C) The MFI values of F4/80 expression of M-MDSC subpopulations, from 3 wpi and 5 wpi mice, were normalized to all enriched 5 wpi M-MDSCs. Histograms (B and C) represent the means of at least three independent experiments with standard deviations: po0.01: ǂversus Ly6Cþ /Lo;#versus Ly6Cþ /Mid.
Umemura et al., 2008). M-MDSCs from LP-BM5 infected mice expressed varying percentages of CCR2, CX3CR1, and CXCR4, but
essentially not CXCR2 (Fig. 7A). CCR2, CX3CR1, and CXCR4 (Fig. 7B),
but not CXCR2 (data not shown), were also observed on all three M-MDSC Quadrant 2 subpopulations, albeit at varying frequencies. The Ly6Cþ /HiM-MDSC subpopulation exhibited the highest percentage of positive cells and expression on a per cell basis (MFI) of CCR2 and CX3CR1 (Fig. 7B), compared to the other two M-MDSC Q2
subpopula-tions. The MFI expression of CXCR4 was also higher on the Ly6Cþ /Hi M-MDSCs compared to the Ly6Cþ /Loand Ly6Cþ /Mid M-MDSC sub-populations (Fig. 7B). InFig. 6A there was no observed phenotypic difference between the Ly6Cþ /Loand Ly6Cþ /MidM-MDSC subpopula-tions, despite their differential suppression of B-cell responses (Fig. 4A). Assessment of chemokine receptor expression by these subpopulations, however, revealed that the Ly6Cþ /MidM-MDSC sub-population displayed increased CCR2 expression on a per cell (MFI) basis (Fig. 7B), compared to the Ly6Cþ /LoM-MDSC subpopulation.
To address the overlap of expression of chemokine receptors within each M-MDSC subpopulation, weﬁrst focused on M-MDSCs expressing only a single chemokine receptor (Fig. 7C). The Ly6Cþ /Lo and Ly6Cþ /Mid M-MDSC subpopulations had larger proportions of cells expressing no chemokine receptors or just the CXCR4 chemokine receptor compared to the Ly6Cþ /HiM-MDSC subpopulation (Fig. 7C). In contrast, the very high level of each individual chemokine receptor frequency in the Ly6Cþ /Hi M-MDSC subpopulation, observed in
Fig. 7B, suggested extensive chemokine receptor co-expression. Indeed, the Ly6Cþ /HiM-MDSC subpopulation expressed the highest proportion of all three chemokine receptors, CCR2þCX3CR1þ
CXCR4þ, and the lowest frequency of CCR2þCXCR4þ dual-expressing cells, all compared to the other two M-MDSC subpopula-tions (Fig. 7D). Collectively these results provide further phenotypic distinction between these three M-MDSC subpopulations—perhaps of
relevance to the ability of these subpopulations to suppress T-cell versus B-cell targets with differential efﬁciencies.
In these studies, we report differential suppression of T- and B-cell responsiveness by quadrant analysis (Q) populations and Q2 sub-populations of M-MDSCs from LP-BM5 retrovirus infected mice, as delineated by the densities of CD11b and/or Ly6C expression. First, M-MDSCs from all four quadrant populations suppressed B-cell respon-siveness, but only cells from Quadrant 2 signiﬁcantly suppressed T-cell responses (Fig. 2). Upon analysis of this Q2 population, a highly suppressive Ly6Cþ /Hi M-MDSC subpopulation was identiﬁed. The Ly6Cþ /HiM-MDSC subpopulation was signiﬁcantly more suppressive of both T- and B-cell responsiveness, as compared to any other single Q2 subpopulation (Figs. 4 and 5), and had the highest co-expression of cell surface F4/80 (Fig. 6A), CCR2, and CX3CR1 (Fig. 7B). Second,
suppression of B-cell responses by the Ly6Cþ /MidM-MDSC subpopu-lation was undetectable (Fig. 4), although these cells consistently and substantially suppressed T-cell responses (e.g. 34%, Fig. 5B) and comprised30% of all enriched M-MDSCs (Fig. 3). The suppression of T-cell responses by the Ly6Cþ /Mid M-MDSC subpopulation was essentially independent of iNOS/NO (Fig. 5), which served to further distinguish this subpopulation. Other observations made here were also in keeping with a dichotomy, under certain circumstances, of MDSC suppression of T- versus B-cell responses. For example we identiﬁed several M-MDSC populations with little/no suppression of T-cell responses: Q1, Q3, and Q4 (Fig. 2C) and pooled Q1,3,4 (Fig. 5A) populations, whereas these same M-MDSCs suppressed B-cell respon-siveness. Additionally, using M-MDSCs derived from different kinetic stages of LP-BM5 retrovirus infection, it was determined that the
Fig. 7. Chemokine receptor expression further delineates M-MDSCs from LP-BM5 infected mice. (A and B) Representativeﬂow cytometry histograms and average frequencies, from four independent experiments, of CCR2, CX3CR1, CXCR2, and CXCR4 on enriched M-MDSCs (A) and M-MDSC subpopulations (gating scheme fromFig. 3)
(B). (B) The meanﬂuorescent intensities (MFI) of CCR2, CX3CR1, and CXCR4 expression by M-MDSC subpopulations were normalized to the expression by all enriched
M-MDSCs. (C and D) The average frequency of expression of (C) none or one only, or (D) two or three chemokine receptors by M-MDSC subpopulations. Histograms (A–D) represent the means of four independent experiments with standard deviations. Fluorescence minus one (FMO). po0.05: ǂversus Ly6Cþ /Lo;#versus Ly6Cþ /Mid; §versus
ability to suppress B-cell (versus T-cell) responses developed earlier post infection (Fig. 1 A and C), despite no observed phenotypic differences among prominent MDSC-associated markers, including expression of F4/80 (Fig. 6B and C).
Our results thus identiﬁed a Ly6Cþ /HiM-MDSC subpopulation,
robustly suppressive of both T- and B-cell responsiveness, and displaying high levels of both Ly6C and F4/80 (Fig. 6A). Unfortu-nately, the functions of Ly6C and F4/80 remain poorly deﬁned, and to our knowledge, there is no agreed-upon ligand for F4/80 (Gordon et al., 2011; Lee et al., 2013; Lin et al., 2005, 2010). It is possible that the differential expression of Ly6C by M-MDSC populations and subpopulations from LP-BM5 infected mice may allow further subdivision on the basis of expression of Ly6C1 and/ or Ly6C2, which currently are indistinguishable using available antibodies (Lee et al., 2013). Macrophages from F4/80 knockout mice display normal macrophage development and no overt impairment of macrophage functions (Lin et al., 2005; Schaller et al., 2002). However, F4/80 may be important in the develop-ment and/or function of M-MDSCs, particularly the Q2 Ly6Cþ /Hi subpopulation, during LP-BM5 retrovirus infection, but presently this possibility remains unclear.
A variety of factors produced by tumor cells and/or activated T-cells, including, but not limited to GM-CSF, IFN
γ, 10, 6, IL-12, and IL-13, are implicated in the activation and/or expansion of MDSCs for a variety of tumor models (Gabrilovich and Nagaraj, 2009). Relative to the LP-BM5 retroviral infection system, changes in the cytokine proﬁle in the ﬁrst week of infection, reported to be characterized by Th1 (IFN
γ) and Th2 cytokines (IL-15, IL-4, IL-10) (Gazzinelli et al., 1992), versus the predominately Th2 cytokines (IL-4, IL-10, IL-6) observed at later stages of infection (Gazzinelli et al., 1992), may inﬂuence M-MDSC accumulation and/or func-tion. Related to the results here, it is possible that a lack of“full” MDSC activation may explain why some M-MDSC populations/ subpopulations have a relatively decreased dependence on iNOS/ NO for suppression (Figs. 1D, 4D, and 5D), and consequently decreased suppression of T-cell responses (Figs. 1C,2C). Similarly, other local combinations of cytokines and other micro-environmental factors may explain the alternative deﬁciency in suppressing B-cell targets (Fig. 4A) versus sufﬁciency in suppres-sing T-cell targets (Fig. 5A) by the same Ly6Cþ /Mid M-MDSC subpopulation. Which retroviral and/or infection-induced host factors speciﬁcally drive M-MDSC expansion and/or activation in the LP-BM5 retroviral model remain unknown.
Several chemokine receptors are found on immature myeloid subsets, including MDSC subpopulations (Hart et al., 2014; Highﬁll et al., 2014; Zhu et al., 2014; Gallina et al., 2006; Umemura et al., 2008; Grifﬁth et al., 2014; Movahedi et al., 2008; Wang et al., 2015; Youn et al., 2008), and are important for migration of cells out of the bone marrow and/or into sites of tumor or infection (Highﬁll et al., 2014; Obermajer et al., 2011; Sawanobori et al., 2008; Shi and Pamer, 2011; Auffray et al., 2007; Huang et al., 2007). A very recent study of murine hepatocellular carcinoma compared the chemokine receptor gene expression proﬁles of splenic M-MDSC and G-MDSC (aka PMN-MDSCs) subsets (Zhao et al., 2015). These authors observed roughly equivalent CXCR4 expression by both M-MDSCs and G-MDSCs, but upregulated expression of CCR2 and CX3CR1 on
M-MDSCs, versus increased CXCR2 expression by G-MDSCs (Zhao et al., 2015). Our data further support these ﬁndings, as splenic M-MDSCs from LP-BM5 infected mice expressed CCR2, CX3CR1, and
CXCR4, but not CXCR2 (Fig. 7A), albeit to varying degrees on the different M-MDSC Q2 subpopulations. It was speciﬁcally of interest to observe that the highest chemokine receptor expression of CCR2 and CX3CR1 (Fig. 7B) and highest frequencies of CCR2þCX3CR1þCXCR4þ
cells (Fig. 7D) were observed for the highly suppressive Ly6Cþ /Hi M-MDSC subpopulation, compared to all other M-M-MDSCs subpopula-tions. Phenotypic expression of chemokine receptors further
distinguished the M-MDSC subpopulations (Fig. 7B and D), and these differences in chemokine receptor expression may help to elucidate the M-MDSC type(s) needed to suppress speciﬁc cellular targets (e.g. T-cell versus B-cell). Further studies are needed to determine whether these chemokine receptors play a role in M-MDSC suppressive activity (including cellular targets) per se and/or M-MDSC recruit-ment to the spleen in LP-BM5 infected mice.
In addition, our lab and others have published extensively on the cellular and molecular requirements for LP-BM5 induced pathogenesis, in the overall context that LP-BM5 co-opts immune cells and normal immune interactions as requisites for pathogen-esis, including the profound and broad immunodeﬁciency. CD4þT
cells and B-cells (Cerny et al., 1990; Mosier et al., 1985; Jolicoeur, 1991; Simard et al., 1997), interactions between CD40 on B-cells and CD40L on T-cells (Green et al., 1998, 1996, 2001, 2002), and recruitment of TNF receptor-associated factor (TRAF) to the TRAF6 binding site on the CD40 cytoplasmic tail (Green et al., 2004) are required for LP-BM5 pathogenesis. These results and others (Li and Green, 2007, 2006) have provided evidence for a central role of pathogenic CD4þT-cells in driving disease. M-MDSCs may expand and/or become activated as a consequence of these interactions and then reciprocally down modulate T cells and/or B cells—either those T/B cells necessary for LP-BM5 disease pathogenesis or as the target cells of the effector mechanisms of the ultimate severe immunodeﬁciency of advanced MAIDS.
MDSCs from HIV-infected patients and SIV-infected macaques suppress T-cell responses, but, to our knowledge, suppression of B-cell responses by these cells remains under reported (Garg and Spector, 2014; Qin et al., 2013; Sui et al., 2014; Vollbrecht et al., 2012). How HIV-infection-associated MDSCs suppress T-cell responses remains unclear: for example, one study reports an arginase-dependent, iNOS/ROS-independent mechanism (Qin et al., 2013), while another report implicates an arginase-inde-pendent, iNOS/ROS-dependent mechanism (Garg and Spector, 2014). In the context of LP-BM5 infection, we have previously shown, both by using multiple speciﬁc inhibitors and iNOS/ mice, that enriched but unsorted M-MDSCs suppress T- and B-cell responses in an arginase-independent, iNOS-dependent manner, albeit with varying levels of iNOS/NO dependency (Green et al., 2013). Here, the Ly6Cþ /HiM-MDSC Q2 subpopulation also exhib-ited complete iNOS-dependent suppression of T- (Fig. 5C and D), and partial iNOS-dependent suppression of B-cell (Fig. 4C and D), responses. In contrast, other M-MDSC subsets suppressed only T-cells (Ly6Cþ /Midsubpopulation) or B- and T- cells (Q1,3,4 popula-tion) with little dependence on iNOS/NO (Figs. 4C, D and5C, D).
How this apparent conundrum can be explained is unclear, but there are two broad possibilities. First, it may well be that the MDSC populations and subpopulations represent relatively stable phenotypic and functional subsets that either inherently, or after activation, display differential suppressive mechanisms, depend-ing on the susceptibility of the T- versus B-cell targets to the various M-MDSC inhibitory mechanism. Alternatively, or in addi-tion to, there may be important cross-regulaaddi-tion of immunosup-pressive mechanisms by certain populations/subpopulations of M-MDSCs. Cross-talk between tumor-associated MDSCs with macro-phages or dendritic cells (Ostrand-Rosenberg et al., 2012), and/or tumor tissue (Jayaraman et al., 2012), can enhance immunosup-pression. Therefore, it can be hypothesized that cross-talk between MDSC subsets may also exist to enhance the overall level of inhibitory activity, and the diversity of molecular mechanisms employed, to achieve more global immunosuppression in tumor and retroviral models. For example, MDSCs can release chemokines which enhance tumor growth by recruitment of Tregs to the tumor site (Schlecker et al., 2012; Luther and Cyster, 2001), but may also inﬂuence the recruitment of other MDSC subsets. Nitric oxide can be a direct regulator of gene expression
(Yamanaka and O’Connor, 2011) and cellular activation (Connelly et al., 2003), therefore one can speculate that NO production by one MDSC subpopulation may inﬂuence the gene expression and/ or post-transcriptional activation of another MDSC subset(s). This cross-regulation may inﬂuence the overall suppressive capacity of the latter MDSC subpopulation, as well as its mechanism(s) of suppression, similar to autocrine—paracrine iNOS polarization in macrophages (Rath et al., 2014). As an example, M-MDSCs when tested individually in isolation as in our sorting experiments, such as the Q1,3,4 populations or Ly6Cþ /Midsubpopulation, may lack exposure to exogenous factors from other M-MDSC subsets to drive iNOS-mediated suppression—factors that they are normally exposed to in the unsorted ex vivo M-MDSC preparation (and in vivo). This lack of normal M-MDSC cross-talk may explain why suppression by certain isolated M-MDSC subsets appear to be iNOS-independent (Figs. 4 and 5). Analogous or alternative kinds of cross-talk, but between M-MDSCs and polyclonally activated responder cells, such as IFN
γproduction by T-cells or IL-6 by B-cells, may also contribute to the differential iNOS-dependence in the M-MDSC suppression of T- and B-cells. For example, in some tumor systems, IL-6 (Chen et al., 2014) and IFN
γ(Gallina et al., 2006) can inﬂuence MDSC expression of arginase and iNOS.
The current understanding of suppression of B-cell responses by MDSCs is very limited to only a few recent reports (Green et al., 2013; Crook et al., 2015), but the threshold for susceptibility to iNOS-mediated (and iNOS-independent-mediated) suppression of T-cells versus B-cells may vary. The ability of a given M-MDSC population or subpopulation to produce sufﬁcient NO to achieve these thresholds may be regulated by arginine (L-Arg) levels available to the iNOS and/or arginase metabolic pathways within the M-MDSC. Several cross-inhibitory interactions have been reported to exist between these two pathways (Rath et al., 2014), and they can also synergize to generate reactive nitrogen and oxygen species (ROS), such as peroxynitrites and H2O2(Bronte
and Zanovello, 2005). Low levels of arginine within the cell, due to increased iNOS activity and/or decreased L-Arg uptake into the cell, can substantially inﬂuence these alternative metabolic path-ways. In addition, only the iNOS pathway can be“rescued,” during L-Arg unavailability, by cytosolic enzymes via conversion of citrul-line into arginine; in contrast, the arginase pathway lacks such a “rescue” mechanism (Rath et al., 2014). Additionally, low levels of arginine can promote the generation of ROS (Bronte and Zanovello, 2005). The single-most dominant mechanism of suppression of B-cell responses by unsorted M-MDSCs from LP-BM5 infected mice is iNOS/NO; but production of ROS, normally a minor contributor to suppression also occurs and may become a sig-niﬁcant compensatory mechanism in the absence of iNOS/NO (Rastad JL and Green WR. Manuscript in preparation). Therefore, arginase, ROS, and/or other alternate mechanisms may also con-tribute to suppression of T- versus B-cell responses by M-MDSC subpopulations, especially when iNOS/NO is limited.
One of most unexpected results from these studies was the consistent lack of detectable suppression of B-cell responses by the
Ly6Cþ /Mid subpopulation. This ﬁnding may complement two
intriguing and very recent observations from our lab: (1) enriched M-MDSCs from LP-BM5 infected mice, whose CD4 T-cell compart-ment has been depleted of FoxP3þT regulatory cells (nTregs) prior to their transfer intro TCR
α/recipients, in an adoptive transfer model of LP-BM5 induced disease, exhibit a signiﬁcant increase in the proportion of the Ly6Cþ /MidQ2 M-MDSC subpopulation; and (2) consistent with the ﬁndings here, M-MDSCs isolated from these nTreg-depleted mice display increased suppression of T-cell, but not B-cell, responsiveness (O’Connor et al., in press). Several possibilities may explain why the Ly6Cþ /Mid subpopulation is unable to suppress B-cell responses: (1) in isolation, these Ly6Cþ /Midcells may lack the required cross-regulation provided
by other cells types, including other M-MDSCs, to become active suppressors of B-cell responses; (2) an alteration in autocrine and/ or paracrine cytokine signaling could alter iNOS expression and/or other activated functions within the Ly6Cþ /Midcells, resulting in decreased NO production, and thus potentially increasing alter-native mechanism(s) of suppression; (3) the non-iNOS/NO mechanism(s) available to the Ly6Cþ /Midsubpopulation may only efﬁciently target a pathway or receptor found in/on T-cells, similar to CD3
ζdown regulation on T-cells following extracellular arginase-induced L-Arg depletion (Rodriguez et al., 2003); and/or (4) the Ly6Cþ /Mid subpopulation, as a newly deﬁned M-MDSC subset, may represent a stable population with a distinct immu-nosuppressive mechanism(s) and target cell speciﬁcity. Future analysis of how this Ly6Cþ /MidM-MDSC subpopulation differs— functionally, phenotypically, and/or epigenetically—from the other Q2 subpopulations, and its potential lineage relationships to these subsets, may be key to understanding why these Ly6Cþ /Mid M-MDSCs display differential suppression of T- versus B-cell responses.
In conclusion, our studies contribute to a more incisive under-standing of retrovirus-induced M-MDSCs, and of MDSCs in gen-eral, by highlighting distinct M-MDSC populations and subpopulations with partially overlapping, but also distinguishing, mechanisms of suppression. Future studies, identifying the genetic and metabolic processes driving M-MDSC effector function, may help deﬁne the differential involvement of iNOS/NO dependent and independent mechanisms of suppression. Further character-ization of T- versus B-cell target speciﬁcity by M-MDSC, as well as G-MDSC, subsets and their suppressive mechanism(s) utilized are needed to accurately identify and selectively target MDSCs, which contribute to the local and/or global immunosuppression that either promotes pathogenesis directly, and/or interferes with the development and maintenance of protective immunity in viral diseases, tumor systems, and autoimmunity.
Materials and methods Mice
C57BL/6 (B6, w.t.) mice were purchased from the National Cancer Institute (NCI; Bethesda, MD) or Charles River (Wilming-ton, MA) and housed in the Center for Comparative Medicine and Research (CCMR) at the Geisel School of Medicine at Dartmouth. All animal experiments were done with the approval of the Institutional Animal Care and Use Committee of Dartmouth College, and in conjunction with the Dartmouth CCMR, an AALAC approved animal facility.
LP-BM5 virus inoculation
LP-BM5 retrovirus was prepared as previously described (Green et al., 1998; Klinken et al., 1988). Mice were given an intraper-itoneal injection of 5 104plaque forming units (pfu) of LP-BM5 at
6–8 weeks of age. M-MDSCs were enriched from pooled spleno-cytes 3–6 weeks post infection (wpi).
Surface staining was performed as previously described (O’Connor and Green, 2013b). Cells were stained with FITC-, PerCP-, PE-, APC-, Pe-Cy7, APC-Cy7-, or Brilliant Violet 421- con-jugated antibodies and analyzed by a MACSQuantﬂow cytometer (Miltenyi Biotec; Auburn, CA) to detect the expression of murine TLR-4 (MTS510), IL-4R
α(CD124) (mIL4R-M1), F4/80 (BM8), Fc
γRIII/ II (93), CD11b (M1/70), Ly6C (HK1.4), GR-1 (RB6-8C5), CXCR4
(L276F12), CXCR2 (SA045E1), CX3CR1(SA011F11), or CCR2 (475301)
(BioLegend; San Diego, CA; BD Biosciences; San Jose, CA; R&D Systems; Minneapolis, MN). Positive gates were selected based on isotype orﬂuorescence minus one (FMO) controls and data were analyzed using FlowJo software (Tree Star, Inc.). All representative ﬂow cytometry dot plots and subsequent phenotypic analyses are of unsorted M-MDSCs.
M-MDSC isolation and cell sorting
M-MDSCs were negatively selected for Ly6G, then positively selected for CD11b using paramagnetic beads and subsequent MACS column puriﬁcation (Miltenyi Biotec) from pooled spleno-cytes (n¼3–10) of infected mice, as previously described (Green et al., 2013). Enriched cells, deﬁned by isotype controls, were sorted using a FACS Aria (Miltenyi Biotec) into serum coated tubes. Unsorted (U) and sorted M-MDSCs were used as suppressor cells in suppression assays.
Responders cells (R) from naïve w.t. mice were cultured with suppressor cells (S; M-MDSCs) at a responder:suppressor (R:S) ratio of 3:1, unless otherwise noted, with supplemented medium and a ﬁnal concentration of either 10
μg/ml lipopolysaccharide (LPS) (B-cell), 0.75
μg/ml concanavalin A (ConA) (T-cell), or 2.5
μg/ml anti-CD3 and 1
μg/ml CD28 (T-cell) stimulations, as previously described (Green et al., 2013). Suppression assays were left untreated or treated with 100
μM of the iNOS/NOS2 inhibitor L-NIL (Enzo Life Sciences) for the duration of the assay. Plates were pulsed with 1
thymidine in the last 6 h of incubation, and assessed at 72 h for thymidine incorporation. Percent suppression was calculated from the control response, as previously described (Green et al., 2013). NO production
NO production was measured from cell supernatants of sup-pression assays (described above) after 65–72 h of culture, using the Griess reagent for nitrite (Sigma-Aldrich; St. Louis, MO), as previously described (O’Connor et al., in press).
Statistical analyses between groups were tested using Student’s t-test, and the Holm–Bonferroni method was used to correct for multiple testing. (ns) indicates no statistical difference.
We thank David Leib, Edward Usherwood, Brent Berwin, Mary Jo Turk, David Mullins, Yina Huang, Cynthia Stevens, Jessica Rastad, Patrick Lizotte, and Shannon Steinberg for many helpful discus-sions and technical assistance.
This work was supported by Public Health Service Grants from the National Institutes of Health: CA-50157 and a pilot grant from P30 GM10345, both to WRG. MAO was supported by NIH T32 AI-007363 and through The American Association of Immunologists Careers in Immunology Fellowship Program. Flow cytometry was performed at the DartLab: Immunoassay and Flow Cytometry Shared Resource at the Geisel School of Medicine at Dartmouth and the Norris Cotton Cancer Center is supported in part by Core Grant CA23108 from the NIH and NIH/NIGMS COBRE P30GM103415. All animal experiments were done with the approval of the Institutional Animal Care and Use Committee of Dartmouth College, and in conjunction with the Dartmouth Center
for Comparative Medicine and Research, an AALAC approved animal facility. This institution has an Animal Welfare Assurance onﬁle (A3259-01).
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