MYC-expressing lymphomas are infiltrated by large numbers of activated Tregs that express high levels of immunosuppressive markers

To characterize the Tregs infiltrating the lymphoma microenvironment in a convenient, rapid and standardized model, we took advantage of MYC-expressing lymphoma cells from Eµ-MYC mice, which, on transplantation into syngeneic immunocompetent C57BL/6 recipients, form tumors in the LNs, spleen and bone marrow within 10–12 days post-transplant; tumor cells can be readily identified flow cytometrically by their CD19, intermediate CD45 and Ki67 expression.28 MYC-driven lymphomas in the LNs (and spleens, data not shown) are highly enriched for Foxp3⁺ Tregs relative to control LNs (figure 1A). The extent of Treg infiltration correlates strongly with the tumor burden (figure 1B). Tregs infiltrate the tumor mass and are found in close proximity to tumor B-cells (figure 1C). Spectral flow cytometry using a panel of 14 antibodies specific for relevant Treg markers identified five Treg clusters that were predominantly found in normal LNs and consisted of naïve cells (CD62L⁺, CD44^-) with low to intermediate expression of functional/immunosuppressive markers (CD39, CD73, GITR) (figure 1D–G, online supplemental figure S1A,B); the immunophenotype of normal LN Tregs described here is consistent with the published markers of thymus-derived Tregs in murine secondary lymphoid organs.40 In contrast, Tregs within tumors showed downregulation of CD62L and enhanced expression of the immunosuppressive markers CD39, CD73 and GITR, of neuropilin-1 (Nrp-1) and of the interleukin-2 (IL-2) receptor α-subunit CD25 (figure 1D–G, online supplemental figure S1B), all of which are attributes of effector Tregs.41 The largest changes in the expression between control and tumor-infiltrating Tregs were observed in CD62L, Nrp-1, CD25 and CD73/CD39, two enzymes that convert immunostimulatory ATP into immunosuppressive adenosine (online supplemental figure S1B). Only one, numerically minor cluster of (tumor-infiltrating) Tregs was positive for Ki67, indicating active proliferation (figure 1G). Tumor-infiltrating Tregs appeared in tumors of wild-type as well as T-cell-receptor (TCR)-transgenic OTII recipients, in which the TCRs of all CD4⁺ T-cells, including Tregs, are specific for the chicken ovalbumin peptide 323–339; this result indicates that Treg recruitment and/or local expansion is not dependent on binding to cognate antigen (figure 1H). Regular orally administered doses of the sphingosine-1-phosphate analog fingolimod, which prevents lymphocyte egress from lymphoid tissues, did not prevent the appearance of effector Tregs within the transplanted lymphoma, indicating that these cells arise as a consequence of local differentiation or expansion, rather than recruitment from the circulation or from distant lymphoid organs (figure 1I). Indeed, the adoptive transfer of >95% pure, Foxp3/YFP^-, naïve CD45.2⁺ CD4⁺ T-cells sorted from Foxp3^YFP reporter mice into tumor-bearing CD45.1⁺ congenic recipients led to their efficient conversion into Foxp3/YFP⁺ Tregs in tumor-bearing organs, which was not seen in non-tumor-bearing control mice (figure 1J). Furthermore, co-culturing of sorted, highly pure (>95%) tumor cells with naïve Tregs isolated from non-tumor-bearing animals’ LNs for 3 days resulted in their increased expression of Ki67, CD44, CD27, Tbet, GITR, CD39 and other markers characteristic of tumor-infiltrating effector Tregs (figure 1K). Interestingly, co-culturing not only affected the immunophenotype of the Tregs, but also boosted the proliferation of the tumor cells, as assessed in a total of eight independent co-cultures (figure 1L). The combined data suggest that naïve T-cells differentiate into Foxp3⁺ Tregs and acquire properties of effector Tregs in the lymphoma microenvironment, a process that appears to be tumor antigen-independent, locally driven and proportional to the tumor burden.

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Figure 1

Regulatory T-cells (Tregs) in MYC-driven lymphomas differ immunophenotypically from normal lymph node (LN) Tregs. (A–G) C57BL/6 mice were intravenously injected with 1 Mio >90% pure lymphoma cells isolated from the spleens of Eμ-Myc-transgenic donors; frequencies of tumor cells and Tregs in pooled axillary and inguinal LN preparations were determined by flow cytometry at the study endpoint (A, B; 10–11 days post injection). Ki67⁺ tumor B-cells and Foxp3⁺ Tregs were visualized by immunofluorescence microscopy (C; scale bar: 50 mm). A subset of mice was subjected to multidimensional flow cytometry using a panel of 14 intracellular and surface markers that allowed the identification and quantification of the 12 indicated naïve and effector Treg populations. Individual marker UMAPs ("uniform manifold approximation and projection") are shown in (D) and UMAPs showing the FlowSOM-guided manual metaclustering of the 12 indicated control and tumor-infiltrating Treg clusters is shown for six control and five tumor-bearing mice in (E) alongside their quantification in (F). Naïve, transitional CD62L^int and effector Tregs are indicated. The heatmaps in (G) show the median expression of the indicated markers (value range: 0–1) of control and tumor-infiltrating Treg clusters. Data in (A) and (B) are pooled from three studies; data in (D)–(G) are from one study, and representative of at least three independently conducted studies. (H) CD45.1⁺ (Ly5.1⁺) OTII-TCR-transgenic (OTII⁺) and their WT (OTII^-) littermates were intravenously injected with 1 Mio MYC-expressing lymphoma cells and frequencies of Tregs and tumor B-cells were determined in LN single cell suspensions by flow cytometry at the study endpoint. (I) C57BL/6 mice were injected with 1 Mio splenic MYC-overexpressing lymphoma cells and treated intraperitoneally every second day with 20 µg fingolimod or the diluent dimethyl sulfoxide (DMSO) only, starting 1 day before tumor cell inoculation. Frequencies of Tregs and tumor cells were determined by flow cytometry at the study endpoint. (J) CD45.1⁺ (Ly5.1⁺) tumor-bearing or non-tumor-bearing mice were adoptively transplanted with 1 Mio sorted CD45.2⁺ FoxP3/YFP^- CD4⁺ T-cells on day 9 post tumor cell transplantation; the frequencies of converted CD45.2⁺ FoxP3⁺ Tregs among all CD4⁺ T-cells were determined in the spleens 2 days later. (K) Splenic tumor cells were sorted by fluorescence-activated cell sorting (FACS) from lymphoma-bearing mice and naïve splenic FoxP3/YFP⁺ Tregs from healthy mice were FACS-sorted and ex vivo co-cultured for 3 days. Activation and immunosuppressive Treg marker were examined by flow cytometry. (L) Sorted splenic tumor cells were cultured either alone or with naïve splenic Tregs isolated from healthy mice; tumor cell proliferation was measured by flow cytometry after 4 days of co-culture. Data in (H), (I) and (K) show individual experiments that are representative of at least two independently conducted studies; data in (J) and (L) are pooled from two experiments. Horizontal lines indicate medians throughout. Statistical comparisons were performed either by ordinary one-way analysis of variance or by unpaired t-test (Mann-Whitney). ns, not significant; *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Single cell RNA-sequencing uncovers the transcriptional signature of lymphoma-infiltrating Tregs

To address how lymphoma-infiltrating Tregs differ from their normal LN counterparts using a complementary technique, we took advantage of Foxp3^GFP reporter mice to sort and single cell-sequence 20,000 Foxp3^GFP-positive Tregs per condition. Dimensionality reduction followed by Seurat clustering revealed nine Treg clusters that differed strongly in their relative abundance in tumor-bearing versus control LNs (figure 2A). Three clusters were exclusively present in lymphoma-bearing LNs, and the remaining six were vastly more abundant in the normal LNs (figure 2A). Hierarchical clustering of the top 80 most differentially expressed transcripts allowed the segregation of the nine populations (figure 2B) and confirmed several of the differentially regulated markers that were identified by flow cytometry, in addition to uncovering new ones. Several transcripts were highly intratumoral Treg-specific (among them the IL-7 receptor-encoded by Il7r), or at least predominantly expressed by tumor-infiltrating Tregs (Tigit, Icos, Pdcd1-encoding PD-1-, Ifngr and Ctla4) (figure 2B,C). Other transcripts (one example being Stat1) were exclusively expressed by control Treg clusters or were expressed by both control and tumor-infiltrating Treg clusters (Bcl2, Stat4; figure 2B,C). The combined data suggest substantial differences in Treg gene expression as a result of lymphoma infiltration, of which several could be confirmed also at the protein level (figure 2D). We further performed a TCR repertoire analysis on tumor-infiltrating versus control Tregs; this analysis uncovered hundreds of T-cell receptor α-chain (TRA) clusters in each sample of pooled Tregs from three to four mice per condition (309 TRA clusters in the control Tregs, 189 in the tumor Tregs; cut-off of >85% amino acid sequence similarity used for assignment to TRA cluster), which together accounted for ~2/3 of all Tregs. A total of 849 cells (control Tregs, vs 463 tumor Treg cells) were assigned to the most numerically dominant cluster; TRA-based Treg clusters did not segregate with the Treg clusters identified by scRNA sequencing of non-TCR transcripts, but rather mapped more or less randomly to the Seurat clusters shown in figure 2A,E. The top 20 most abundant TRA clusters were found at similar frequencies among control and tumor-infiltrating Tregs (figure 2F). The combined data indicate that Tregs infiltrating MYC-driven lymphomas are not subject to clonal expansion, but show a very distinct (non-TCR) gene expression profile that clearly distinguishes them from normal LN Tregs.

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Figure 2

Lymphoma-infiltrating regulatory T-cells (Tregs) differ from normal lymph node (LN) Tregs with respect to their transcriptional signature. (A–C) Foxp3^YFP mice were intravenously injected with 1 Mio splenic MYC-overexpressing lymphoma cells. Foxp3/YFP⁺ Tregs from pooled axillary and inguinal LNs of three tumor-bearing and four non-tumor-bearing mice were sorted by fluorescence-activated cell sorting (FACS) at the study end point (day 11), labeled with antibody-conjugated hashtags and subjected to scRNA-Seq. UMAPs ("uniform manifold approximation and projection") (A) and heatmaps (B; 80 top differentially expressed genes) of the transcriptional profiles of 9 populations of tumor-infiltrating and control Tregs are shown alongside the expression of 10 representative transcripts in (C). (D) Single cell LN suspensions of tumor-bearing and control mice were subjected to flow cytometric analysis of the indicated surface markers; three differentially expressed surface markers are shown alongside a marker (ISOS) whose expression is unchanged. (E) TCR α-chain (TRA) sequences were extracted from the scRNAseq data and the top 10 most abundant clonotypes were mapped onto the Seurat clusters shown in (A). (F) Alluvial bar plots showing the proportions of the top 20 most abundant clonotypes among control and tumor-infiltrating Tregs. Statistical comparisons in (D) were performed by unpaired t-test (Mann-Whitney) and horizontal lines indicate medians throughout. ns, not significant; **p<0.01, ***p<0.005.

Tregs infiltrate some but not all subtypes of human DLBCL

To assess whether the level of Treg infiltration observed in our model is reflective of Treg infiltration in human DLBCL cases, we stained 105 prospectively collected specimens from newly diagnosed patients with DLBCL in Switzerland (two independent cohorts, one unpublished and one published36) for FoxP3 and subjected their tumor DNA to amplicon sequencing of the 68 most recurrently mutated DLBCL genes. We found FoxP3-positive Tregs in close proximity to tumor cells, with some tumor cell/Treg pairs forming what appeared to be direct contacts (figure 3A). Roughly one half of cases exhibited evidence of substantial (>5%) FoxP3⁺ Treg infiltration, whereas the other half contained negligible numbers of Tregs (figure 3B,C). GCB-DLBCL and ABC-DLBCL—distinguished using the Hans classification—were similarly likely to be infiltrated by Tregs (figure 3C). A comparison of the spectrum of mutations in DLBCL driver genes revealed that several mutations were over-represented in the Treg-infiltrated cases; among these were SOCS1, ACTB and EZH2 (online supplemental figure S2A-C). Other mutations were over-represented in the Treg-negative cases; among these were CD79B, KMT2D and DTX1 (online supplemental figure S2A-C). The parallel analysis of Treg infiltration of 206 DLBCL patient datasets publicly available through TCGA using cibersort42 demonstrated that the ‘MCD’ genetic subtype (defined by co-occurring MYD88 and CD79 mutations) had the lowest Treg infiltration and FOXP3 transcript levels, whereas the other three genetic subtypes introduced by Schmitz et al all had high Treg infiltration (figure 3D,E); over-represented mutations in the Treg-infiltrated cases included the TNFRSF14 variant, whereas Treg^neg/low cases were more likely to carry mutations in MYD88, PIM1 and CD79B (figure 3F, online supplemental figure S2D). The combined data suggest that DLBCL exhibiting the characteristic ‘MCD’ subtype mutations are largely devoid of Treg infiltration; the Treg-infiltrated cases are less uniform in their mutational spectrum and include cases with canonical GCB-DLBCL mutations but also cases of the ‘N1’ genetic subtype.

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Figure 3

Regulatory T-cells (Tregs) infiltrate human diffuse large B-cell lymphoma (DLBCL) to variable extents. (A) Human fresh-frozen DLBCL cryosections were stained with anti-human FoxP3 and anti-human Ki67 antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). White circles mark FoxP3⁺ Tregs in close contact with Ki67⁺ lymphoma cells. A representative section is shown of five independently stained sections. (B, C) Human DLBCL tissue microarrays (TMAs) comprising 105 cases were stained with anti-human FoxP3 antibody; FoxP3/Treg-negative and FoxP3/Treg-positive representative images are shown in (B) alongside the quantification of all cases in (C), also stratified by cell-of-origin (COO) status. Scale bar in (A) and (B), 50 µm. (D) Treg proportions (in %) infiltrating the four main DLBCL genetic subtypes were determined by performing cibersort on 206 DLBCL cases publicly available through TCGA and are shown stratified based on genetic subtype.6 (E) FOXP3 transcript levels of the cases shown in (D). (F) Co-oncoplot of the top and bottom quartiles of Treg^high and Treg^low cases (assigned as described in D), showing the most common and differentially present mutations across the two groups. **p<0.01, ***p<0.005 indicate significantly or non-significantly (ns) differentially present mutations.

Treg depletion reduces lymphoma growth and boosts anti-lymphoma effector T-cell responses

Given the high frequencies of effector Tregs within the lymphoma microenvironment in our experimental model, we set out to functionally assess the consequences of Treg depletion for lymphoma growth and anti-lymphoma immunity. Mice expressing the diphtheria toxin (DT) receptor specifically in the Treg compartment (Foxp3^DTR mice) received DT every 3 days starting on the day of lymphoma cell injection; this treatment led to a >90% reduction of Tregs in LNs, blood and other lymphoid and non-lymphoid tissues (online supplemental figure S3A and data not shown). Treg depletion resulted in a strongly reduced lymphoma burden in the LNs and spleen (figure 4A, online supplemental figure S3B); DT administration to wild-type mice had no effect (online supplemental figure S3C). The residual tumor cell population in Treg-depleted Foxp3^DTR mice failed to proliferate as determined by Ki67 staining (figure 4B). Continuous Treg depletion extended the lifespan of tumor-bearing mice by at least 3 days past the usual humane endpoint (day 11) of the experimental protocol, and measurably reduced the tumor burden as late as 14 days post injection (online supplemental figure S3D). The reduced lymphoma growth on Treg depletion was accompanied by strongly elevated frequencies of intratumoral CD4⁺ and CD8⁺ T-cells and by elevated interferon-γ (IFN-γ) production by both NK and CD4⁺ T-cells, but not CD8⁺ T-cells (online supplemental figure S3E-J). Spectral flow cytometry of the CD4⁺ and CD8⁺ T-cell compartments of Treg-replete and Treg-depleted tumor-bearing and control LNs further revealed large differences in these T-cell populations, with samples segregating into the three treatment groups (online supplemental figure S3K,L). Whereas CD4⁺ and CD8⁺ T-cells in control LNs were naïve (CD62L^hi, CD44^neg) and expressed the co-inhibitory receptor CTLA-4, their tumor-resident counterparts had downregulated CD62L and CTLA-4 but for the most part remained negative for CD44; only after Treg depletion, both compartments were fully activated and expanding as judged by their CD44, ICOS and Ki67 expression, and had differentiated into Tbet-positive type I cells; CD8⁺ T-cells additionally upregulated the activation/exhaustion markers TIM3 and LAG3 (figure 4C–H, online supplemental figure S3M,N). To address whether the emergence of CD8⁺, CD4⁺ T-cells or NK cells is functionally relevant for the reduction of lymphoma growth associated with Treg depletion, all three populations were depleted by suitable antibodies alongside the depletion of Tregs by DT in Foxp3^DTR mice. Neither CD8⁺ T-cell nor NK cell depletion reversed the effects of Treg depletion on lymphoma growth, despite a >90% reduction of the targeted populations; in contrast, the depletion of CD4⁺ T-cells—in addition to Tregs—resulted in the restoration of lymphoma growth (figure 4I, online supplemental figure S3O-Q).

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Figure 4

Lymphoma-infiltrating regulatory T-cells (Tregs) suppress CD4⁺, but not CD8⁺ T-cell or natural killer (NK) cell responses to support lymphoma growth. (A–H) FoxP3^DTR/GFP mice (‘DTR’) were intravenously injected with 1 Mio splenic MYC-overexpressing lymphoma cells and intraperitoneally injected or not, starting on the same day and continued every 3 days thereafter, with 20 ng/g body weight diphtheria toxin (+DT). Mice were examined at the study endpoint (11 days post injection) for their lymphoma burden in lymph nodes (LNs) (frequencies and absolute numbers (A)), their LN frequencies of Ki67⁺ proliferating tumor cells (summary plot and representative plots (B)) and the immunophenotypes of their LN CD4⁺ (C–E) and CD8⁺ (F–H) T-cell compartments, relative to non-tumor-bearing controls (no tumor). Marker UMAPs ("uniform manifold approximation and projection") are shown in (C) and (F); condition UMAPs are shown for 14 CD4⁺ T-cell and for 15 CD8⁺ T-cell populations in (D) and (G), alongside the quantification of the most differentially present clusters in all examined mice (n=5–7) in (E) and (H). Naïve, CD62L^int transitional and effector populations are marked in (D) and (G). Data in (A) and (B) are pooled from three experiments; data in (C)–(H) are from one experiment, but representative of three. (I) Mice were transplanted with tumor cells as described in (A)–(H), and intraperitoneally injected with an initial dose of 300 µg of anti-mouse CD4, CD8 and NK1.1 neutralizing antibody or isotype control antibody followed by twice weekly injections of 200 µg intraperitoneal either alone or together with 20 ng/g diphtheria toxin intraperitoneal. Mice were sacrificed on day 11 and the tumor burden in axillary and inguinal lymph node preparations was assessed. (J, K) C57BL/6 mice were transplanted with lymphoma cells as described in (A)–(H), and intraperitoneally injected, starting on the same day, with an initial dose of 300 µg of anti-mouse (α)CD25, αCD73, αTIM3, αTIGIT (100 µg/dose), αGITR agonist, αPD-L1 and αCTLA4 neutralizing antibody, isotype control antibody or the indicated combination of antibodies, which was followed by 200 µg maintenance doses twice a week. Tumor burden (J) and Treg frequencies (K) were determined by flow cytometry. Results in (I)–(K) are pooled from two to three experiments per experimental group. Statistical comparisons were performed by ordinary one-way analysis of variance. ns, not significant; *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. Horizontal lines indicate medians.

Various monoclonal antibodies that target the immunosuppressive activities of Tregs are currently in clinical development; among the most promising are antibodies blocking TIM3, TIGIT, CD73 and CD25 as well as a GITR agonistic antibody, especially when combined with immune checkpoint inhibitors such as anti-PD-L1.43 To address whether any of the listed antibodies in clinical development would phenocopy the effects of DT-mediated Treg ablation, we administered each antibody, alone and/or in combination with anti-PD-L1 or anti-CTLA-4. Neither the agonistic GITR-specific antibody, nor the TIGIT-blocking or CD25-blocking antibodies reduced the lymphoma burden relative to isotype control antibodies; in contrast, robust effects were obtained with anti-CD73 and anti-TIM3 blocking antibodies, alone and together with PD-L1 neutralization (figure 4J). The reduction of lymphoma growth was accompanied by a reduction of Treg infiltration under all effective treatments (figure 4K), with the latter being quite predictive of treatment success. Whether Treg depletion is a cause or consequence of the reduction in the lymphoma burden cannot be deduced from these data. The combined results suggest that Treg depletion effectively reduces the tumor burden, which can be attributed to reinvigorated CD4⁺ effector T-cell, but not to CD8⁺ T-cells or NK cell responses. Further experimental evidence suggests that Tregs suppress CD4⁺ T-cells via their expression of the ATP hydrolyzing enzymes CD39 and CD73.

Tregs promote early stages of lymphoma engraftment and growth

To study the dynamics of lymphoma engraftment and growth, and of Treg support of these processes in more detail, we first tracked and quantified lymphoma cells and Tregs in the LNs and spleen, and also in the blood and bone marrow. Tumor cells were first detectable in blood, spleen and bone marrow at 6 days post injection, and appeared in the LNs 2 days later (figure 5A, online supplemental figure S4A). Treg frequencies among all CD4⁺ T-cells differed substantially from one examined tissue to the other (being highest in the bone marrow at 50%, and lowest in the LNs and blood at <10%), and increased Treg frequencies were detected in all tissues on lymphoma cell injection (figure 5B, online supplemental figure S4B). The kinetics of increased Treg frequencies coincided with the kinetics of lymphoma cell appearance in the three tissues (day 8 in the LNs, day 6 in bone marrow and spleen), but not in blood, where only modest Treg increases were visible, and only with a 3-day delay at day 9 (figure 5A,B, online supplemental figure S4A,B). To address at which point in time Treg depletion would have the strongest impact on the lymphoma burden, we initiated DT treatments either 2 days before, or on days 1, 2, 3, 4 or 5, or on day 9 post tumor cell injection, and continued the treatment until the study endpoint. Only the early depletion of Tregs—initiated up until day 3, but not later depletion effectively reduced the lymphoma burden, and lymphoma cell proliferation as assessed by Ki67 staining (figure 5C,D). Early, but not late Treg depletion further also boosted the intratumoral CD4⁺ and CD8⁺ effector T-cell responses that coincided with tumor control (online supplemental figure S4C-E). Indeed, a few doses of DT administered up until day 4 (resulting in the restoration of Tregs back to normal levels by the end of the study) were as effective as continuous Treg depletion until the study endpoint at suppressing the lymphoma burden (figure 5E–G). This was accompanied by increased CD4⁺ T-cell activation, as indicated by elevated levels of IFN-γ, Ki67 and PD-1 (figure 5H). The combined results indicate that Tregs facilitate lymphoma cell engraftment and growth in our experimental model, likely by affecting CD4⁺ effector T-cell priming, and have a critical role long before elevated Treg levels (and lymphoma cells) appear in affected tissues and the bloodstream.

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Figure 5

Regulatory T-cells (Tregs) act during early stages of lymphomagenesis. (A, B) C57BL/6 mice were intravenously injected with 1 Mio splenic MYC-overexpressing lymphoma cells and examined with respect to the lymph node (LN) lymphoma burden (A) and intratumoral Treg frequencies (B) at the indicated time points post transplantation. Means±SD are shown for three mice per group. (C, D) FoxP3^DTR/GFP mice were intravenously injected with 1 Mio splenic MYC-overexpressing lymphoma cells and 20 ng/g diphtheria toxin was administered intraperitoneal starting either 2 days prior to tumor cell inoculation, on the day of inoculation, or at 2, 3, 4, 5 and 9 days post inoculation; treatments were continued until the study endpoint (11 days post injection) with injections every 3 days. Tumor burden (C) and frequencies of Ki67⁺ proliferating tumor cells (D) were determined by flow cytometry. (E–H) FoxP3^DTR/GFP mice were intravenously injected with 1 Mio splenic MYC-overexpressing lymphoma cells; 20 ng/g diphtheria toxin was injected intraperitoneal on the day of tumor cell inoculation followed by a second and final injection on day 4, or on days 4, 7 and 10. Tumor burden (E), frequencies of Ki67⁺ proliferating tumor cells (F), Treg frequencies (G) and frequencies of IFNγ⁺, Ki67⁺ and PD-1⁺ CD4⁺ T-cells (H) were determined by flow cytometry. Horizontal lines indicate medians. Statistical comparisons were performed by ordinary one-way analysis of variance. ns, not significant; *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Treg-specific IL-2 starvation reduces Treg infiltration and lymphoma growth

As IL-2 is a key cytokine driving Treg differentiation by promoting Foxp3 expression in naïve T-cells,44 we asked whether Treg-specific IL-2 starvation might phenocopy the effects of Treg ablation in Foxp3^DTR mice, and whether tumor B-cells might directly act as a source of IL-2. Indeed, we found that MYC-expressing lymphoma cells produce large amounts of IL-2, which is not the case for normal B-cells in the same LNs (figure 6A). Human DLBCL cell lines vary in their IL-2 expression, with some expressing enormous amounts, others modest amounts and yet others no IL-2 whatsoever (online supplemental figure S5A). To specifically starve Tregs, but not effector T-cells of IL-2, we took advantage of an antibody that selectively prevents IL-2 binding by the α-chain of the IL-2 receptor, CD25, which is expressed by Tregs at much higher levels than by effector T-cells.45 Anti-IL-2 antibody treatment reduced Treg frequencies without affecting CD4⁺ or CD8⁺ effector T-cell frequencies, and dose-dependently reduced the lymphoma burden (figure 6B and C, online supplemental figure S5B); CD25 expression was indeed much higher on tumor-infiltrating Tregs than on intratumoral CD4⁺ and CD8⁺ T-cells (online supplemental figure S5C). The small residual Treg population that escaped starvation did not immunophenotypically differ from tumor-infiltrating Tregs in isotype-treated tumor-bearing mice as determined by spectral flow cytometry (figure 6D, online supplemental figure S5D). Anti-IL-2 treatment had no effect on the tumor burden in T-cell (and Treg)-deficient RAGγc^-/- mice (figure 6E), suggesting that the beneficial effects of IL-2 neutralization are probably not tumor cell-intrinsic. Interestingly, IL-2 neutralization had no effect on the quantity or immunophenotype of normal Tregs in control mice, the surface marker expression and frequencies of which were unchanged; indeed, Tregs in non-tumor-bearing LNs expressed much less CD25 than their tumor-infiltrating counterparts (figure 6F,G, online supplemental figure S5E,F). The combined results provide experimental evidence for the efficacy of Treg-specific IL-2 starvation in controlling lymphoma growth in this MYC-driven model.

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Figure 6

Anti-IL-2 treatment reduces regulatory T-cell (Treg) infiltration and lymphoma growth. (A) C57BL/6 mice were injected intravenously with 1 Mio splenic MYC-overexpressing lymphoma cells. Interleukin-2 (IL-2) expression by tumor cells and normal B-cells in single cell lymph node (LN) preparations was determined by intracellular cytokine staining (summary plot and representative flow cytometry plots are shown in (A) at the study endpoint. (B–D) Mice were treated Intraperitoneally twice weekly with either 50 µg or 100 µg anti-IL-2 antibody or isotype control antibody starting on the day of tumor cell inoculation. Tumor burden (summary plot and representative plots, (B) and Treg frequencies (C) were determined by flow cytometry. Condition UMAPs ("uniform manifold approximation and projection") of Tregs from anti-IL-2 (100 µg) or isotype control antibody-treated mice are shown in (D). (E) RAGγc^-/- mice were injected with lymphoma cells and treated with 100 µg of anti-IL-2 antibody or isotype control antibody as described in (B)–(D). Frequencies of tumor cells, and Ki67⁺ tumor cells were determined at the study endpoint (day 10). (F, G) Non-tumor-bearing C57BL/6 mice were treated as described in (E) and examined on day 10 with respect to their LN Treg frequencies (F) and Treg immunophenotypes as determined by spectral flow cytometry (G). Data in (A) and (B) are pooled from three studies; data in (E) and (F) are from one study. Statistical comparisons were performed either by ordinary one-way analysis of variance or by unpaired t-test (Mann-Whitney). ns, not significant; **p<0.01, ***p<0.005, ****p<0.001. Horizontal lines indicate medians.

IL-2 neutralization synergizes with CD40 activation in promoting durable responses

As Treg starvation alone is unlikely to confer long-term survival benefits in our aggressive lymphoma model, we asked whether anti-IL-2 therapy would synergize with other immunotherapies in promoting durable responses beyond the usual 10-day timeframe of our model. As we found aberrantly low expression of the co-stimulatory surface marker CD40 on tumor B-cells relative to normal B-cells (figure 7A), and detected a strong increase in CD40 expression on tumor B-cells but not normal B-cells on Treg depletion (figure 7B), we set out to determine whether targeting CD40 using an agonistic antibody would reduce the lymphoma burden, with and without additional Treg depletion by IL-2 neutralization. Twice-weekly injection of a CD40-specific agonistic antibody effectively reduced the lymphoma burden and lymphoma cell proliferation (figure 7C,D). In fact, a single anti-CD40 injection administered on the day of tumor cell injection was sufficient to prevent lymphoma growth until the humane endpoint was reached for all isotype antibody-treated mice (figure 7C,D). As CD40 activation led to a strong Fas upregulation on MYC-expressing lymphoma cells (online supplemental figure S6A), and simultaneously to Fas ligand (FasL) upregulation on NK cells (online supplemental figure S6B), we asked whether the tumoricidal activity of the CD40 agonistic antibody required the cytotoxic activities of T-cells or NK cells, or of FasL. CD40 activation was as effective in RAG2γc^-/- mice-lacking T-lymphocytes and B-lymphocytes and NK cells—as in wild-type C57BL/6 mice at reducing lymphoma growth (figure 7E) and could not be counteracted by FasL blockade (online supplemental figure S6C). A single dose of CD40 agonist administered 1 day prior to the study endpoint was sufficient to reduce the lymphoma burden (figure 7E) and allowed us to determine that tumor cells became annexin-V-positive under treatment, indicating apoptotic cell death induced by CD40 activation (figure 7F). Normal B-cells in the same lymph nodes did not undergo increased apoptosis on CD40 activation, and even appeared to proliferate more (online supplemental figure S6D,E).

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Figure 7

Regulatory T-cell (Treg) depletion and CD40-driven cytotoxicity synergize for sustained durable responses. (A) C57BL/6 mice were injected intravenously with 1 Mio splenic MYC-overexpressing lymphoma cells. CD40 surface expression of tumor cells and normal B-cells in single cell lymph node (LN) preparations was determined by flow cytometry. (B) FoxP3^DTR/GFP mice were injected with lymphoma cells; 20 ng/g diphtheria toxin was injected intraperitoneally every 3 days. CD40 expression on tumor cells and normal B-cells was evaluated by flow cytometry on day 10 post transplantation. (C, D) C57BL/6 mice were treated with either a single 100 µg dose of anti-CD40 agonistic antibody or by twice weekly injections of anti-CD40 or isotype control antibody. Frequencies of lymphoma cells (C) and Ki67⁺ lymphoma cells (D) were determined by flow cytometry. (E) RAGγc^-/- or C57BL/6 mice were injected with lymphoma cells and treated with 100 µg anti-CD40 agonistic antibody or isotype control antibody either at from the day of tumor cell inoculation or only on day 9 (1 day prior to the study endpoint). Absolute numbers of tumor cells are shown in (E). (F) AnnexinV⁺ apoptotic lymphoma cells were quantified by flow cytometry on single dose (day 9) αCD40 or isotype control antibody treatment; a summary plot and representative plots are shown. (G–I) Human FFPE DLBCL specimens spotted on tissue microarrays (TMAs) were stained for CD40; representative images are shown for each score (0–3; G) and the scores for all 110 analyzed cases are shown in (H).The frequencies of FoxP3+ and FoxP3– cases are shown in (I) for each CD40 score. (J–L) C57BL/6 mice were injected with lymphoma cells and treated either with anti-CD40 agonistic antibody, isotype control antibody or with a combination of anti-CD40 and anti-IL-2 antibody (100 µg/dose) for the indicated durations. Mice were sacrificed on either day 10 or on day 26, and the tumor burden (summary plot and representative plots; J) was determined by flow cytometry. Kaplan-Meier curves and Treg frequencies are shown in (L). Data in (B), (C), (D), (J) and (K) are pooled from two studies, all others are from one representative study of two. Statistical comparisons were performed either by ordinary one-way analysis of variance or by unpaired t-test (Mann-Whitney). ns, not significant; **p<0.01, ***p<0.005, ****p<0.001. Horizontal lines indicate medians.

To address how common CD40 expression is among patients with DLBCL, we stained 110 samples from the two prospectively collected patient cohorts mentioned earlier for CD40. CD40 expression varied radically across patients, with some tumors expressing no CD40 whatsoever and others expressing copious amounts (figure 7G). Roughly one-fourth of cases expressed high, moderate, low levels of, or no CD40, respectively (figure 7H). No bias towards ABC-DLBCL or GCB-DLBCL subtype was observed (figure 7H). A similar variability of CD40 expression was observed in a panel of DLBCL cell lines (online supplemental figure S6F). Treg infiltration and CD40 expression appeared to be independent variables in the 94 patients with DLBCL for whom both CD40 and FoxP3 immunohistochemical stainings were evaluable (figure 7I).

Although highly efficient in the short term at reducing the lymphoma burden, CD40 activation alone did not sustain durable responses, neither if the treatment was continued for another 2 weeks, nor if it was discontinued at the usual study endpoint of 10 days (figure 7J,K). However, when CD40 activation was combined with IL-2 neutralization, with both treatments beginning on the day of tumor cell injection, all mice on the dual treatment were symptom-free and had a minimal or undetectable tumor burden at the study endpoint (figure 7J,K). Treg infiltration was reflective of the tumor burden, and lowest in the absence of tumor and under IL-2 neutralization (figure 7L). The combined results implicate CD40 as a promising target enabling direct killing by apoptosis of a subset of CD40-expressing DLBCL; the response to CD40-directed therapy is further improved by Treg depletion.