In‐vitro effect of pembrolizumab on different T regulatory cell subsets

S M Toor; A S Syed Khaja; I Alkurd; E Elkord

doi:10.1111/cei.13060

. 2017 Nov 3;191(2):189–197. doi: 10.1111/cei.13060

In‐vitro effect of pembrolizumab on different T regulatory cell subsets

S M Toor ^1,², A S Syed Khaja ², I Alkurd ³, E Elkord ^1,^2,^4,^✉

PMCID: PMC5758372 PMID: 28963773

Summary

Programmed death‐1 (PD‐1) and interactions with PD‐ligand 1 (PD‐L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour‐infiltrating T cells and regulatory T cell (T_reg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti‐PD‐1 antibody, pembrolizumab, on various T_reg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD‐1 expression in both cohorts. We found that PD‐1 was expressed mainly on CD4⁺CD25⁺ T cells and pembrolizumab had a greater effect on PD‐1 expression in CD4⁺CD25⁻ T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of T_reg‐related markers, including cytotoxic T lymphocyte antigen‐4 (CTLA‐4), CD15s, latency‐associated peptide (LAP) and Ki‐67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)⁻Helios⁺ T_reg in HD, but it is expressed on FoxP3⁺Helios⁻ T_reg subset in addition to FoxP3⁻Helios⁺ T_reg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/−Helios^+/− T_reg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T_reg or change their phenotype or function but rather blocks signalling via the PD‐1/PD‐L1 axis in activated T cells.

Keywords: cancer, regulatory T cells, T cells, tumour immunology

Introduction

Immune checkpoint proteins such as cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4) and programmed death 1 (PD‐1) play important roles in peripheral tolerance by regulating negatively the activation of T cell‐mediated immune responses. CTLA‐4, a homologue of CD28, is expressed on activated T cells, where it outcompetes CD28 in binding to CD80 and CD86, dampens the stimulatory signals and attenuates T cell activation 1, 2. Similarly, PD‐1 is expressed on activated T cells, and upon binding to its ligands (PD‐L1 and PD‐L2) inhibits T cell functions 3.

In non‐pathological conditions, T cells are activated on antigenic stimulation, resulting in the generation of antigen‐specific T effector cells, which then target cells expressing that specific antigen. As a control mechanism, activation of T cells also results in the up‐regulation of PD‐1 on their surfaces, which then binds to PD‐L1 and PD‐L2, relays inhibitory signals to activated T cells and down‐modulates their activation, and thus helps in controlling autoimmunity 1. However, in chronic infections and pathological conditions such as cancer, due to prolonged and consistent antigenic stimulation, PD‐1 expression on T cells remains elevated, leading to ‘T cell exhaustion’, identified by decreased cytokine production, proliferation and cytotoxic activity 4, 5. This ‘hyporesponsive’ state results in their inability to mount immunological responses against the identified antigens 6. The PD‐1/PD‐L pathway is thus exploited by various cancers to evade tumour‐specific host immune responses, and blockade of these interactions using PD‐1 and/or PD‐L1‐specific antibodies results in partial restoration of T effector cell functions. PD‐1 blockade using monoclonal antibodies also results in down‐regulation of forkhead box protein 3 (FoxP3) expression in regulatory T cells (T_reg) and prevented T_reg‐mediated suppression of melanoma antigen‐specific cytotoxic T lymphocytes 7.

Recently, immune checkpoint blockade has emerged as an important approach in developing effective cancer immunotherapies, and the success of recent clinical trials using antibodies targeting CTLA‐4 (ipilimumab) 8, PD‐1 (pembrolizumab) 9, lambrolizumab 10, nivolumab 11, 12 and PD‐L1 (atezolizumab) 13 in various cancers has elicited strong interest supporting this strategy 14, 15, 16. Ipilimumab, the first approved immune checkpoint inhibitor targeting CTLA‐4, improved overall survival in a Phase III clinical trial in metastatic melanoma patients 8, 17. Pembrolizumab (Keytruda) is a humanized monoclonal immunoglobulin (Ig)G4k antibody 18, with a strong binding affinity to PD‐1 19. It evokes a strong anti‐tumour immune response by binding to PD‐1 and blocking its interactions with its ligands. Pembrolizumab has been approved for the treatment of metastatic melanoma and has also shown positive responses in patients with triple‐negative breast cancer, non‐small‐cell lung cancer, gastric cancer, advanced bladder cancer and head and neck cancer 16. However, the effects of pembrolizumab on various T cell subsets and their markers have not yet been investigated fully.

In this study, we investigated the effect of pembrolizumab on different T_reg subsets in peripheral blood of healthy donors (HD) and primary breast cancer (PBC) patients. Pembrolizumab effectively blocked PD‐1 expression but did not affect the expression of other T_reg‐related markers. Additionally, we show that pembrolizumab‐mediated blockade only affects CD4⁺CD25⁻ non‐T_regs. Our results suggest that pembrolizumab may reverse immune escape through blockage of PD‐1/PD‐L1 interaction, and not by altering T_reg phenotype or function.

Materials and methods

Isolation of peripheral blood mononuclear cells

The study was performed after ethical approval from Al Ain Medical District Research Ethics committee, Al Ain, United Arab Emirates (Protocol no. 13/23‐CRD 244/13). All patients and HD provided written informed consent prior to sample collection and all experiments were performed in accordance with relevant guidelines and regulations. Peripheral blood was collected in heparinized tubes from eight HD and eight PBC patients.

Peripheral blood mononuclear cells (PBMC) were isolated from fresh whole blood by density‐gradient centrifugation using Histopaque‐1077 (Sigma‐Aldrich, St Louis, MO, SA). PBMC were frozen in cryovials at a density of 5 million cells per 1 ml freezing media [50% fetal calf serum (FCS), 40% RPMI‐1640 media and 10% dimethylsulphoxide (DMSO)] to be used in batches for subsequent staining and analysis.

PBMC treatment with pembrolizumab

Freshly thawed PBMC were suspended at 2 × 10⁶ cells/well in 2 ml complete medium (RPMI‐1640 supplemented with 2 mM L‐glutamine, 10% FCS and 1% penicillin/streptomycin); 24‐well non‐treated culture plates were precoated with plate‐bound 2 μg/ml anti‐CD3 antibody [muromonab‐CD3 (OKT3) clone; eBioscience, San Diego, CA, USA] and 2 μg/ml anti‐CD28 antibody (CD28.2 clone; eBioscience) for 2·5 h at 37°C. PBMC were either plated as ‘non‐activated’ in non‐coated wells or ‘activated’ in precoated wells. Plated cells were then treated with anti‐PD‐1 monoclonal antibody, Keytruda (pembrolizumab; Merck, Kenilworth, NJ, USA) at different concentrations (0, 1, 2, 5 and 10 µg/ml), and were incubated for 24 h in a humidified incubator at 37°C and 5% CO₂.

Multi‐parametric flow cytometry

After treatment, cells were collected in fluorescence activated cell sorter (FACS) tubes, washed and resuspended in 100 µl staining buffer [phosphate‐buffered saline (PBS) with 2% FCS and 0·1% sodium azide]. Cells were then blocked for Fc receptor using FcR Blocker (Miltenyi Biotec, Bergisch Gladbach, Germany), and surface and intracellular staining were performed as described previously 20. To gate out dead cells, Fixable Viability Dye eFluor^® 660 (FVD 660; eBioscience) was used. The following antibodies were used for staining: CD3‐allophycocyanin (APC)‐H7 (clone SK‐7; BD Biosciences, Oxford, UK), CD4‐Alexa Fluor 700 (clone RPA‐T4, BioLegend, San Diego, CA, USA), CD25‐phycoerythrin cyanin 7 (PE/Cy7; clone M‐A251, BioLegend), latency‐associated peptide‐phycoerythrin (LAP‐PE) (clone Tw4‐2F8; BioLegend), PD‐1‐PE/Dazzle™ 594 (clone EH12.2H7; BioLegend), PD‐1‐peridinin chlorophyll (PerCP)‐eFluor^® 710 (clone J105; eBioscience), cytotoxic T lymphocyte antigen 4 (CTLA‐4)‐PerCP‐eFluor^® 710 (clone 14D3; eBioscience), CD15s‐APC (clone CSLEX1; BD Biosciences), Ki‐67‐PerCP/Cy5.5 (clone B56; BD Biosciences), forkhead box protein 3 (FoxP3)‐PE‐Cy7 (clone PCH101; eBioscience) and Helios‐fluorescein isothiocyanate (FITC) (clone 22F6; BioLegend).

All data were acquired with a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Biosciences) and analysed on BD FACSuite software (BD Biosciences).

Statistical analyses

All statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) and Microsoft Excel (Microsoft Corporation, Washington DC, USA). The Shapiro–Wilk normality test followed by paired/Wilcoxon's matched‐pairs signed‐rank test or unpaired/Mann–Whitney tests were used to examine the differences within groups or between groups, respectively. Data are represented as mean ± standard error of the mean (s.e.m.). A P‐value < 0·05 was considered statistically significant.

Results

Pembrolizumab effectively blocks PD‐1 but does not affect CTLA‐4 expression in non‐activated and activated T cells

We investigated the effect of pembrolizumab on CD4⁺ T cell markers in steady and activated states. We first tested different concentrations of pembrolizumab in non‐activated and activated PBMC for 24 and 48 h drug exposure (Supporting information, Fig. S1). We found that drug exposure for 24 h at a concentration of 2 µg/ml reduced PD‐1 expression significantly. This concentration did not affect the viability of T cells (data not shown). Therefore, we used this concentration and duration of drug exposure for further investigations.

Representative flow cytometric plots of PD‐1 and CTLA‐4 expressions in non‐activated and activated PBMC from HD and PBC are shown in Fig. 1a,d. Within CD4⁺ T cells, PD‐1 expression was significantly higher in PBC patients compared with HD in steady state (Fig. 1a,b). Pembrolizumab treatment reduced PD‐1 expression significantly in CD4⁺ T cells in both cohorts in non‐activated and activated PBMC (Fig. 1b,e). Treatment with pembrolizumab did not affect CTLA‐4 expression in steady and activated states (HD, 8·9 ± 0·8 versus 7·4 ± 1·3 and PBC, 14·8 ± 1·5 versus 14·3 ± 1·7; Fig. 1c,f).

Effect of pembrolizumab on programmed death 1 (PD‐1) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4) expression in CD4⁺ T cells in healthy donors (HD) and primary breast cancer patients (PBC). Peripheral blood mononuclear cells (PBMC) from HD and PBC cultured for 24 h (with plate‐bound anti‐CD3 and anti‐CD28 antibodies for activated cells) and treated with 2 μg/ul pembrolizumab (for treated cells) were stained for CD4, PD‐1 surface markers and intracellular expression of CTLA‐4. Live cells were gated using FVD 660 viability dye first. Representative flow cytometric plots of PD‐1 and CTLA‐4 expression with effect of pembrolizumab on non‐activated and activated CD4⁺ T cells in HD and PBC are shown (a,d). Scatter‐plots showing drug effect on PD‐1⁺CTLA4^+/− in HD and PBC in non‐activated (b) and activated states (e). Scatter‐plots showing effect of pembrolizumab on PD‐1^+/−CTLA4⁺ T cells in HD and PBC in non‐activated (c) and activated states (f). [Colour figure can be viewed at wileyonlinelibrary.com]

PD‐1 is expressed mainly on CD4⁺CD25⁺ T cells and blockade by pembrolizumab is mainly within CD4⁺CD25⁻ T cells

We investigated PD‐1 expression within CD4⁺CD25^+/− T cells to determine which T cell subset is affected by pembrolizumab. Representative histogram plots for PD‐1 expression in CD4⁺CD25^+/− T cells in HD and PBC are shown in Fig. 2a. We found that PD‐1 levels were significantly higher in CD4⁺CD25⁺ T cells compared to CD4⁺CD25⁻ T cells in both HD and PBC patients. Pembrolizumab treatment resulted in a significant reduction in PD‐1 expression in both subsets (Fig. 2b). PD‐1 was affected greatly by pembrolizumab in CD4⁺CD25⁻ T cells compared to CD4⁺CD25⁺ populations in HD and PBC patients (Fig. 2a,b). There was nearly 90% reduction in PD‐1 expression in CD4⁺CD25⁻ T cells after pembrolizumab treatment compared to approximately 20% reduction in PD‐1 expression in CD4⁺CD25⁺ T cells after treatment in HD and PBC patients (Fig. 2c).

Effect of pembrolizumab on programmed death 1 (PD‐1) expression in CD4⁺CD25^+/− T cell subsets in healthy donors (HD) and primary breast cancer patients (PBC). Peripheral blood mononuclear cells (PBMC) from HD and PBC were stained for CD4, CD25, PD‐1 and CTLA4. Representative flow cytometric histogram plots show PD‐1 expression in CD25^+/− T cell subsets within CD4⁺ cells from HD and PBC patients (a). Bar plot shows effect of pembrolizumab on PD‐1 expression in CD25^+/− cells in HD and PBC (b). Bar plots showing pembrolizumab has a greater effect on PD‐1 expression in CD25⁻ T cells compared to CD25⁺ T cells in HD and PBC (c). [Colour figure can be viewed at wileyonlinelibrary.com]

Levels of different subsets of FoxP3^+/−Helios^+/− T_reg are not affected by pembrolizumab

FoxP3 is an established marker for T_reg 21, and Helios expression is essential for their suppressive characteristics 22, 23, 24. Representative flow cytometric plots for FoxP3 and Helios expression in CD4⁺ T cells in non‐activated and activated PBMC from HD and PBC are shown in Fig. 3a and d, respectively. We have recently reported increased levels of circulating and tumour‐infiltrating FoxP3⁺Helios⁺ T_reg in breast cancer patients 20. Pembrolizumab did not affect the levels of FoxP3⁺Helios^+/− and FoxP3^+/−Helios⁺ T_reg in HD and PBC in non‐activated (Fig. 3b,c) and activated states (Fig. 3e,f).

Effect of pembrolizumab on forkhead box protein 3 (FoxP3) and Helios expression in CD4⁺ T cells in healthy donors (HD) and primary breast cancer patients (PBC). Representative flow cytometric plots show intracellular staining for FoxP3 and Helios expression in CD4⁺ T cells from HD and PBC with effect of pembrolizumab in non‐activated and activated cells (a,d). Scatter‐plots showing effect of pembrolizumab in HD and PBC on FoxP3⁺Helios^+/− and FoxP3^+/−Helios⁺ subsets in non‐activated cells (b,c) and activated cells (e,f) in HD and PBC. [Colour figure can be viewed at wileyonlinelibrary.com]

Ki‐67 expression is detected mainly in CD25⁺ and FoxP3⁺Helios⁻, FoxP3⁺Helios⁺ and FoxP3⁻Helios⁺ T cell subsets

We investigated Ki‐67 expression in CD4⁺ T cells, which is widely established as a marker of proliferation. We found a reduction in Ki‐67 expression, which did not reach statistical significance following pembrolizumab treatment (Supporting information, Fig. S2a). We then looked for Ki‐67 expression in CD4⁺CD25^+/− T cell subsets and found that CD25⁺ T cells had significantly higher Ki‐67 expression than CD25⁻ T cells (Supporting information, Fig. S2b). This significantly higher expression of Ki‐67 in CD25⁺ populations then led us to investigate which subset of FoxP3 and Helios populations within CD4⁺ T cells showed the highest Ki‐67 expression. Ki‐67 expression was very low in FoxP3⁻Helios⁻ T cells, whereas its expression was similar in FoxP3 and/or Helios‐expressing T_reg subsets (Supporting information, Fig. S2c). Pembrolizumab treatment lowered its expression in these subsets, but the decrease was not statistically significant (Supporting information, Fig. S2c). Taken together, pembrolizumab did not have any significant effect on Ki‐67 expression in non‐activated and activated states.

Differential expression of CD15s in different FoxP3‐ and Helios‐expressing T_reg subsets in HD and PBC patients, which was not affected by pembrolizumab

Next, we investigated CD15s and LAP expression, which serve as functional markers for T cell suppressive activity 22, 25 on CD4⁺ T cells. There was no reduction in CD15s or LAP expression in CD4⁺ T cells in HD and PBC patients in steady and activated states following pembrolizumab treatment (Supporting information, Fig. S3).

We then investigated CD15s and LAP expression in different FoxP3^+/−Helios^+/− T_reg subsets in HD and PBC patients. Representative flow cytometric plots of CD15s and LAP expression in different T_reg subsets are shown in Fig. 4a. CD15s expression in HD was observed mainly in the FoxP3⁻Helios⁺ T_reg subset (36·8 ± 7·3%), which was significantly higher compared with its expression in FoxP3⁺Helios⁺ (11·2 ± 1·2%) and FoxP3⁺Helios⁻ (7·7 ± 1·4%) T_reg subsets in the non‐activated state (Fig. 4a).

Latency‐associated peptide (LAP) and CD15S expression in different forkhead box protein 3 (FoxP3) and Helios subsets in healthy donors (HD) and primary breast cancer patients (PBC). Representative flow cytometric plots show LAP and CD15s co‐expression in different FoxP3^+/−Helios^+/− subsets in non‐activated and activated peripheral blood mononuclear cells (PBMC) from HD and PBC, followed by bar plots showing CD15s expression in respective T cell subsets (a). Bar plots show effect of pembrolizumab on CD15s expression in different FoxP3 and Helios subsets in HD and PBC in non‐activated and activated cells (b). [Colour figure can be viewed at wileyonlinelibrary.com]

Interestingly, the CD15s expression pattern was different in PBC patients compared to HD (Fig. 4a,b). In PBC patients, CD15s expression was observed mainly in the FoxP3⁺Helios⁻ T_reg subset (20·7 ± 6·4%), followed by FoxP3⁻Helios⁺ (16·6 ± 5·8%) and FoxP3⁺Helios⁺ (12·1 ± 3·5%) T_reg subsets in a non‐activated setting. Pembrolizumab did not have any effect on CD15s expression in different FoxP3/Helios T_reg subsets in HD or PBC patients in both non‐activated and activated settings (Fig. 4b). LAP expression also remained unaffected in different FoxP3/Helios subsets in non‐activated and activated settings in the study cohorts (data not shown).

Discussion

T cell‐mediated immune response relies primarily on antigen recognition by the T cell receptor (TCR). Various co‐stimulatory and inhibitory pathways are involved in the regulation of T cell responses under normal pathological conditions. Interactions between PD‐1 and its ligand PD‐L1 regulate peripheral tolerance via down‐regulation of effector T cell levels, induction of apoptosis of tumour‐infiltrating T cells and promoting CD4⁺ T cell differentiation into suppressive FoxP3⁺ T_reg 26, thereby providing an immune permissive environment for tumours to progress. Effective blockade of PD‐1/PD‐L1 can eradicate these deleterious effects of T cell‐mediated immunity in cancer progression 2, 27.

Pembrolizumab was the first anti‐PD‐1 antibody approved by the Food and Drug Administration (FDA) given to patients with metastatic melanoma 9. PD‐L1 expression was associated with poor prognosis in a study conducted by Qin et al. 28, who assessed correlations between PD‐L1 expression and patient clinical characteristics in 870 breast cancer patients, and recently a clinical trial has assessed the safety profile and anti‐tumour efficacy of pembrolizumab in triple‐negative breast cancer patients that have shown acceptable safety and promising anti‐tumour effects 29, with Phase II trials currently under way (clinicaltrials.gov identifier: NCT02447003). Additionally, Adams et al. 30 reported a remarkable response to combination therapy consisting of pembrolizumab and Nab‐paclitaxel in a patient with metaplastic breast cancer, associated commonly with over‐expression of PD‐L1 31, as part of an ongoing clinical trial (clinicaltrials.gov identifier: NCT02752685).

In the present study, we investigated the effect of pembrolizumab on various T_reg‐associated molecules, and compared between HD and PBC patients. T_reg are comprised mainly of thymic‐derived T_reg and peripheral T_reg, which are broadly recognized by CD25 and FoxP3 expression 22. FoxP3 is a key transcription factor responsible for their functional characteristics, as shown by the development of autoimmune/inflammatory disease, following FoxP3⁺ T_reg depletion 32. The prognostic significance of FoxP3⁺ T_reg in breast cancer is contradictory; however, as the majority of studies in solid malignancies have shown reduced survival with expanded FoxP3 T_reg, some studies have reported a favourable association between T_reg expansion and survival; for instance, in oestrogen receptor‐negative breast cancer 33, 34. We have recently reported an expansion of CD4⁺FoxP3⁺Helios⁺ T_reg in the tumour microenvironment (TME) of breast cancer patients and have suggested a negative prognosis based on the co‐expression of immune inhibitory molecules, including CTLA‐4 and PD‐1 20. Additionally, we reported that tumour‐infiltrating CD4⁺ and CD8⁺ T cells show similar PD‐1 expression in PBC patients 20. In the present study, we found that PD‐1 expression on circulating CD4⁺ T_reg in PBC patients is reduced effectively by pembrolizumab.

PD‐1 is expressed on exhausted T cells, which results in inhibition of T cell proliferation, cytokine secretion and cytotoxic effects through various pathways 1, while CTLA‐4 is expressed constitutively on T_reg and is up‐regulated on activated T cells. CTLA‐4 affects T cell‐mediated immunosuppression and peripheral tolerance greatly, as shown by the spontaneous T_reg proliferation and development of autoimmune disorders in CTLA‐4‐deficient mice 35. Furthermore, genetic polymorphisms in PD‐1 and CTLA‐4 have been associated with increased risk of developing breast cancer 36. The unaffected CTLA‐4 expression in T cells following pembrolizumab treatment in our study confirmed the distinct pathways employed by PD‐1 and CTLA‐4 in immune inhibition.

We have reported that PD‐1 is expressed mainly on CD4⁺CD25⁺ T cells, and pembrolizumab has a greater effect on PD‐1 expression in CD4⁺CD25⁻ T cells that are comprised of non‐T_reg and/or non‐activated T cells. Peripheral T_regs are induced from naive T_regs via peripheral antigen presentation 21. Su et al. 37 suggested blocking of naive CD4⁺ T cell recruitment as an effective mechanism to reverse immunosuppression in breast cancer. Additionally, Plitas et al. 38 showed an expansion of suppressive intratumoral T_regs in breast cancer with distinct gene expression compared to circulating T_regs. In addition to FoxP3, the Ikaros zinc finger transcription factor, Helios and CD15s (Sialyl Lewis x) have been linked to the suppressive potential of T_regs. Miyara et al. 25 suggested CD15s⁺FoxP3^high as highly suppressive T_regs, because depletion of CD15s⁺CD4⁺ T cells from human blood enhanced in‐vitro anti‐tumour and anti‐viral antigen responses. Moreover, Helios has been reported previously as a vital marker for highly suppressive T_regs in the activated state, which express LAP and GARP (glycoprotein A repetitions predominant) and secrete interleukin (IL)‐10 23, 39. We found LAP expression mainly in Helios⁺ T_reg in both HD and PBC. Additionally, we found CD15s expression mainly in FoxP3⁻Helios⁺ T_reg in HD, but PBC showed CD15s expression in FoxP3⁺Helios⁻ T_reg. Therefore, our results suggest that expanded CD4⁺ T cells in PBC are highly suppressive, with PD‐1 expression indicative of their suppressive potential and contributing towards worse prognosis. However, the overall levels of FoxP3 and Helios‐expressing T_reg remained unchanged in both cohorts following pembrolizumab treatment. Therefore, pembrolizumab does not affect the potentially highly suppressive T_regs in the breast cancer setting. Additionally, we have shown Ki‐67 expression is mainly in FoxP3 and/or Helios‐expressing T_reg subpopulations and not in FoxP3⁻Helios⁻ populations, which is indicative of T_reg proliferation in situ. Tumour‐infiltrating CD25⁺FoxP3⁺ T_regs have also been reported previously with high Ki‐67 expression in other human cancers and indicated efficient in‐situ proliferation.

Recently, Ribas et al. 40 also reported no change in T_regs, defined as CD45⁺CD3⁺CD4⁺CD25^highCD127^low in melanoma patients treated with pembrolizumab, and suggested expansion of CD8⁺ effector T cells following therapy. The cytotoxic T cell : T_reg ratio may be considered as pivotal in immune checkpoint inhibitory therapies, as CD8⁺ T cell expansion is associated with better overall survival in various malignancies treated with CTLA‐4 and PD‐1 blockade 41, 42. Yamaki et al. 43 recently reported significantly poorer survival of PD‐L1⁺ compared to PD‐L1⁻ in pancreatic ductal adenocarcinoma patients presenting with CD8⁺ tumour infiltration. Moreover, PD‐1 expression in tumour‐associated macrophages is associated with disease progression in various human malignancies, while PD‐1/PD‐L1 blockade favours macrophage phagocytic activity and suppresses tumour progression 44. Our results suggest that pembrolizumab exerts its therapeutic effects by inhibiting negative signalling via the PD‐1 pathway and not by altering the phenotype or function of T_regs.

Disclosure

The authors declare no conflicts of interest.

Author contributions

S. T. performed experimental work, data analysis and wrote the paper. A. S. S. K. assisted in experimental work, data analysis and reviewing the paper. I. A. reviewed and corrected the paper. E. E. conceived the idea, designed the study, obtained funds, supervised the project, analysed and interpreted data and wrote and revised the paper. All authors were involved in the final approval of the paper.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. Effect of different concentrations and duration of pembrolizumab exposure on programmed death 1 (PD‐1) expression in CD4⁺ T cells. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD), non‐activated and activated by plate‐bound anti‐CD3 and CD28 antibodies for 24 and 48 h were checked for PD‐1 expression with different drug concentrations. Line graph shows effect of different concentrations of pembrolizumab on PD‐1 expression in non‐activated cells after 24 h (a). PD‐1 expression in activated cells treated with different concentrations of pembrolizumab for 24 and 48 h is shown (b,c).

Fig. S2. Effect of pembrolizumab on Ki‐67 expression. Bar plot shows Ki‐67 expression and effect of pembrolizumab on non‐activated and activated cells from healthy donors (HD) (a). Bar plot shows effect of pembrolizumab on Ki‐67 expression in different subsets of CD25^+/− cells within CD4⁺ T cells from HD (b). Bar plot shows Ki‐67 expression and effect of pembrolizumab in different forkhead box protein 3 (FoxP3) and Helios subsets within CD4⁺ non‐activated T cells from HD (c).

Fig. S3. Effect of pembrolizumab on CD15s and latency‐associated peptide (LAP) expression in CD4⁺ cells in healthy donors (HD) and primary breast cancer patients (PBC). Bar plots show effect of pembrolizumab on CD15s expression in CD4⁺ T cells from HD and PBC in non‐activated (a) and activated cells (b). Bar plots show effect of pembrolizumab on LAP expression in CD4⁺ T cells from HD and PBC in non‐activated (c) and activated cells (d).

Click here for additional data file.^{(95.2KB, docx)}

Acknowledgements

We are thankful to all participating individuals for donation of their samples. We are also grateful to Dr Mohammed Jaloudi from Tawam Hospital, Al Ain, United Arab Emirates and Merck & Co., NJ, USA for providing us with Keytruda (pembrolizumab) for our research. This work was supported by grants from United Arab Emirates University Program of Advanced Research (31M190) and the Terry Fox Foundation (21M094).

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19. Na Z, Yeo SP, Bharath SR et al Structural basis for blocking PD‐1‐mediated immune suppression by therapeutic antibody pembrolizumab. Cell Res 2017; 27:147–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Syed Khaja AS, Toor SM, El Salhat H et al Preferential accumulation of regulatory T cells with highly immunosuppressive characteristics in breast tumor microenvironment. Oncotarget 2017; 8:33159–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008; 133:775–87. [DOI] [PubMed] [Google Scholar]
22. Chaudhary B, Elkord E. Regulatory T cells in the tumor microenvironment and cancer progression: role and therapeutic targeting. Vaccines (Basel) 2016; 4:E28. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Elkord E, Abd Al Samid M, Chaudhary B. Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP. Oncotarget 2015; 6:20026–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Kim HJ, Barnitz RA, Kreslavsky T et al Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. Science 2015; 350:334–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Miyara M, Chader D, Sage E et al Sialyl Lewis x (CD15s) identifies highly differentiated and most suppressive FOXP3high regulatory T cells in humans. Proc Natl Acad Sci USA 2015; 112:7225–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Zitvogel L, Kroemer G. Targeting PD‐1/PD‐L1 interactions for cancer immunotherapy. Oncoimmunology 2012; 1:1223–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Yao S, Zhu Y, Chen L. Advances in targeting cell surface signalling molecules for immune modulation. Nat Rev Drug Discov 2013; 12:130–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Qin T, Zeng YD, Qin G et al High PD‐L1 expression was associated with poor prognosis in 870 Chinese patients with breast cancer. Oncotarget 2015; 6:33972–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Nanda R, Chow LQ, Dees EC et al Pembrolizumab in patients with advanced triple‐negative breast cancer: phase Ib KEYNOTE‐012 study. J Clin Oncol 2016; 34:2460–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Adams S. Dramatic response of metaplastic breast cancer to chemo‐immunotherapy. NPJ Breast Cancer 2017; 3:8. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Joneja U, Vranic S, Swensen J et al Comprehensive profiling of metaplastic breast carcinomas reveals frequent overexpression of programmed death‐ligand 1. J Clin Pathol 2017; 70:255–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Sakaguchi S, Ono M, Setoguchi R et al Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self‐tolerance and autoimmune disease. Immunol Rev 2006; 212:8–27. [DOI] [PubMed] [Google Scholar]
33. Shang B, Liu Y, Jiang SJ, Liu Y. Prognostic value of tumor‐infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta‐analysis. Sci Rep 2015; 5:15179. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. West NR, Kost SE, Martin SD et al Tumour‐infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor‐negative breast cancer. Br J Cancer 2013; 108:155–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Klocke K, Sakaguchi S, Holmdahl R, Wing K. Induction of autoimmune disease by deletion of CTLA‐4 in mice in adulthood. Proc Natl Acad Sci USA 2016; 113:E2383–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Janakiram M, Abadi YM, Sparano JA, Zang X. T cell coinhibition and immunotherapy in human breast cancer. Discov Med 2012; 14:229–36. [PMC free article] [PubMed] [Google Scholar]
37. Su S, Liao J, Liu J et al Blocking the recruitment of naive CD4+ T cells reverses immunosuppression in breast cancer. Cell Res 2017; 27: 461–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Plitas G, Konopacki C, Wu K et al Regulatory T cells exhibit distinct features in human breast cancer. Immunity 2016; 45:1122–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Abd Al Samid M, Chaudhary B, Khaled YS, Ammori BJ, Elkord E. Combining FoxP3 and Helios with GARP/LAP markers can identify expanded Treg subsets in cancer patients. Oncotarget 2016; 7:14083–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Ribas A, Shin DS, Zaretsky J et al PD‐1 blockade expands intratumoral memory T cells. Cancer Immunol Res 2016; 4:194–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Kvistborg P, Philips D, Kelderman S et al Anti‐CTLA‐4 therapy broadens the melanoma‐reactive CD8+ T cell response. Sci Transl Med 2014; 6:254ra128. [DOI] [PubMed] [Google Scholar]
42. Tumeh PC, Harview CL, Yearley JH et al PD‐1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014; 515:568–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Yamaki S, Yanagimoto H, Tsuta K, Ryota H, Kon M. PD‐L1 expression in pancreatic ductal adenocarcinoma is a poor prognostic factor in patients with high CD8+ tumor‐infiltrating lymphocytes: highly sensitive detection using phosphor‐integrated dot staining. Int J Clin Oncol 2017; 22:726–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Gordon SR, Maute RL, Dulken BW et al PD‐1 expression by tumour‐associated macrophages inhibits phagocytosis and tumour immunity. Nature 2017; 545:495–99. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[cei13060-bib-0031] 31. Joneja U, Vranic S, Swensen J et al Comprehensive profiling of metaplastic breast carcinomas reveals frequent overexpression of programmed death‐ligand 1. J Clin Pathol 2017; 70:255–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0032] 32. Sakaguchi S, Ono M, Setoguchi R et al Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self‐tolerance and autoimmune disease. Immunol Rev 2006; 212:8–27. [DOI] [PubMed] [Google Scholar]

[cei13060-bib-0033] 33. Shang B, Liu Y, Jiang SJ, Liu Y. Prognostic value of tumor‐infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta‐analysis. Sci Rep 2015; 5:15179. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[cei13060-bib-0035] 35. Klocke K, Sakaguchi S, Holmdahl R, Wing K. Induction of autoimmune disease by deletion of CTLA‐4 in mice in adulthood. Proc Natl Acad Sci USA 2016; 113:E2383–92. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0036] 36. Janakiram M, Abadi YM, Sparano JA, Zang X. T cell coinhibition and immunotherapy in human breast cancer. Discov Med 2012; 14:229–36. [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0037] 37. Su S, Liao J, Liu J et al Blocking the recruitment of naive CD4+ T cells reverses immunosuppression in breast cancer. Cell Res 2017; 27: 461–82. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0038] 38. Plitas G, Konopacki C, Wu K et al Regulatory T cells exhibit distinct features in human breast cancer. Immunity 2016; 45:1122–34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0039] 39. Abd Al Samid M, Chaudhary B, Khaled YS, Ammori BJ, Elkord E. Combining FoxP3 and Helios with GARP/LAP markers can identify expanded Treg subsets in cancer patients. Oncotarget 2016; 7:14083–94. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0040] 40. Ribas A, Shin DS, Zaretsky J et al PD‐1 blockade expands intratumoral memory T cells. Cancer Immunol Res 2016; 4:194–203. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0041] 41. Kvistborg P, Philips D, Kelderman S et al Anti‐CTLA‐4 therapy broadens the melanoma‐reactive CD8+ T cell response. Sci Transl Med 2014; 6:254ra128. [DOI] [PubMed] [Google Scholar]

[cei13060-bib-0042] 42. Tumeh PC, Harview CL, Yearley JH et al PD‐1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014; 515:568–71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0043] 43. Yamaki S, Yanagimoto H, Tsuta K, Ryota H, Kon M. PD‐L1 expression in pancreatic ductal adenocarcinoma is a poor prognostic factor in patients with high CD8+ tumor‐infiltrating lymphocytes: highly sensitive detection using phosphor‐integrated dot staining. Int J Clin Oncol 2017; 22:726–33. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cei13060-bib-0044] 44. Gordon SR, Maute RL, Dulken BW et al PD‐1 expression by tumour‐associated macrophages inhibits phagocytosis and tumour immunity. Nature 2017; 545:495–99. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

In‐vitro effect of pembrolizumab on different T regulatory cell subsets

S M Toor

A S Syed Khaja

I Alkurd

E Elkord

Summary

Introduction

Materials and methods