Novel expression of Neuropilin 1 on human tumor-infiltrating lymphocytes in colorectal cancer liver metastases.

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Original Research

Novel expression of Neuropilin 1 on human tumor-infiltrating lymphocytes in colorectal cancer liver metastases

1.

Introduction

2.

Materials and methods

3.

Results

Belal Chaudhary & Eyad Elkord†

4.

Discussion

†

5.

Conclusions

United Arab Emirates University, College of Medicine and Health Sciences, Al Ain, UAE

Objectives: Neuropilin 1 (NRP1) is a transmembrane protein with diverse roles in physiological and pathological settings. NRP1 expression has been reported on T cells in inflammatory microenvironments and in secondary lymphoid tissue. Tumor-infiltrating lymphocytes (TILs) play an important role in cancer prognosis. In this study, we investigated NRP1 expression on TILs and peripheral blood mononuclear cells (PBMCs) from colorectal cancer liver metastases (LI/CRC). Methods: TILs from LI/CRC and PBMCs from healthy donors and patients were analyzed for expression of NRP1, in addition to other Treg-related markers. PBMCs were co-cultured in vitro with tumor tissue and analyzed for NRP1 expression. Results: We report for the first time that NRP1 is highly expressed on CD3+CD4+ TILs compared to PBMCs. NRP1 expression correlated closely with CD25 expression in TILs. NRP1 was expressed on both Helios+ and Helios-FoxP3-expressing Tregs and on a FoxP3--Helios-- T cell subset. It was also induced on PBMCs following in vitro co-culture with tumor tissue. Conclusions: NRP1 is upregulated on TILs and can be induced on PBMCs by tumor tissue. Further studies are warranted to define the function of NRP1 on human TILs. As a therapeutic target, NRP1 may allow selective targeting of TIL subsets including suppressive Tregs. Keywords: colorectal cancer liver metastases, expression, Neuropilin 1, regulatory T cells, tumor-infiltrating lymphocytes Expert Opin. Ther. Targets (2015) 19(2):147-161

1.

Introduction

Tumor-infiltrating lymphocytes (TILs) play an important role in cancer progression and predicting disease outcome [1,2]. Tumor-specific CD8+ cytotoxic T cells are able to infiltrate and attack tumors, reducing tumor burden and inducing tumor remission. They have been utilized successfully in adoptive cell transfer therapies [3]. Within the tumor microenvironment (TME), however, CD8+ cytotoxic T cells are often functionally impaired exhibiting an ‘exhausted’ phenotype and may also be unable to effectively infiltrate tumor tissue [4]. In contrast, immunosuppressive regulatory T cells (Tregs) effectively migrate into tumor tissue in response to tumor-secreted factors where they are able to expand in situ via a number of mechanisms, comprising a significant proportion of the TILs population [2]. Tregs have been reported to accumulate in tumors and at peripheral sites in numerous cancers, where, in conjunction with other immunosuppressive subsets, they contribute to the establishment of a tolerogenic TME, suppressing tumor-specific immune responses and accelerating tumor development [5] -- although in certain cases, Treg infiltration has been reported to correlate with increased overall survival [2,6]. The ratio of tumor 10.1517/14728222.2014.977784 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

147


B. Chaudhary & E. Elkord

infiltrating CD8+ cytotoxic T cells to Tregs and other tumorinfiltrating subsets has proved particularly important in determining disease outcome [2]. Treg depletion prior to cancer immunotherapy and cancer vaccine administration greatly enhances the efficacy of these therapies [7,8]. Tregs are broadly divided into thymic-derived tTregs and peripheral-induced pTregs -- referred to in previous reports as nTregs and iTregs, respectively. Defining effective Treg markers, especially for tumor-infiltrating subsets, is key to understanding their immunobiology and for developing methods to manipulate them in a therapeutic setting. A number of promising Treg markers including CD26, Glycoprotein Repetition A Predominant (GARP), latencyassociated peptide (LAP) and immune checkpoint molecules have recently been reviewed [2]. Two markers of particular interest are Neuropilin 1 (NRP1) and Helios. NRP1 is a Type I transmembrane protein first identified as a selective marker of murine tTregs. There have been conflicting reports regarding NRP1 expression patterns in murine and human immune systems as discussed in a recent perspective [9]. In humans, NRP1 is expressed on plamacytoid dendritic cells (pDCs), arterial and tumor vasculature, and on small populations of Tregs isolated from secondary lymph nodes [9-12]. NRP1 plays a variety of roles in physiological and pathological settings, including neuronal and axonal guidance, angiogenesis, tumor progression and cell migration [9]. These functions are mediated through NRP1 interactions with a diverse array of ligands: TGF-b, semaphorins, plexins, various growth factors and their receptors, including VEGF receptors & VEGF isoforms [9]. NRP1 can be induced on lymphocytes in vivo by inflammatory microenvironments, as has been reported in mice and humans [13-17]. NRP1 is also overexpressed on tumor vasculature where it is implicated in promoting tumor growth, angiogenesis and recruitment of immune cells [18-20]. Given these diverse interactions, NRP1 offers a valuable therapeutic target. Early pre-clinical and Phase I trials have shown positive results where NRP1-blocking antibodies synergized with bevacizumab in patients with advanced solid tumors and NRP1-targeted therapeutics exhibited increased tumor tissue penetration and enhanced anti-tumor efficacy in murine models [21-25]. The Ikaros zinc finger transcription factor, Helios, is another marker whose expression and role in the immune system is still not fully defined. Helios was first identified as a murine tTreg marker by gene microarray analyses [26]. Following this, Helios has been characterized in humans on pTregs, tTregs, TILs and activated T cells. Despite the uncertainty regarding Helios expression patterns and its functional contribution on immune cells, it is clear that Helios+ Tregs comprise a distinct functional subset that might exhibit enhanced immunosuppressive activity, accumulate selectively in tumors and influence prognosis [27-29]. In this study we investigated the expression of NRP1 on TILs extracted from liver metastases of colorectal cancer 148

patients compared with peripheral blood mononuclear cells (PBMCs) from patients and healthy donors. We report novel expression of NRP1 on TILs from liver metastases. In contrast, PBMCs from both patients and healthy donors exhibited low levels of NRP1 expression. We found that PBMCs co-cultured with tumor tissue highly upregulated NRP1 expression. We also investigated the expression of other Treg markers, CD25, the forkhead box P3 transcription factor (FoxP3) and Helios, with NRP1 on TILs and PBMCs. 2.

Materials and methods

Blood and tissue samples Written consents were obtained from all patients and healthy donors before blood or tissue sampling using the research protocol approved by the National Research Ethical Committee, Salford Research Ethics Committee and the Local Research and Development departments. Blood and tissue samples were collected from patients undergoing surgical resection of colorectal cancer liver metastases (LI/CRC) at the North Manchester General Hospital, UK. Blood was taken from patients prior to surgery, whereas resected LI/CRC was obtained during surgery. In addition, blood samples were collected from healthy donors as controls. Blood samples were collected in a 50 ml Falcon tube (BD Biosciences, Oxford, UK) containing 200 µl (1000 IU/ml) heparin. Liver metastases were collected in sterile tubes supplemented with RPMI-1640 and kept on ice until arrival at the laboratory to be processed immediately. 2.1

Cell isolation and tumor tissue storage PBMCs were isolated from healthy donors and CRC patients using Ficoll-Hypaque density gradient centrifugation. Cells were then frozen at 5 -- 10 106 cells/ml in cryovials in 1 ml of freezing media (50% FCS, 40% RPMI-1640 and 10% DMSO) and stored in liquid nitrogen (LN) for later use. Trypan blue was used for PBMC viability testing and counting. LI/CRC tissue pieces were frozen in freezing media and stored in LN. 2.2

Extraction of tumor-infiltrating lymphocytes TILs were isolated by explant culture with IL-2, or by enzymatic disaggregation, as previously described [30]. For explant culture, liver metastases were dissected into pieces 2 -- 3 mm in length. Each fragment was cultured individually in a single well in a treated 24-well tissue culture plate with 2 ml of complete media (10% FCS in RPMI-1640 supplemented with L-glutamine and streptomycin and penicillin) and 600 IU/ml of recombinant IL-2. TILs were supplemented with IL-2 to maintain IL-2 levels, and were counted by trypan blue staining. TILs were collected for flow cytometric analysis when sufficient cell numbers were reached. 2.3

Expert Opin. Ther. Targets (2015) 19(2)


PBMC co-culture with tumor tissue Cryopreserved LI/CRC tissue was thawed and cultured for up to 30 days in complete media with no IL-2 in order to allow the growth of tumor cells but not lymphocytes. Allogeneic PBMCs were then thawed and cultured with the LI/CRC cells in 1 ml complete media and 50 IU/ml IL-2 at a concentration of 106 PBMC/ml in treated 24-well tissue culture plates. PBMCs were stained prior to co-culture directly after thawing and were also collected after 48 h and 96 h of co-culture for flow cytometric analysis. 2.4

PBMC culture in IL-2 To simulate TILs extraction conditions, 106 PBMCs from patients and healthy donors were cultured in treated 24-well tissue culture plates. PBMCs were cultured in 1 ml complete media with 600 IU/ml of recombinant IL-2, supplemented with IL-2 to maintain levels, and recovered after 96 h for flow cytometric analysis.


2.5

Flow cytometric analysis Cryopreserved PBMCs were thawed and stained for surface and intracellular markers. The previous work has shown that Treg levels decrease after freezing -- when analyzing samples, either fresh or frozen samples should consistently be used [31]. TILs and PBMCs from in vitro culture experiments were collected and stained directly. Flow cytometric analysis of samples was carried out on a BD FACSVerse with BD FACSuite software to analyze data. 2.6

Surface marker staining Cells were washed and labeled with surface markers: antihuman CD3 FITC (BD Pharmingen, Oxford, UK), antihuman CD4 PerCP-Cy5.5 (eBioscience, Hatfield, UK), anti-human CD25 PE-Cy7 (BD Pharmingen) and antihuman CD304 (NRP1) APC (Biolegend, Cambridge, UK). For each sample, two tubes were used: i) a ‘partially stained’ tube stained for CD3, CD4 and CD25 but not CD304 and ii) a ‘fully stained’ tube stained for CD3, CD4, CD25 and CD304. This allowed for accurate determination of NRP1 expression and exclusion of background staining. 2.6.1

Intracellular staining Cells were washed and labeled with surface markers: antihuman CD3 APC-H7 (BD Pharmingen), anti-human CD4 PerCP-Cy5.5 (eBioscience), anti-human CD25 PE-Cy7 (BD Pharmingen) and anti-human CD304 APC (Biolegend). Cells were subsequently fixed and permeabilized using fixation and permeabilization buffers (eBioscience). Cells were then blocked using rat serum (eBioscience) and mouse serum (SLI Ltd., West Sussex, UK) before staining with either rat anti-human FoxP3 PE (clone PCH101, eBioscience) and Armenian hamster anti-mouse/human Helios FITC (Clone 22F6, Biolegend) or rat anti-human IgG2a-PE isotype negative control (eBioscience). 2.6.2

3.

Results

NRP1 is expressed at higher levels on tumor-infiltrating lymphocytes

3.1

NRP1 expression has been reported in inflammatory microenvironments in mice and in human secondary lymph nodes, including both healthy donor lymph nodes and metastatic tumor-draining lymph nodes (TDLN) [10,11,13-15]. NRP1 expression on TILs however has not previously been investigated. We analyzed the expression of NRP1 on PBMCs from LI/ CRC patients and healthy donors compared with TILs extracted from LI/CRC by explant culture. TILs isolation via explant culture is beneficial since cells are left with their surroundings to more accurately mimic their in vivo environment. NRP1 was expressed at significantly higher levels on CD3+CD4+ TILs compared to their counterparts isolated from peripheral blood of both LI/CRC patients and healthy donors (mean ± SD: TILs; 5.56% ± 0.94, LI/CRC PBMC; 0.18% ± 0.10, HD PBMC; 0.36% ± 0.29) (Figure 1). This difference was highly significant in patients (p = 0.0064) and healthy donor PBMC (p = 0.0008) (Figure 1B). There was no significant difference in NRP1 expression on healthy donors and patients PBMCs. One LI/CRC patient exhibited exceptionally high NRP1 expression in TILs (> 21%; Figure 1D). NRP1 is expressed mainly on CD25-expressing CD3+CD4+ TILs

3.2

We then investigated the co-expression of NRP1 with CD25 on CD3+CD4+ TILs and PBMCs from healthy donors and patients (Figure 2). Interestingly, in TILs, NRP1 expression correlates closely with CD25 expression (Figure 2A) CD25HI TILs exhibited the highest levels of NRP1 expression compared to CD25-- and CD25INT TIL subsets. Similar findings have been reported earlier on human T cells isolated from secondary lymph nodes [11]. NRP1 is expressed on FoxP3+ and FoxP3-- T cell subsets

3.3

FoxP3 is critical for the development and suppressive function of Tregs. In order to further elucidate the nature of NRP1 expression, we investigated NRP1 expression on FoxP3+ and FoxP3-- CD4+ T cell subsets (Figure 3A). NRP1 was mainly expressed on TILs: on FoxP3+ Tregs and FoxP3-- CD4+ T cells, although NRP1 seemed to be expressed at higher levels on FoxP3+ Tregs. NRP1 was expressed at much lower levels on PBMCs from healthy donor and patients, on both FoxP3+ and FoxP3-- T cell subsets (Figure 3A). NRP1 is expressed on Helios+ and Helios-- FoxP3expressing TILs 3.4

Despite current uncertainty whether Helios is a specific marker of tTregs, it is clear that Helios+ Tregs comprise a


149


A.

B.

Gated on CD3+CD4+ T cells

NRP1 upregulated on TILs

0.20%

CD4

0.41%

% NRP1 expression

LI/CRC PBMC

0.05%

0.0008*** 0.0064**

LI/CRC TILS LI/CRC PBMC HD PBMC

6

4

2 ns

0.27% 0 LS

C

C

TI

C. 5.77%

R

C

NRP1

CD4

D.

M

PB

D

H

R

/C

LI

PB

NRP1 expression on TILs vs PBMC 8

% NRP1 expression

6.38%

C

M

/C

LI

LI/CRC TILs


Healthy donors PBMC

8

LI/CRC TILS LI/CRC PBMC

6

4

2

21.59% 0 nt

tie

Pa

NRP1

1

nt

Pa

tie

2

nt

3

tie

Pa

Figure 1. NRP1 expression on CD3+CD4+ TILs. (A) Representative flow cytometric plots showing NRP1 expression on CD3+CD4+ TILs isolated by explant culture compared with CD3+CD4+ PBMCs from both healthy donors and LI/CRC patients. (B) Bar graph comparing mean expression of NRP1 on CD3+CD4+ TILs and PBMCs from patients and healthy donors. (C) Bar chart comparing NRP1 expression on TILs and autologous PBMCs from 3 LI/CRC patients. (D) TILs from one patient showed exceptionally very high levels of NRP1 expression. LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

distinct functional subset that accumulate in tumors and exhibit enhanced suppressive activity [2,32,33]. In agreement with previous studies, Helios was expressed on the majority of FoxP3+ PBMCs, but not on CD4+FoxP3-- PBMCs (Figure 3A). Helios and NRP1 co-expression have been characterized on murine Tregs induced in vitro and in vivo [14]. We determined NRP1 expression on FoxP3+Helios+ and FoxP3+Helios-- Treg subsets (gating shown in Figure 3B). Our results show that NRP1 is expressed on both FoxP3+Helios+ and FoxP3+Helios-- TILs (Figure 3A top panel and Figure 3B). Interestingly, in TILs, Helios and NRP1 are co-expressed only in FoxP3+ Tregs but not in FoxP3-T cells (Figure 3A) 150

NRP1 and Helios are co-expressed on CD25+FoxP3+ TILs

3.5

To further define Tregs based on co-expression of FoxP3 and CD25, we gated four CD25+/--FoxP3+/-- T cell sub-populations as shown in Figure 4. NRP1 was mainly expressed on TILs; this NRP1 expression was confined to CD25-expressing FoxP3+ and FoxP3-- TIL subsets (Figure 4A). NRP1 was expressed at much lower levels on PBMCs, regardless of FoxP3 and CD25 expression (Figure 4B and C). Of note, in PBMCs, Helios was mainly expressed in FoxP3-expressing CD25+ and CD25-- subsets (Figure 4B and C). We also examined NRP1 and Helios co-expression on different CD25+/--FoxP3+/-- T cell subsets from PBMC and TILs



A.

93.7%

6.30%

39.13%

60.87%

98.78%

1.22%

89.41%

10.59%

99.46.%

0.54%

96.29%

3.71%

CD25HI

Gated on CD3+CD4+ TILs

CD4

17.21%

16.08%

CD25–


CD25

CD25INT

66.71%

CD4

NRP1

B. CD25–NRP1+

CD25INTNRP1+

CD25HINRP1+

TILs

0.22% ± 0.29

0.23% ± 0.05

4.18% ± 2.05

LI/CRC PBMC

0.034% ± 0.47

0.098% ± 0.076

0.026% ± 0.021

HD PBMC

0.096% 0.17

0.15% ± 0.17

0.04 ± 0.0374

Figure 2. NRP1 expression with CD25 expression. (A) Representative flow cytometric plots showing expression of NRP1 on CD3+CD4+ TILs gated for different levels of CD25 expression: CD25 negative (CD25--), CD25 intermediate (CD25INT) and CD25 high (CD25HI). NRP1 expression correlates with higher CD25 expression. TILs from one patient that showed exceptionally high NRP1 expression (Figure 1D) were also analyzed for co-expression with CD25 (Figure 2A, right column). (B) Mean expression ± SD of NRP1 on CD25--, CD25INT and CD25HI TILs and PBMCs from healthy donors and patients. LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

(Figure 4). NRP1 and Helios were only co-expressed on CD25+FoxP3+ Tregs from TILs. Modulation of NRP1 expression by IL-2 To confirm whether the NRP1 expression observed on TILs might be due to culture with IL-2 we cultured healthy donor and patient PBMCs in the presence of IL-2, simulating conditions utilized for TILs extraction. NRP1 was slightly upregulated in some PBMC samples but remained significantly lower than TILs after 96 h culture (Figure 5A). We further analyzed expression of NRP1 and Helios in the four CD25+/--FoxP3+/-- T cell sub-populations following 96 h culture (as shown in Figure 5B). In contrast to TILs, NRP1 was mainly confined to CD25 negative subsets within FoxP3+ and FoxP3-- T cell subsets (Figure 5C). Within the FoxP3+CD25-- T cell subset, the majority of NRP1+ T cells

co-expressed Helios, whereas NRP1+ T cells from the FoxP3--CD25-- T cell subset did not co-express Helios (Figure 5C).

3.6

NRP1 is induced on PBMC following co-culture with tumor tissue

3.7

In order to further investigate the role of the TME and tumor-associated factors in inducing NRP1 expression, we co-cultured allogeneic PBMCs for up to 96 h with LI/CRC tissue and analyzed PBMCs for NRP1 expression (Figure 6). Prior to co-culture, NRP1 was not expressed on PBMCs (Figure 6A). NRP1 was induced on PBMCs following 48 h of co-culture, and NRP1 expression further increased after 96 h of co-culture (Figure 6A). We then investigated expression of NRP1 on various CD25-expressing sub-populations as shown in Figure 6B. Similar to TILs isolated by explant


151


+

Healthy donors PBMC

+

LI/CRC TILs

72.62%

0.77%

81.09%

26.47%

0.14%

17.30%

1.30%

35.31%

15.73%

Helios

Gated on CD4+FoxP3+ T cells FoxP3

11.93%

0.37%

19.93%

29.02%

NRP1

CD4

2.19%

0.01%

3.88%

97.22%

0.58%

95.40%

0.05%

0.40%

2.08%

Helios

Gated on CD4+FoxP3– T cells

88.07%

0.68%

76.45%

21.07%

NRP1

B. Healthy donor PBMC

88.38%

3.17% FoxP3

LI/CRC PBMC

0.85%

TILs

0.45%

9.43%

Helios

7.24%

Gated on FoxP3+Helios– T cells

1.21%

Gated on FoxP3+Helios– Tregs


Helios


LI/CRC PBMC

Gated on CD3 CD4 T cells

A.

1.62%

0.97%

6.51%

NRP1

Figure 3. NRP1 expression on Helios+ and Helios-- TILs and PBMCs. (A) Representative flow cytometric plot from LI/CRC patient showing CD4 and FoxP3 gating strategy: FoxP3+ and FoxP3-- subsets were gated from CD3+CD4+ T cells and analyzed for NRP1 co-expression with Helios in healthy donor PBMCs, LI/CRC patient PBMCs and TILs. (B) Representative flow cytometric plot from LI/CRC patient showing FoxP3 and Helios gating strategy: FoxP3-expressing Helios+ and Helios-- subsets were gated and analyzed for NRP1 co-expression with Helios in healthy donor PBMCs, LI/CRC patient PBMCs and TILs. LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

culture (Figure 2A), NRP1 was expressed at the highest level on the CD25HI T cells, at lower levels on CD25INT T cells and at negligible levels on CD25-- T cells (Figure 6B). 3.8

NRP1 and Helios expression on CD8+ T cells

We also analyzed NRP1, Helios and FoxP3 expression on CD3+CD4- (CD8+) T cells (Figure 7). There was no expression of NRP1 on CD8+ T cells from healthy donor or patient PBMCs, whereas in two patient samples NRP1 was expressed on CD8+ TILs (Figure 7A). CD8+ T cells from all samples expressed significant levels of Helios but not FoxP3 (Figure 7B). On TILs, NRP1 was expressed on both Helios+ and Helios-- CD8+ T cell subsets (Figure 7C). Of note, Helios was upregulated on CD8+ TILs and PBMCs of LI/CRC patients compared to healthy donors (Figure 7B and C). 152

4.

Discussion

In this study, we report novel expression of NRP1 on lymphocytes isolated from liver metastases of colorectal cancer patients. NRP1 was highly upregulated on CD3+CD4+ TILs compared to PBMCs isolated from the peripheral blood of patients and healthy donors. NRP1 was expressed on a significant proportion of FoxP3+ TILs and was represented on both Helios+ and Helios-- subsets. In the peripheral blood of humans, NRP1-expressing lymphocytes comprise < 0.5% of the total PBMC population [11,34-36]. We also report low levels of NRP1 expression on PBMCs isolated from LI/CRC patients and healthy donors. The only significant populations of NRP1-expressing lymphocytes identified in humans so far are from secondary



B.

LI/CRC TILs 5.22%

73.64%

(iii)

20.89%

0.25%

(ii)

(iv)

(iii)

Helios

2.04%

21.62%

(ii)

(iv)

(iii) 0.91%

65.60%

2.09%

67.03%

35.09%

2.09%

15.44%

17.90%

31.93%

0.41%

(iii)

(iv)

0.03%

69.8%

(i)

(ii)

Helios

0.91%

(i)

FoxP3

FoxP3

(i)

73.68%

7.71%

CD25

(i)

FoxP3

3.79%

25.78%

8.55%

CD25

(iv)


22.33%

Healthy donor PBMC

(ii)

CD25

(i)

C.

LI/CRC PBMC

6.18%

0.62%

(ii)

97.04%

0.84%

29.44%

1.09%

0.01%

49.48%

0.14% 0.74%

(iii)

(iv)

0%

80.78%

(i)

Helios

A.

0.21%

(ii)

93.60%

0.22%

18.42%

0.39%

2.19%

0.01%

54.0%

0.91%

(iii)

(iv)

No cells

87.59%

98.51%

9.96%

0.39%

NRP1

49.30%

0.48%

NRP1

97.75%

0.05%

44.63%

0.46%

NRP1

Figure 4. NRP1 expression on FoxP3+/--CD25+/-- T cell subsets. Four T cell sub-populations were gated from CD3+CD4+ lymphocytes and defined as follows: (i) FoxP3--CD25+; (ii) FoxP3+CD25+; (iii) FoxP3+CD25--; and (iv) FoxP3-- CD25--. Each sub-population was analyzed for NRP1 and Helios co-expression from TILs (A), LI/CRC patient PBMCs (B), and healthy donor PBMCs (C). LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

lymph nodes and tonsils, including TDLN of cervical cancer patients where NRP1 expression was significantly upregulated in metastatic TDLN (mTDLN) compared to metastasis-free TDLN [10-12,35,37]. We report for the first time that TILs isolated by explant culture from liver metastases of CRC patients highly expressed NRP1. The exact phenotype of NRP1+ lymphocytes varies considerably between studies. NRP1-expressing lymphocytes isolated from secondary lymph nodes and tonsils in one study were mainly CD25--FoxP3--, although small NRP1+FoxP3+ sub-populations were also present, making up to 0.42 and 0.33% of the entire CD4+ PBMC population from secondary lymph nodes and tonsils, respectively [35]. Another study reported that the majority of NRP1-expressing lymphocytes isolated from human lymph nodes in healthy donors were CD25HIFoxP3+ and exhibited suppressive activity in vitro suggesting that they were Tregs [11]. The same group identified a subset of NRP1-expressing Tregs that accumulated in

the mTDLN of cervical cancer patients and influenced the efficacy of chemoradiation therapy [10]. These Tregs were defined as CD4+CD25HICD45RO+FoxP3+GITR+NRP1+. NRP1-expressing FoxP3+ Tregs have also been isolated from the synovium of rheumatoid arthritis (RA) patients -- a highly inflammatory microenvironment [16]. In a separate study, CD4+NRP1+ T cells were reported to be expanded in the peripheral blood of RA patients compared to healthy donors. mRNA analysis showed these NRP1-expressing cells expressed high levels of FoxP3 mRNA and IL-10 compared to CD4+NRP1-- T cells from the same donors, suggesting that they were NRP1+ Tregs [38]. In addition, two groups have reported that NRP1 expression defines follicular T helper cells (Tfh) isolated from human secondary lymphoid tissue [12,39]. Tfh expressed CD57, CD69 and PD-1 among other Tfh markers, but did not express CD25 or FoxP3. To further elucidate the nature of NRP1 expression on lymphocytes, we studied the expression of Treg-related


153


0h 99.51%

0.49%

99.75%

LI/CRC PBMC

0.63%

96 h

0h

CD4

Healthy donors PBMC

99.37%

96 h

0.15%

1.48%

NRP1

NRP1

7.25%


98.52%

CD4

A.

7.61%

CD25

B.

84.0%

1.14% FoxP3

FoxP3–CD25–

FoxP3–CD25+ 0.04%

1.69%

FoxP3+CD25+

0.31%

33.33%

44.10%

73.21%

0.81%

1.69%

20.48%

2.09%

25.83%

0.15%

Healthy donors PBMC

6.72%

FoxP3+CD25–

0.96%

96.31%

5.91%

0.36%

1.03%

0.09%

36.36%

22.22%

67.24%

4.01%

86.42%

7.31%

97.00%

1.89%

38.39%

3.03%

26.71%

2.04%

LI/CRC PBMC

Helios

92.28%

NRP1

Figure 5. Modulation of NRP1 by IL-2. Healthy donor PBMCs and LI/CRC patient PBMCs were cultured with 600IU/ml of IL-2 simulating conditions utilized for TILs extraction from tumor tissue. (A) PBMCs were analyzed for expression of NRP1 at 0 h and after 96 h culture with IL-2. (B) PBMCs were also gated on CD3+CD4+CD25+/--FoxP3+/-- T cell subsets, as shown, and analyzed for NRP1 and Helios co-expression. LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

154



A. 48 h

100%

0%

98.78%

0%

0%

0%

96 h 1.22%

96.33%

3.67%

CD4


0h

0%

0%

0%

B. 85.70%

14.30%

CD25HI

96 h co-culture with tumor tissue

0%

0%

97.57%

2.43%

78.36%

CD4

CD25INT

6.45%

CD25

0%

0%

99.26%

15.19%

0.74%

CD25–


NRP1

CD4 0%

0%

NRP1

Figure 6. Induction of NRP1 on PBMCs by co-culture with tumor tissue. (A) Cryopreserved PBMCs were thawed and directly stained (0 h). PBMCs were also co-cultured with allogeneic tumor tissue and analyzed for NRP1 expression after 48 h and 96 h. NRP1 expression was analyzed on CD3+CD4+ T cells. (B) PBMCs co-cultured with allogeneic tumor tissue for 96 h were analyzed for NRP1 expression on CD3+CD4+ T cells gated for different levels of CD25 expression: CD25 negative (CD25--), CD25 intermediate (CD25INT) and CD25 high (CD25HI). LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

markers, FoxP3 and Helios, with NRP1 on TILs. NRP1 was identified on both FoxP3+ and FoxP3-- TIL subsets although NRP1 was expressed on a greater proportion of FoxP3+ TILs compared to FoxP3-- TILs. The FoxP3+NRP1+ TILs comprised both Helios+ and Helios-- subsets, whereas

FoxP3--NRP1-- TILs did not co-express any Helios. We also studied CD25 and FoxP3 co-expression with NRP1 to better define Tregs. NRP1 was highly expressed on the FoxP3+CD25+ TIL subset, suggesting that they are more likely to be Tregs. These FoxP3+CD25+NRP1+ Tregs also


155


Healthy donors PBMC A.

0.09%

99.87%

LI/CRC TILs

0.13%

97.99%

1.97%

CD3

99.91%

LI/CRC PBMC

10.70%

0.04%

34.17%

0.06%

31.93%

0.76%

89.18%

0.08%

65.73%

0.04%

66.92%

0.38%

3.06%

Helios

B.

Gated on CD3+CD4– T cells

FoxP3

C.

10.12%

0.01%

34.05%

0.27%

32.39%

89.66%

0.21%

64.97%

0.71%

61.57%

Helios


NRP1

2.98%

NRP1

Figure 7. NRP1, Helios and FoxP3 expression on CD8+ T cells. We analyzed NRP1, Helios and FoxP3 expression on CD3+CD4(CD8+) T cells. Representative flow cytometric plots showing expression of (A) NRP1 (B) FoxP3 with Helios and (C) NRP1 with Helios on CD3+CD4- T cells from TILs, healthy donor PBMCs and LI/CRC patient PBMCs. LI/CRC: Colorectal cancer liver metastases; NRP 1: Neuropilin 1; PBMCs: Peripheral blood mononuclear cells; TILs: Tumor-infiltrating lymphocytes.

consisted of two populations: one that co-expressed Helios and one that did not. There did not seem to be any direct correlation between expression of NRP1 and Helios. We also identified NRP1 on a subset of FoxP3--CD25+ TILs and to a lesser extent on FoxP3--CD25-- TILs and PBMCs from LI/CRC patients. These subsets, however, did not co-express Helios. A small yet significant FoxP3-- Helios+NRP1-- T cell subset was also present in all sample groups; this population comprised both CD25+ and CD25-- T cells. The role of Helios in T cells is still not clear. It is possible that Helios defines activated or antigen-experienced Tregs. More recently, Helios-- Tregs have been suggested to define 156

a distinct population of inducible pTregs with a greater capacity to ‘adapt’ and alter their suppressive activity according to their microenvironment [40]. Helios has also recently been implicated in a key role maintaining the Treg phenotype by repressing IL-2 expression [41]. No link however can be made between NRP1 and Helios expression as of yet. A population of CD4+FoxP3--Helios--NRP1+ T cells was also present (< 1%) in patients TILs and PBMCs but not healthy donor PBMCs. On a side note, we also found that Helios was upregulated on CD8+ TILs and PBMCs from patients compared to healthy donors. FoxP3 was not expressed in any significant amount in any of the sample




groups, whereas NRP1 was expressed at low levels on Helios+ and Helios-- CD8+ TILs and patient PBMCs. The significance of this is not yet clear. We also defined different T cell populations according to their expression of CD25. Interestingly, we found that NRP1 was mainly expressed on CD25+ TILs, with the highest levels of NRP1 expression being observed on CD25HI subsets. Similar findings have been reported on NRP1-expressing T cells isolated from lymph nodes [11]. Our findings suggest that rather than defining Tregs, NRP1 might correlate best with CD25 as a marker of late-stage activation. The same report found that NRP1 correlated with expression of GITR and HLA-DR, both molecules associated with late-stage activation [11]. NRP1 has also been induced at significant levels on CD4+ and CD8+ T cells following in vitro polyclonal activation in the presence of IL-2 [35]. The majority of TIL samples in this study highly upregulated CD25 due to culture with IL-2. To determine the effect of IL-2, if any, on NRP1 expression, we set up a control culturing healthy donor and patients PBMCs with IL-2. NRP1 expression remained low in all samples and slightly increased in one patient sample following PBMC culture with IL-2. Although NRP1 was expressed at almost negligible levels on PBMCs, we found that NRP1 was expressed at a higher level on FoxP3+Helios+ PBMCs than FoxP3+Helios-- PBMCs following in vitro culture with IL-2. This might be indicative of increased proliferative activity of the Helios+ subset rather than specific upregulation of NRP1. This has previously been reported in human studies both in vitro and in vivo where FoxP3+Helios+, but not FoxP3+Helios--, T cell populations were expanded following treatment with IL-2 [27,42]. The mechanism by which NRP1 expression might be induced has been investigated previously. One group showed that intercellular transfer of NRP1 between NRP1+ DCs and NRP1-- resting T cells by trogocytosis might account for NRP1 expression [43]. A study by Milpied et al. reported NRP1 was induced on T cells following in vitro polyclonal activation [35]. Interestingly, T cells magnetically sorted to exclude any monocytes or DCs still expressed NRP1 following polyclonal activation. This shows that NRP1 can be synthesized and expressed by T cells independently. A recent study reported negligible NRP1 expression on lymphocytes isolated from peripheral blood, healthy colon tissue and CRC tissue [34]. TILs were extracted by physical means without culture beforehand. We obtained similar results in TILs isolated from LI/CRC by enzymatic disaggregation. This raises an interesting question regarding NRP1 expression since our results show that NRP1 is selectively upregulated on TILs isolated by explant culture. Our control confirms that IL-2 seems to have no direct influence on NRP1 expression suggesting that it is the TME and tumorassociated factors that might induce NRP1 expression. In this study, we detected NRP1 expression on CD4+NRP1-- PBMCs following 96 h co-culture with tumor tissue. In mice with EAE, NRP1 is upregulated on Tregs,

whereas in humans NRP1 has been shown to be upregulated on Tregs isolated both from the synovium and peripheral blood of RA patients [14,16,38]. The TME is a highly inflammatory environment and tumor-associated or inflammatory factors might upregulate NRP1 or even ‘prime’ lymphocytes for NRP1 expression following in vitro culture. Recent work has identified several key roles that NRP1 may play in the immune system. In mice, NRP1 expression enhances Treg suppressive activity in a cell-to-cell contact manner and blockade of NRP1 abrogates suppressive activity [44]. In human, NRP1+ Tregs have been reported to exhibit greater suppressive activity than NRP1-- Tregs although the mechanisms by which this suppressive activity is exerted are not clear [11]. NRP1 blockade did not affect Treg suppressive activity in vitro suggesting that NRP1 is not directly involved in Treg suppressive mechanisms. Homophilic interactions between NRP1 on T cells and DCs have been shown to prolong T cell-DC interactions and enhance immunological synapse formation thus aiding in T cell activation [44,45]. This is likely not to be important for T cell activation since the majority of T cells and antigen-presenting cells (APCs) do not express NRP1. The only immune cell subset to constitutively express NRP1 is pDCs, found mainly within peripheral lymphoid organs. The significance of NRP1 expression on Tregs, specifically in CRC liver metastases, is not immediately clear. There are a number of mechanisms by which NRP1+ Tregs might influence cancer progression in CRC. pDCs have been reported to promote Treg development and to accumulate in cancers where they contribute to establishment of a tolerogenic environment. An increased pDC:myeloid dendritic cell (mDC) ratio has been reported in the mTDLN of cervical and colorectal cancers [37,46]. In both cancers the increased pDC: mDC ratio correlated with increased infiltration of mTDLN by FoxP3+ Tregs. In cervical cancer, these Tregs were defined specifically as FoxP3+NRP1+ Tregs also expressing the activation markers HLA-DR and CD69. Together NRP1+ T cells and pDCs can contribute to T cell anergy, Treg development and a tolerogenic microenvironment thus preventing effective immune responses, as recently discussed [9]. NRP1+ Tregs could also interact directly with NRP1 on tumor cells via homophilic interactions. Tumor cells have been shown to induce Treg generation in a contact-dependent manner [47]. Similar to prolonged cell interactions described earlier between NRP1-expressing DCs and T cells, the NRP1 homophilic interaction might contribute to prolonged Treg-tumor cell contacts aiding in Treg development. NRP1 is overexpressed on various cancers, including metastatic solid tumors. NRP1 is also able to bind and activate the latent form of TGF-b, LAP-TGF-b [48]. The interaction between semaphorin-4A (SEMA4A) has been shown to be critical for maintaining the stability of intra-tumoral Tregs in mice -possibly providing a novel therapeutic target [49]. Perhaps most interestingly, in murine models, NRP1 has been shown to mediate Treg migration into tumor tissue through


157



interactions with tumor-derived VEGF [50,51]. This has not yet been confirmed in human studies. However, NRP1 has been shown to be important in tumor cell migration, differentiation and adhesion, and also for migration of tumorassociated macrophages into hypoxic tumor regions in response to VEGF and other growth factors [52,53]. Whereas these mechanisms have been described in various settings, further investigations are required to confirm whether they are actual pathways by which NRP1 expression on T cells or Tregs can contribute to tumor progression. As a therapeutic target, NRP1 has already been targeted on tumor vasculature - this could be especially effective since NRP1 is overexpressed on tumor tissue and correlates with tumor progression and invasiveness [20,54]. NRP1-targetted therapeutics including cell-penetrating peptides and NRP1targetted liposomes and gold nanoparticles enhance therapeutic activity and penetration into tumor tissue while allowing targeted drug delivery [9,21,55]. In addition anti-NRP1 antibodies synergize with bevacizumab and are effective in inhibiting angiogenesis as reported in a recent clinical trial [23]. In the TDLN of cervical cancer patients, a selective reduction of NRP1+ Tregs correlated directly with increasing efficacy of chemoradiation therapy and reduction in tumor mass, whereas NRP1-- Treg levels remained stable [10,11]. In another study, NRP1+ Tregs were increased in the TDLN of cervical cancer patients treated with high-dose chemoradiation and correlated with impaired anti-tumor immunity [10]. In chronic lymphocytic leukemia, NRP1+ Tregs were decreased following successful thalidomide therapy, although this was also attributed to reduced VEGF serum levels [56]. Utilizing currently existing NRP1-targetted therapeutics might allow for selective targeting and depletion of NRP1+ Tregs. This preliminary work shows that NRP1 is a possible target for Treg depletion in cancer. Further studies however must be undertaken with several possible future directions: i) functional studies to define the role of NRP1 on lymphocytes;

158

ii) Further define the correlation between NRP1+ Treg infiltration and prognosis, and also whether it is directly involved or it is a prognostic marker; iii) To investigate any beneficial anti-tumor immune responses by targeting NRP1+ Tregs in cancer. 5.

Conclusions

NRP1 is upregulated on TILs from colorectal cancer liver metastases. These include both CD4+FoxP3+Helios+/-- Tregs and to a lesser extent CD4+FoxP3-- T cell subsets. NRP1 might be upregulated by tumor-associated factors in vivo as we showed in vitro. NRP1 might also be a marker of late-stage activation as it correlated well with CD25 expression. Further studies are warranted especially regarding the function of NRP1 on immune cells. As a therapeutic target, NRP1 may allow selective targeting of TIL subsets including suppressive Tregs that impair anti-tumor immunity and contribute to cancer progression. Utilizing currently existing NRP1-targetted therapeutics might allow for selective targeting of tumorinfiltrating NRP1+ Tregs especially in highly inflammatory or metastatic cancers.

Acknowledgements We are grateful to Basil Ammori and Yazan Khaled for their help in providing samples from patients and healthy donors.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.



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Affiliation

Belal Chaudhary1,2 & Eyad Elkord†1,2,3 † Author for correspondence 1 United Arab Emirates University, College of Medicine and Health Sciences, PO Box 17666, Al Ain, UAE Tel: +971 37137527; Fax: +971 37671966; E-mail: [email protected]; [email protected] 2 University of Salford, School of Environment and Life Sciences, Biomedical Research Centre, Manchester, UK 3 University of Manchester, Institute of Cancer Sciences, Manchester, UK


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