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Myeloid-derived suppressor cells (MDSCs) were initially identified in humans and mice with cancer where they profoundly suppress T cell– and NK cell–mediated antitumor immunity. Inflammation is a central feature of many pathologies and normal physiological conditions and is the dominant driving force for the accumulation and function of MDSCs. Therefore, MDSCs are present in conditions where inflammation is present. Although MDSCs are detrimental in cancer and conditions where cellular immunity is desirable, they are beneficial in settings where cellular immunity is hyperactive. Because MDSCs can be generated ex vivo, they are being exploited as therapeutic agents to reduce damaging cellular immunity. In this review, we discuss the detrimental and beneficial roles of MDSCs in disease settings such as bacterial, viral, and parasitic infections, sepsis, obesity, trauma, stress, autoimmunity, transplantation and graft-versus-host disease, and normal physiological settings, including pregnancy and neonates as well as aging. The impact of MDSCs on vaccination is also discussed.

Human cytomegalovirus (CMV) is a widespread persistent herpes virus requiring lifelong immune surveillance to maintain latency. Such long-term interactions with the immune system may be associated with deleterious effects including immune exhaustion and senescence. Regarding the COVID-19 pandemic, we asked whether CMV-specific cellular and humoral activity could influence immune responses toward SARS-CoV-2 and/or disease severity. All adults with mild (n = 15) and severe (n = 14) COVID-19 were seropositive for anti-CMV IgG, but negative for IgM antibodies. Antibody titers did not correlate with COVID-19 severity. Six patients presented elevated frequencies of CMV-specific CD4 + and CD8 + T cells producing IFNγ, IL-17, and TNFα, designated as CMV high responders (hiT CMV). In comparison to low CMV responders, hiT CMV individuals exhibited higher frequencies of SARS-CoV-2-specific CD4 + IL-17 + and CD8 + IFNγ + , IL-17 + or TNFα + T cells. These results indicate that high frequencies of CMV-specific T cells may be associated with a SARS-CoV-2-reactive profile skewed toward Th17-dominated immunity.

PDF file - 69K, Mutation details.

PDF file - 81K, Detailed clinical course of disease in representative patients.

XLSX file - 82K, Change in frequency of cytokine-releasing T cells at different time points.

PDF file - 51K, Promoter hypermethylation events.

L19–IL2 is a recombinant protein comprising the cytokine IL2 fused to the single-chain monoclonal antibody L19. In previous studies, intralesional injection with IL2 has shown efficacy for the locoregional treatment of cutaneous/subcutaneous metastases in patients with advanced melanoma. The objectives of this study were to investigate whether (i) intralesional delivery of a targeted form of IL2 would yield similar results, with reduction of injection frequency and treatment duration; and (ii) systemic immune responses were induced by the local treatment. Patients with stage IIIB/IIIC melanoma and cutaneous/subcutaneous injectable metastases received weekly intratumoral injections of L19–IL2 at a maximum dose of 10 MIU/week for 4 consecutive weeks. Tumor response was evaluated 12 weeks after the first treatment. Twenty-four of 25 patients were evaluable for therapy-induced responses. A complete response (CR) by modified immune-related response criteria (irRC) of all treated metastases was achieved in 6 patients (25%), with long-lasting responses in most cases (5 patients for ≥24 months). Objective responses were documented in 53.9% of all index lesions [44.4% CR and 9.5% partial responses (by irRC)], and 36.5% of these remained stable, while 9.5% progressed. Toxicity was comparable with that of free IL2, and no serious adverse events were recorded. A significant temporary increase of peripheral regulatory T cells and natural killer cells, sustained increase of absolute CD4+ lymphocytes, and decrease of myeloid-derived suppressor cells were observed upon treatment. Finally, we recorded encouraging data about the progression time to distant metastases and overall survival. Cancer Immunol Res; 2(7); 668–78. ©2014 AACR.

PDF file - 945K, Frequency and severity of common adverse events.

PDF file - 128K, Primer sequences.

PDF file - 43K, Copy number gains.

PDF file - 119K, List of lesions present at screening and overall patient responses (modified irRC and RECIST).

PDF file - 74K, Mutations and homozygous deletions.

PDF file - 870K, Detailed gating strategy of all the analyzed immune cell subsets.

Melanoma genomes contain thousands of alterations including: mutations, copy number alterations, structural aberrations, and methylation changes. The bulk of this variation is stochastic and functionally neutral, with only a small minority representing “drivers” that contribute to the genesis and maintenance of tumors. Drivers are often directly or inversely correlated across tumors, reflecting the molecular and regulatory signaling pathways in which they operate. Here, a profile of genetic and epigenetic drivers in 110 human melanoma cell lines was generated and searched for non-random distribution patterns. Statistically significant mutual exclusivity was revealed among components of each of the p16INK4A-CDK4-RB, RAS-RAF-MEK-ERK and PI3K-AKT signaling pathways. In addition, an inverse correlation was observed between promoter hypermethylation of retinoic acid receptor β (RARB) and CDKN2A alterations affecting p14ARF (P < 0.0001), suggesting a functional link between RARβ signaling and the melanoma-suppressive activities of p14ARF. Mechanistically, all-trans retinoic acid (ATRA) treatment increased the expression of p14ARF in primary human melanocytes and the steady-state levels of p14ARF in these cells were shown to be regulated via RARβ. Furthermore, the ability of ATRA to induce senescence is reduced in p14ARF-depleted melanocytes, and we provide proof-of-concept that ATRA can induce irreversible growth arrest in melanoma cells with an intact RARβ-p14ARF signaling axis, independent of p16INK4A and p53 status.Implications: These data highlight the power of mutual exclusivity analysis of cancer drivers to unravel molecular pathways and establish a previously unrecognized cross-talk between RARβ and p14ARF with potential implications for melanoma treatment. Mol Cancer Res; 11(10); 1166–78. ©2013 AACR.

Supplemental Figure 2. Glioblastoma patient overall survival.

Antibody panels used for flow cytometry* FoxP3 buffer (BD) was used to fix and permeabilize cells. Only frequencies of frequencies of CD4+, CD8+ T cells from Treg panel 1 and, CD14+ cells from MDSC panel 1 were included. 1 Panel 1 (n=14), 2 Panel 2 (n=68).

PDF file - 65K, Summarized percentages of cytokine-producing cells within the CD4 and CD8 compartment respectively for patients with antigen-specific T-cells.

Calibration of combination models. Calibration was calculated after 12 and 24 months using the calibrate function in the rms package of R for combination model 1 (A) and combination model 2 (B). Bootstrapping (1000 repeats) was performed to obtain bias-corrected estimates of predicted vs. observed values. Non-convergence reduced the number of included bootstrapping steps for combination model 2 to 981 or 990 after 12 or 24 months, respectively. "Predicted" survival probabilities at 12 or 24 months are those predicted by the Cox model, and "observed" refers to the corresponding Kaplan-Meier survival estimate at the given time-point. Mean absolute error in predictions, the mean squared error, and the 0.9 quantile of the absolute error is reported. "Error" refers to the difference between the predicted values and the corresponding bias-corrected calibrated values. Mean error was

Purpose: Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance.Experimental Design: The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2− cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay.Results: The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2− cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2− Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity.Conclusions: Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities. Clin Cancer Res; 17(9); 2668–78. ©2011 AACR.

Purpose: To analyze the prognostic relevance and relative impact of circulating myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) compared with functional tumor antigen–specific T cells in patients with melanoma with distant metastasis.Experimental Design: The percentage of CD14+CD11b+HLA-DR−/low MDSCs, CD4+CD25+FoxP3+ Tregs, and the presence of NY-ESO-1- or Melan-A–specific T cells was analyzed in 94 patients and validated in an additional cohort of 39 patients by flow cytometry. Univariate survival differences were calculated according to Kaplan–Meier and log-rank tests. Multivariate analyses were performed using Cox regression models.Results: NY-ESO-1–specific T cells, the M-category, and the frequency of MDSCs were associated with survival. The absence of NY-ESO-1–specific T cells and the M-category M1c independently increased the risk of death. In a second Cox model not considering results on antigen-specific T cells, a frequency of >11% MDSCs showed independent impact. Its association with survival was confirmed in the additional patient cohort. Median survival of patients with a lower frequency of MDSCs was 13 months versus 8 months for others (P < 0.001, combined cohorts). We observed a strong correlation between high levels of MDSCs and the absence of melanoma antigen–specific T cells implying a causal and clinically relevant interaction. No prognostic impact was observed for Tregs.Conclusions: Circulating CD14+CD11b+HLA-DR−/low MDSCs have a negative impact on survival and inversely correlate with the presence of functional antigen–specific T cells in patients with advanced melanoma. Our findings provide a rationale to investigate MDSC-depleting strategies in the therapeutic setting especially in combination with vaccination or T-cell transfer approaches. Clin Cancer Res; 20(6); 1601–9. ©2013 AACR.

Supplemental Figure 5. Confirmation of gut microbiota depletion.

Impact of delayed increases in % of CD8+ T cells on overall survival. Kaplan-Meier analysis of overall survival according to delayed changes (increase vs. decrease) of % of CD8+ T cells observed during ipilimumab treatment within patients showing early increases in ALC and delayed increase in % of CD4+ T cells. Censoring is indicated by vertical lines; p-values were calculated by log rank statistics.

Increases of CD4+, CD8+ and CD3- cell counts according to changes in ALC and overall survival according to changes of CD4+, CD8+ and CD3- cell counts. Absolute lymphocyte counts (ALC). *Early defines changes between baseline (BL) and a second time-point, ranging from 2-8 weeks after start of ipilimumab. #Delayed defines changes between BL and a third time-point, ranging from 8-14 weeks after start of ipilimumab. The percentage of patients identified with an increase in absolute numbers of CD4+, CD8+ T cells or CD3- lymphocytes was defined within patients showing an early increase or decrease in ALC (A). Kaplan-Meier analysis of overall survival according to early and delayed changes (increase vs. decrease) of absolute CD4+ T cells (B, E), CD8+ T cells (C, F) and CD3- lymphocytes (D, G) observed during ipilimumab treatment. Censoring is indicated by vertical lines; p-values were calculated by log rank statistics.

PDF file - 43K, Univariate analysis using an alternative follow-up time, calculated from the date of diagnosed stage IV disease to the date of last follow-up or death.

Multivariate Models including only patients receiving 3 mg/kg ipilimumab. Lactate dehydrogenase (LDH), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), hazard ratio (HR). * Relative lymphocyte count, absolute monocyte count, absolute and relative eosinophil count

PDF file - 67K, Kaplan-Meier survival curves of the combined cohort of non-resectable stage IV patients.

Supplementary Materials and Methods

Discriminatory ability of combination models. The concordance index (c-index, y-axis) was calculated for the combination of factors with independent impact according to Cox regression analysis (model 6.1) and 13 alternative combination models considering 5, 7, or 8 factors (A). The numbers refer to the rows in A. The c-indices are presented according to the number of combined factors (B). The combination model with highest discriminatory ability (7.4), which considered regulatory T cells in addition to the 6 factors with independent impact according to Cox regression analysis was chosen as combination model 1. No further increase of the c-index compared to combination model 1 was observed if one of the 3 remaining factors was additionally considered (models 8.1, 8.2, 8.3). C-indices were calculated for different combination models accounting for the number of unfavorable values of all factors considered in the given model (C). All possible models derived from combinations of the five routine factors were considered. The c-indices are presented according to the number of considered factors (D). The model with highest discriminatory ability (4.1) was selected.

Supplementary Figures S1-S2 from Association of IFN-γ Signal Transduction Defects with Impaired HLA Class I Antigen Processing in Melanoma Cell Lines

PDF file - 165K, Type of cytokines produced upon antigen stimulation. Proportion of patients with T-cells producing different cytokines in the group of all patients with detectable T-cells responding to NY-ESO-1, Melan-A or Influenza. CD4 (A) and CD8 (B) responses.

PDF file - 506K, Detailed gating strategy for each analyzed immune cell subset.

Changes associated with overall survival and their correlation with clinical responses in sub-cohorts. Analyses were limited to patients with available data at all three time points or limited to patients treated with ipilimumab monotherapy at 3 mg/kg bodyweight. *Early defines a second time-point, ranging from 2-8 weeks after start (BL) of ipilimumab. #Delayed defines a third time-point, ranging from 8-14 weeks after start (BL) of ipilimumab.

PDF file - 174K, Detailed Gating strategy.

Supplemental Figure 6. Characterization of the potent clinical-grade IDO enzyme inhibitor, BGB-7204.

Impact of cell frequencies or counts on OS at single time points. Absolute (Abs.), Baseline (BL), median survival time (MST), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), Relative (Rel.). *Early defines a second time-point, ranging from 2-8 weeks after start (BL) of ipilimumab. #Delayed defines a third time-point, ranging from 8-14 weeks after start (BL) of ipilimumab.

Gating strategy for quantification of subsets of T cells and regulatory T cells (Tregs), monocytes and myeloid-derived suppressor cells (MDSCs). Total cells were selected by gating on Time vs. SSC-A. Duplicates were removed via progressive gating on FSC-H vs. FSC-A and SSC-H vs. SSC-A. Dead cells were excluded by considering only EMA-negative cells. (A) A morphological gate was used to identify the population of lymphocytes. Next, CD3+ cells were selected and further separated into CD4+ and CD8+ cells. Ki67 expression was investigated on CD4+ and CD8+ cells. CD8+ T cells with suppressive potential were defined as CD103+. Previously described phenotypes of Tregs were defined as CD4+CD25+FoxP3+ and CD4+CD127lowCD25+FoxP3+. These were further subdivided into proliferating (Ki67+CD45RA-) and non-proliferating Tregs (Ki67-CD45RA+). (B) A lineage cocktail (CD3, CD19, CD56) was used to avoid cross-contamination. Previously described MDSC populations were identified as Lin-CD14+HLA-DRlow and Lin-CD14-CD15+CD11b+ within the all-cell gate. Overall monocytes were defined as CD14+, while subsets were separated into classical monocytes (Lin-CD14+CD16-HLA-DR+), non-classical monocytes (Lin-CD14-CD16+HLA-DR+) and Lin- CD14-CD16dimHLA-DR+ monocytes within the all-cell gate.