Abstract

   The outcome of patients affected by high-risk or metastatic
   neuroblastoma (NB) remains grim, with ≥ 50% of the children
   experiencing relapse or progression of the disease despite multimodal,
   intensive treatment. In order to identify new strategies to improve the
   overall survival and the quality of life of these children, we recently
   developed and optimized a third-generation GD2-specific chimeric
   antigen receptor (CAR) construct, which is currently under evaluation
   in our Institution in a phase I/II clinical trial ([55]NCT03373097)
   enrolling patients with relapsed/refractory NB. We observed that our
   CAR T-cells are able to induce marked tumor reduction and even achieve
   complete remission with a higher efficiency than that of other CAR
   T-cells reported in previous studies. However, often responses are not
   sustained and relapses occur. Here, we demonstrate for the first time a
   mechanism of resistance to GD2.CAR T-cell treatment, showing how
   polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase
   in the peripheral blood (PB) of NB patients after GD2.CAR T-cell
   treatment in case of relapse and loss of response. In vitro, isolated
   PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of
   different generations of GD2.CAR T-cells. Gene-expression profiling of
   GD2.CAR T-cells “conditioned” with PMN-MDSC shows downregulation of
   genes involved in cell activation, signal transduction, inflammation
   and cytokine/chemokine secretion. Analysis of NB gene-expression
   dataset confirms a correlation between expression of these genes and
   patient outcome. Moreover, in patients treated with GD2.CAR T-cells,
   the frequency of circulating PMN-MDSC inversely correlates with the
   levels of GD2.CAR T-cells, resulting more elevated in patients who did
   not respond or lost response to the treatment. The presence and the
   frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a
   useful prognostic marker to predict the response to GD2.CAR T-cells and
   other adoptive immunotherapy. This study underlines the importance of
   further optimization of both CAR T-cells and clinical trial in order to
   target elements of the tumor microenvironment.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13045-021-01193-0.

   Keywords: Neuroblastoma, Polymorphonuclear myeloid-derived suppressor
   cells, GD2.CAR T-cells, Clinical response, T-cell functionality,
   Long-term response
     __________________________________________________________________

   Chimeric antigen receptor (CAR) T-cell technology has rapidly evolved
   during the past decade, offering unprecedented results and now
   commercially available CAR T-cells targeting CD19 are employed to treat
   patients with B-cell malignancies non-responding to conventional
   therapy [[56]1]. On the contrary, a big challenge remains the treatment
   of refractory solid tumors with CAR T-cells. Indeed, while pre-clinical
   studies showed encouraging results, only partial and transient
   responses have been obtained in clinical trials [[57]2, [58]3]. These
   studies, however, uncovered mechanisms which may explain the limited
   success of CAR T-cell therapy, including lack of CAR T-cell
   persistence, quality of targeted antigen and the suppressive effect of
   the tumor microenvironment (TME) [[59]2, [60]4]. Several strategies to
   overcome these mechanisms have been explored, including the targeting
   of regulatory/suppressive immune cells responsible of the TME-mediated
   inhibition [[61]5–[62]8]. Myeloid-derived suppressor cells (MDSC) are
   immature myeloid cells that arise from bone-marrow (BM) myeloid
   progenitors. Within the MDSC population, two main subsets can be
   identified: monocytic (CD45^+, Lin^−, HLA-DR^−/low, CD33^+, CD11b^+,
   CD14^+, CD66b^− and CD15^−) and polymorphonuclear (PMN) (CD45^+, Lin^−,
   HLA-DR^−/low, CD33^+, CD11b^+, CD14^−, CD66b^+ and CD15^+) cells
   [[63]9]. The expansion of these myeloid cell subsets has been detected
   in several conditions characterized by a high level of inflammation,
   including cancer [[64]10], autoimmune disorders [[65]11, [66]12],
   sepsis and infectious diseases [[67]13, [68]14]. MDSC are recruited by
   solid tumors and consist of immature cells contributing to the
   establishment of an immunosuppressive microenvironment [[69]15].

   MDSC presence has been documented in patients with different tumors and
   their potent immunosuppressive activity is now widely recognized
   [[70]16]. Recently, in a mouse model, the expansion of MDSC has been
   reported to decrease CAR T-cell efficacy in metastatic liver tumors
   [[71]17] and interventions to inhibit MDSC showed anti-tumor benefit in
   a murine sarcoma model [[72]5].

   In this study, we show that PMN-MDSC with inhibitory activity are
   highly enriched in peripheral blood (PB) of Neuroblastoma (NB) patients
   with refractory/relapsed disease after treatment of GD2.CAR-T cells
   (Fig. [73]1A, B, Additional file [74]1: Fig. S1). These patients, in
   whom multi-modal conventional treatments had failed, were enrolled at
   our Institution in a clinical trial exploring the safety/efficacy of a
   novel GD2.CAR T-cell product [[75]18] (Additional file [76]6:
   Supplementary materials and methods). First, we investigated whether
   PMN-MDSC could be involved in GD2.CAR T-cell functional impairment. To
   this end, we showed that allogeneic PMN-MDSC, obtained from
   hematopoietic stem-cell transplantation-donors undergoing mobilization
   with G-CSF [[77]9], display a marked inhibitory effect on several
   second- and third-generation GD2.CAR T-cells (Fig. [78]1C, D,
   Additional file [79]2: Fig. S2, Additional file [80]3: Fig. S3).

Fig. 1.

   [81]Fig. 1
   [82]Open in a new tab

   Presence of PMN-MDSC in PB of NB patients and their effect on GD2.CAR
   T-cells. A, B Mononuclear cells isolated from the PB of NB patients
   were analyzed by flow-cytometry for the expression of specific markers
   allowing the identification of PMN-MDSC subsets. A Percentages of
   PMN-MDSC on CD45^+cells (CD66b^+CD14^−cells) in healthy donors (HD,
   n = 10) and in NB patients (NB pts, n = 9). B A representative gating
   strategy for PMN-MDSC identification by flow-cytometry is shown. C, D
   Second- and third-generation GD2.CAR T-cells were cultured either in
   the absence (w/o, blue bars) or in the presence (cond, red bars) of
   PMN-MDSC collected from stem cell donors given G-CSF and undergoing to
   leukapheresis. After 48h, GD2.CAR T-cells and non-transduced (NT) cells
   (used as control) were collected, purified and co-cultured at the
   effector:target ratio 1:1 with the SH-SY5Y-eGFP^+ NB cell line.
   Percentages of SH-SY5Y-eGFP^+ NB residual live cells at day 5 of
   co-culture with (C) second- (n = 10) and D third- (n = 10) generation
   GD2.CAR T-cells are reported. Patient derived GD2.CAR T-cells
   expressing CD28.4-1BBζ (the same used in our clinical trial) were
   cultured in the presence of PB-derived (n = 8) or BM-derived (n = 3)
   PMN-MDSC or PB-derived neutrophils (n = 3) collected from NB patients.
   After 48h, GD2.CAR T-cells were collected, purified from
   PMN-MDSC/neutrophils as described in supplementary data (Additional
   file [83]6), and co-cultured at an Effector:Target (E/T) ratio 1:1 with
   SH-SY5Y-eGFP^+ NB cell line for 3 days. Percentages of SH-SY5Y-eGFP^+
   NB residual live cells were analyzed. E One representative co-culture
   experiment of GD2.CAR T-cells and PMN-MDSC is shown. F Percentages of
   residual live NB tumor cells in different culture conditions is
   reported. G Correlation between PMN-MDSC and GD2.CAR T-cells in CD45^+
   cells in NB patients (n = 55, different time point after GD2.CAR T-cell
   infusion), r^2 = 0.07 and p value is indicated. Mononuclear cells
   present in the PB of NB patients were analyzed by flow-cytometry.
   Graphs represent percentages of PMN-MDSC and the corresponding absolute
   number/µl of GD2.CAR T-cells in NB patients. H Graphs represent GD2.CAR
   T-cells/µl (blue squares) and the percentages of PMN-MDSC (red dots) in
   CD45^+ cells in responder (n = 5) and non-responder (n = 6) patients
   (multiple comparison with mixed-effect analysis). *p < 0.05,
   **p < 0.01, ***p < 0.005. Where not indicated, data were not
   statistically significant. A Mann–Whitney and C, D, F Wilcoxon
   Student’s t test. Data are shown as mean ± standard error of the mean
   (SEM)

   To corroborate our data, we analyzed the inhibitory activity of
   PMN-MDSC that were collected from PB of GD2.CAR
   T-cell-treated-NB-patients. Our results confirmed the strong inhibitory
   capability of these cells on third-generation GD2.CAR T-cells in vitro,
   while neither autologous PB neutrophils nor BM low-density neutrophils,
   inhibit GD2.CAR T-cell cytotoxicity (Fig. [84]1E, F, Additional file
   [85]1: Fig. S1).

   PMN-MDSC could be detected after GD2.CAR T-cell treatment and,
   importantly, their frequency in PB inversely correlated with that of
   GD2.CAR T-cells (Fig. [86]1G). Although, the number of patients
   analyzed in the present study is limited, we could observe that in
   patients with an early expansion of GD2.CAR T-cells a very low
   percentage of PMN-MDSC was observed. Of note, these patients
   (Responder) were characterized by a good response to therapy,
   reflecting a potent GD2.CAR T-cell activation. In non-responder
   patients, as well as in patients that lost response, high percentages
   of PMN-MDSC were associated with a lack of substantial expansion of
   GD2.CAR T-cells (Fig. [87]1H, Additional file [88]4: Fig. S4).

   To better define the molecular mechanisms of the inhibitory effect, we
   performed a gene-expression profiling on PMN-MDSC-conditioned GD2.CAR
   T-cells. We observed modulation of numerous genes (p ≤ 0.01) involved
   in inflammation, cell activation, signal transduction and
   extra-cellular communication (Fig. [89]2A–C, Additional file [90]5:
   Fig. S5A), as well as cytokine/chemokine secretion (Fig. [91]2D). Three
   genes were found to be significantly upregulated in all conditioned
   T-cells: S100A8, S100A9 and TNFAIP6. These genes are involved in the
   immune response, leukocyte activation, inhibitory pathways induced by
   cell surface receptors, extracellular communications and others.

Fig. 2.

   [92]Fig. 2
   [93]Open in a new tab

   PMN-MDSC induce modulation of the gene-expression profile and inhibit
   cytokine secretion of GD2.CAR T-cells. A–C NT and GD2.CAR T-cells were
   cultured for 48h either in the absence or in the presence (cond.) of
   PMN-MDSC (NT n = 3; GD2.CAR T-cells n = 3). A Gene expression level
   (Rq) represented as mean plot with the 95% of confidence interval for
   the total gene analyzed in NT and GD2.CAR T-cells conditioned or not
   with PMN-MDSC. B Heat-maps representing the unsupervised hierarchical
   clustering of samples analyzed for inflammation, kinome and signal
   transduction gene-expression profile. Columns represent samples (NT and
   GD2.CAR T-cells), rows represent genes. C Volcano plots show
   inflammation, kinome and signal transduction gene expression data. The
   red or green dots indicate genes-of-interest that display highly
   positive or negative fold-change (x axis) and high statistical
   significance (− log10 of p value, y axis). The gene name related to a
   red and green dot is reported in the table (right panel). D Levels
   (pg/ml) of the indicated cytokines/chemokines in the supernatant of NT
   (n = 6) and GD2.CAR T-cells (n = 12) conditioned (red) or not (blue)
   with PMN-MDSC are shown. E Kaplan–Meier curves estimate of overall
   survival of HR-NB patients according to the level of S100A8 or S100A9
   or TNFAIP6 gene expression. Red (low) and blue (high) lines indicate
   the level of gene expression using median cut-off modus. Dataset used
   for the analysis is indicated. One-way ANOVA was used for statistical
   analysis. *p < 0.05, **p < 0.01, ***p < 0.005

   S100A8 and S100A9, belonging to the S100 family of proteins containing
   two canonical EF-hand calcium binding motifs, are involved in the
   calcium dependent control of cell differentiation, cell cycle
   progression and growth. Several studies underlined how these two
   proteins are up-regulated in many cancer patients and demonstrated
   their role in tumor promotion and progression by inducing MDSC [[94]19]
   or generating the protein calprotectin, which has been shown to induce
   T-cell apoptosis and exhaustion [[95]20–[96]22].

   The TNFAIP6 is a secretory protein that contains a hyaluronan-binding
   domain extremely important for T-cell migration within the
   extracellular matrix. Furthermore, TNFAIP6, like the other family
   members of TNFAIP, is involved in immune reactions, inflammatory
   responses, signal transduction, apoptosis, differentiation, material
   transport and other biological functions. They play important roles in
   multiple diseases and in the immune and inflammatory processes of
   cancer, including their overexpression in T-cells [[97]23].

   Interestingly, analysis on the up- and down-regulated genes showed
   upregulation of Th2 cytokines, activation of senescence pathways and
   sensitivity to TNF signaling, as well as down-regulation of genes
   involved in pathways controlling the intracellular signaling, metabolic
   process and inflammasome in conditioned GD2.CAR T-cells as compared to
   un-conditioned GD2.CAR T-cells (Additional file [98]5: Fig. S5B–D).

   To further validate our data and verify the effect of the S100A8,
   S100A9 and TNFAIP6, we queried the public R2 NB gene-expression data
   set and found a strong correlation between upregulation of S100A8,
   S100A9 and poor clinical outcome in high-risk (HR)-NB patients and a
   trend for the TNFAIP6 (Fig. [99]2E). Moreover, the correlation between
   upregulation of S100A8, S100A9 and poor patient outcome has been
   confirmed also on the full NB population (Additional file [100]5: Fig.
   S5E). As previously shown, PMN-MDSC are able to inhibit different
   immune effector cells, including NK [[101]9], αβ- and γδ-T-cells
   [[102]24]. Moreover, inflammatory mediators, such as IL8 and IL1β and
   IL13, drive their accumulation and contribute to their inhibitory
   activity. In addition, MDSC can induce macrophage polarization towards
   a M2 profile, inhibit NK cell-mediated anti-tumor activity and recruit
   regulatory T-cells [[103]25]. Of note, the different immunophenotypic
   characteristics of MDSC and their suppressive mechanisms of MDSCs
   reflect their heterogeneous nature [[104]25, [105]26]. Therefore,
   different approaches are required for targeting human MDSC. Several
   compounds, including ATRA (all-trans-retinoic acid) [[106]27, [107]28],
   vitamin D [[108]29] and paclitaxel [[109]30], have been shown to be
   able to block the MDSC immunosuppressive activity by inducing their
   differentiation. Notably, chemotherapeutic agents (e.g. gemcitabine or
   5-fluorouracil) [[110]31, [111]32] directly reduce the frequency of
   MDSC enhancing the anti-tumor immune activity.

   Overall, our data demonstrate the need to further implement the design
   of both clinical trials and CAR constructs and highlights the
   prognostic relevance of PMN-MDSC, since these cells are able to
   suppress in general anti-tumor effector cells, and not only CAR
   T-cells. Therapeutic agents capable of targeting and neutralizing
   PMN-MDSC might help restoring an effective anti-tumor activity of
   effector cells and protect CAR T-cells from the detrimental action
   displayed by immunosuppressive cells.

   In conclusion, PMN-MDSC may represent a novel target in tumor
   immunotherapy and an important biomarker predicting response to
   immunotherapy-based treatments, not necessarily limited to CAR
   T-cell-based approaches. Whether these cells may interfere with
   conventional chemotherapy is an issue to be investigated in future ad
   hoc designed studies.

Supplementary Information

   [112]13045_2021_1193_MOESM1_ESM.pdf^ (8.3MB, pdf)

   Additional file 1: Fig. S1. Functional test to assess the PMN-MDSC
   inhibitory capability. (A-D) T-cells isolated from healthy donor PB or
   GD2.CAR T-cells were co-cultured either in the absence or in the
   presence of PMN-MDSC derived from PB of NB patient. The proliferation
   capability was assessed by flow-cytometry for T-cells (A) or GD2.CAR
   T-cells (C, n = 6) after 5 or 4 days, respectively. The number of cells
   for each division is indicated. (B and D) TNF-α and IFN-γ production by
   CD8^+ and CD8^− T-cells (B) or GD2.CAR T-cells (D, n = 6) upon
   over-night stimulation with PMA and Ionomycin.
   [113]13045_2021_1193_MOESM2_ESM.pdf^ (2.6MB, pdf)

   Additional file 2: Fig. S2. Schematic representation of the retroviral
   transduction. (A) Schematic representation of the CAR constructs.
   Second-generation CAR T-cell constructs encoding CD28 or OX40 or 4-1BB
   costimulatory molecules. Third-generation CAR T-cell constructs
   encoding CD28.OX40 or CD28.4-1BB costimulatory molecules. All
   transduced CAR T-cells were equipped with the signal endo-domain
   derived from the CD3ζ chain. (B) Retroviral vector codifying for an
   eGFP. Created with Biorender.
   [114]13045_2021_1193_MOESM3_ESM.pdf^ (1.5MB, pdf)

   Additional file 3: Fig. S3. Effect of PMN-MDSC on third-generation
   GD2.CAR T-cells. Third-generation GD2.CAR T-cells were cultured either
   in the absence (w/o PMN-MDSC) or in the presence (with PMN-MDSC, 1:1)
   of PMN-MDSC collected from stem cell donors given G-CSF for
   hematopoietic stem cell mobilization and undergoing to leukapheresis.
   After 48 hours, GD2.CAR T-cells and non-transduced (NT) cells (used as
   control) were collected, purified and co-cultured at the
   effector:target ratio 5:1 with the SH-SY5Y-eGFP NB cell line.
   Percentages of SH-SY5Y-eGFP^+ NB residual live cells at day 3 of
   co-culture third-generation GD2.CAR T-cells (n = 7).
   [115]13045_2021_1193_MOESM4_ESM.pdf^ (1.9MB, pdf)

   Additional file 4: Fig. S4. Schematic representation of how PMN-MDSC
   compromise the GD2.CAR T-cell based therapy. (A) CAR T-cell preparation
   and infusion. (B) Differentiation steps from common myeloid precursors
   (pink cells) towards neutrophils (blue cells). Tumor cells (brown
   cells) and the TME may induce neutrophil accumulation and
   differentiation towards PMN-MDSC (dark grey cells). (C-D) Upon GD2.CAR
   T-cell infusion, patients could either (C) respond to CAR treatment
   (Responders) or (D) could display accumulation of PMN-MDSC which
   inhibit GD2.CAR T-cell expansion/function, thus contributing to the
   lack of efficacy of CAR T-cell therapy (Non-Responders). Created with
   Biorender.
   [116]13045_2021_1193_MOESM5_ESM.pdf^ (11.7MB, pdf)

   Additional file 5: Fig. S5. Expression of informative genes in GD2.CAR
   T-cells upon interaction with PMN-MDSC. (A) Gene expression level (Rq)
   represented as mean plot with the 95% of confidence interval in NT and
   GD2.CAR T-cells conditioned or not with PMN-MDSC for the indicated gene
   arrays: inflammation (n = 607 genes), kinome (n = 828 genes) and signal
   transduction (n = 597 genes). (B) Gene analysis correlation between
   S100A8, S100A9 and TNFAIP6 transcripts and all genes analyzed with
   p ≤ 0.01. (C) Pathway enrichment analysis for S100A8, S100A9 and
   TNFAIP6 genes. Clusters of different pathways were visualized in
   different colors, with the size of rectangles adjusted to reflect their
   p-value. (D) Pathway enrichment analysis on the up- and down-regulated
   genes in CAR/CAR conditioned T-cells. (E) Overall survival of NB
   patients (low, medium and HR) and level of S100A8 or S100A9 or TNFAIP6
   gene expression. Red (low) and blue (high) lines indicate the level of
   gene expression using median cut-off modus. Dataset used for the
   analysis is indicated.
   [117]13045_2021_1193_MOESM6_ESM.docx^ (25.8KB, docx)

   Additional file 6. Supplementary materials and methods.

Acknowledgements