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. Author manuscript; available in PMC: 2019 Jul 15.
Published in final edited form as: J Immunol. 2018 May 25;201(2):814–820. doi: 10.4049/jimmunol.1700755

Human naïve T cells express functional CXCL8 and promote tumorigenesis

Joel Crespo 1,6, Ke Wu 1,2, Wei Li 1,2, Ilona Kryczek 1, Tomasz Maj 1, Linda Vatan 1, Shuang Wei 1, Anthony W Opipari 3, Weiping Zou 1,4,5,6,7
PMCID: PMC6039239  NIHMSID: NIHMS967157  PMID: 29802127

Abstract

Naïve T cells are thought to be functionally quiescent. Here, we studied and compared the phenotype, cytokine profile, and potential function of human naïve CD4+ T cells in umbilical cord and peripheral blood. We found that naïve CD4+ T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8+ naïve T cells were preferentially enriched CD31+ T cells and did not express T cell activation markers and typical T helper effector cytokines including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naïve T cells retained high levels of CXCL8 expression. Furthermore, we showed that naïve T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naïve T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses.

Keywords: Naïve T cell, memory T cell, CXCL8, tumor, tumorigenesis, neutrophil

Introduction

Naïve T cells are thought to be functionally quiescent. These cells are largely in the lymph nodes and may maintain lymph node integrity through lymphotoxin expression and be epigenetically poised in newborns to Th2 type responses (1–6). Interestingly, recent reports have shown that human naive T cells can express chemokine Interleukin-8 (CXCL8) (7, 8). However, the phenotype, cytokine profile, and functional potential of CXCL8+ naïve T cells in tumor are poorly defined in human literature.

CXCL8 is a pro-inflammatory chemokine with a variety of functions in the immune response. In cancer, CXCL8 promotes neutrophil migration, endothelial cell proliferation, wound healing responses, and metastatic potential (9–11). CXCL8 may promote cancer cell growth through increased angiogenesis and tumor cell proliferation (12–15). Such effects are abrogated in the presence of the drugs targeting the CXCL8 protein (14). CXCL8 is highly expressed in the advanced tumor cancer microenvironment and predicts poor prognosis (15). For example, we have shown that in patients with advanced ovarian cancer a subset of regulatory T cells (16) and plasmacytoid dendritic cells (17) can express CXCL8 and contribute to tumor immunopathology. In line with this observation, we observed that naive T cells selectively and spontaneously expressed high levels of CXCL8. Given the role of CXCL8 in tumor and the location of naïve T cells in the lymph nodes, we suggest that naïve T cell-derived CXCL8 may be functionally important in tumorigenesis and particularly in tumor lymphoid metastasis.

Materials and Methods

Human samples, cell isolation and flow cytometric analysis (FACS)

Human tonsil was obtained tissue procurement lab in the University of Michigan. Study was approved by the local Institutional Review Board. Human peripheral blood samples were obtained from healthy volunteers by venipuncture or cytopheresis. Human umbilical cord blood was collected by C.S. Mott Children's Hospital (University of Michigan) in blood collection tubes (Becton Dickinson Biosciences). Mononuclear cells were collected by Ficoll-Hypaque or Lymphoprep density gradient centrifugation. Peripheral blood naïve T cells were isolated by RossetteSep (Stemcell Technologies) followed by CD45RA+ or CD45RO+ microbeads (Myltenyi) column selection. Naïve T cells from umbilical cord blood and human tonsil were isolated by first depleting CD14+ monocytes followed by positive selection of CD4+ T cells using CD14+ monocyte selection EasySep and CD4+ T cell selection EasySep (Stemcell Technologies). CD4+CD45RA+CD31+/− naïve T cells were sorted from single-cell suspensions by high-speed cell sorter (FACSAria, Becton Dickinson Immunocytometry Systems). Neutrophils were isolated as described from adult blood (18). Cellular purity was measured by flow cytometry (LSRII, BD). Surface and cytokine profiles were measured through surface and intracellular staining, respectively, and analyzed by LSRII, DIVA and FloJo software (19–21).

Reagents

Recombinant-human cytokines and chemokines were from R&D systems. Antibodies for flow cytometry were CCR7, CD3, CD4, CD8, CD25, CD31, CD45RA, CD45RO, CD62L, CD69, CD154, CXCR2, CXCR3, CXCL8, IFN-γ, IL-4, IL-10, IL-17, and IL-22 (BD), CD57, CD122, Foxp3 (eBiosciences/ThermoFischer), KLRG-1 (BioLegend). Antibodies for T cell stimulation were CD3, CD28 (BD or Affymetrix). Small-molecule CXCR1/2 chemical inhibitor Reparixin (RPX) was purchased from MedChem Express.

In vitro T cell and OC8 tumor cell line cultures

T cell subsets (1 million cells/mL) were stimulated with αCD3/αCD28 beads (BD Biosciences) for up to two weeks. Cells were supplemented with fresh medium and recombinant-human IL-2 (5 ng/mL) every three days and re-stimulation on days six and twelve. Cytokine expression was measured at three-day time points through FACS. Umbilical cord blood naïve T cells (10 million cells/mL) were cultured for 24 hours in medium with PMA/Ionomycin and supernatants were saved and measured for CXCL8 (R&D Systems) and used in the sphere assay. Human tonsil naïve T cells were cultured in vitro in the presence of IL-7 (10ng/mL) and IL-15 (10ng/mL) for up to twelve days. Human primary OC8 ovarian cancer cells (22, 23) were cultured in vitro in RPMI supplemented with 10% FBS and 1% PennStrep (ThermoFischer).

Flow cytometry and Cytokine Detection

Freshly isolated mononuclear cells were stimulated in vitro with PMA/Ionomycin, GolgiStop and GolgiPlug (BD Biosciences) for 4 hours, then stained against extracellular and intracellular antigens. Single-cell suspensions from tissues were stained for extracellular surface antigens with specific antibodies, followed by fixation and permeabilization using Perm/Fix solution (ThermoFischer), then stained against intracellular antigens. Viable single cells were analyzed.

Tumor model

NOD.Scid.IL-2Rγc−/− (NSG) male mice aged 14–18 weeks (Jackson Laboratories) were used (22, 23). Primary human ovarian cancer cells (OC8) were cultured in RPMI supplemented with 10% FBS (24, 25). NSG mice were subcutaneously co-injected with 1×106 OC8 and 1×106 human T cells for two weeks (25). Mice were received peritoneal injection with (200uL) PBS-solubilized Reparixin daily (5mg/kg) or PBS (26). In a separate experiment, OC8 cells were cultured in the presence of recombinant human CXCL8 (10 ng/mL) for 24 hours and 1×106 single-cells were subcutaneously injected into mice.

Neutrophil migration assay

T cells were cultured in vitro for three days at 1 million/mL in the presence of αCD3/αCD28 beads (BD Biosciences). Supernatants were collected and placed on the bottom of 3-µm-Pore, 12-mm-diameter Transwells while neutrophils were placed on the upper chamber. Reparixin was added as indicated. Neutrophil migration was performed for two hours (18).

Sphere formation assay

Primary ovarian cancer cells (23, 25) were cultured in vitro in X-VIVO medium (Lonza) and ultra-low attachment plates (Corning) for 5d supplemented with 0, 1, or 10(µg/mL) recombinant human CXCL8. Separately, supernatants from umbilical cord blood naïve T cells cultured in vitro from above was mixed 1:1 with X-VIVO medium and cultured as described above at a density of 10,000 viable cells/well. Spheres (>100µm) were counted after 5 days.

Enzyme-linked Immunoassay

ELISA to detect CXCL8 was carried out as per the manufacturer’s protocol (R&D Systems).

Statistical analysis

Student’s T-Test was carried out to determine significant difference between expression levels, tumor weight, and neutrophil migration numbers.

Results

Naïve T cells express CXCL8

To determine whether naïve T cells are functionally quiescent, we studied and compared the cytokine profiles of naïve and memory CD4+ T cells in fresh umbilical cord blood and adult peripheral blood. CD45RA+CD45RO−CD62L+CCR7+ naïve T cells and CD45RA−CD45RO+ memory T cells were defined by flow cytometric analysis (FACS) (Fig. 1A). We found that umbilical cord blood naïve CD4+ T cells spontaneously expressed 22% CXCL8, and less than 1% IFN-γ, IL-4, IL-17, IL-10 and IL-22 (Fig. 1B). The expression level of CXCL8 was the highest among detectable cytokines in umbilical cord blood naïve CD4+ T cells (Fig. 1B). Next we compared the cytokine expression levels in cord blood and adult peripheral blood (Fig. 1C, D). Furthermore, naïve T cells expressed higher levels of CXCL8 than memory T cells (Fig. 1C, D). In contrast, the expression levels of IFN-γ and IL-17 were higher in memory T cells than naive T cells (Fig. 1C, E). Thus, human naïve T cells spontaneously and selectively express CXCL8.

Figure 1.

Figure 1

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Human naïve T cells spontaneously express CXCL8. (A) Gating strategy used to identify T cell subsets. Naïve T cells: CD3+CD4+CD45RA+CD45RO−CD62L+CCR7+. Memory T cells: CD3+CD4+CD45RA−CD45RO+CD62L−CCR7−. (B–E) Cytokine profile of cord blood and peripheral T cells. Intracellular cytokines were analyzed by FACS. Representative FACS plots and percentages are shown (B, C). (D–E) Graphical summary of CXCL8 (D) and IFN-γ (E) FACS values in human T cell subsets. n = 4–8, *, P < 0.05.

CXCL8+ naïve T cells are enriched in CD31+ T cells

We defined naïve T cells based on CD45RA+CD45RO−CD62L+CCR7+ surface phenotype. We could not exclude whether CXCL8+ naïve T cells were activated and/or differentiated in vivo. To address this possibility, we analyzed the activation and differentiation markers in CXCL8+ naïve T cells. CXCL8+ naïve T cells expressed neither of several activation markers including CD69, CD122, CD154, and CXCR3 (Fig. 2A) nor differentiation markers including CD57 and KLRG1 (Fig. 2B). We previously reported that a subset of regulatory T cells expressed in the tumor microenvironment (16). We found that CXCL8+ naïve T cells did not express Foxp3 and CD25 (Fig. 2C). Thus, CXCL8+ naïve T cells are not regulatory T cells. Interestingly, CXCL8+ naïve T cells expressed minimal levels of CXCR2, the receptor for CXCL8 (Fig. 2D), suggesting that CXCL8+ naïve T cells may not consume CXCL8 either from their own or from the microenvironment. CD31 is a marker for recent thymic emigrant cells (27). Adult peripheral blood naïve T cells can be divided into CD31+ cells and CD31− mature cells (27). We sorted CD31+ and CD31− naïve cells. We found that the levels of CXCL8 were higher in CD31+ T cells than CD31− T cells (Fig. 2E). Thus, peripheral blood CXCL8+ T cells are enriched in CD31+ naïve T cells.

Figure 2.

Figure 2

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CXCL8+ naïve T cells are enriched in CD31+ T cells. (A–D) Fresh CXCL8+ umbilical cord blood naïve T cells were analyzed for expression of surface and intracellular marks of activation and differentiation. Representative FACS plots and percentages are shown. Gated on CXCL8+ naïve T cells. (E) Graphical summary of CXCL8 expression in CD31+ and CD31− T cells (E). n = 4–8, *, P < 0.05.

Activated naive T cells remain to express CXCL8

CXCL8+ naïve T cells are enriched in CD31+ T cells. It suggests a potential dynamic expression of CXCL8 in naïve T cells. To test this, we activated naïve T cells from umbilical cord and adult peripheral blood with αCD3/αCD28 stimulation and kinetically analyzed CXCL8 expression. We found that TCR engagement initially maintained and subsequently stimulated CXCL8 expression in naïve T cells from umbilical cord blood (Fig. 3A) and adult peripheral blood (Fig. 3B) naïve CD4+ T cells. As expected, the levels of IFN-γ expression were low and gradually increased following T cell activation in CD4+ naïve T cells from umbilical cord blood (Fig. 3C) and adult peripheral blood (Fig. 3D). Next, we cultured CD4+ naïve T cells enriched from human tonsil with homeostatic cytokines IL-7 and IL-15 and kinetically analyzed CXCL8 expression. We showed that homeostatic cytokines initially maintained and subsequently stimulated CXCL8 expression in these cells (Fig. 3E, 3F). However, homeostatic cytokines did not stimulate IFN-γ expression in human tonsil CD4+ naïve T cells within 12 days stimulation (Fig. 3G). The data suggest that CXCL8 expression may be a functional feature for naïve T cells.

Figure 3.

Figure 3

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Human naïve T cells retain CXCL8 expression in vitro under homeostatic and activation conditions. (A–D) Results are shown as the kinetic CXCL8 (A, B) and IFN-γ (C, D) expression in naïve T cells in cord blood (A, C) and peripheral blood (B, D) upon activation with αCD3/αCD28 beads. (E) Expression plots for CXCL8 and IFN-γ following in vitro activation. (F–G) Results are shown as the kinetic CXCL8 (F) and IFN-γ (G) expression in naïve T cells in the presence of IL-7 and IL-15 from human tonsil. Average of 3–4 experiments is shown.

Naïve T cell-derived CXCL8 is biological active

To study the potential role of naïve CD4+ T cell-derived CXCL8, we carried out an in vivo model of tumor growth in the NSG mice. We inoculated primary human ovarian cancer cells into the NSG mice with or without umbilical cord blood naive CD4+ T cells at a 1:1 ratio. We treated the mice with Reparixin, a small-molecule chemical inhibitor for CXCL8 receptors, CXCR1 and CXCR2 (28, 29). We observed that mice that received umbilical cord blood naïve CD4+ T cells showed enhanced tumor volume and weight (Fig. 4A, 4B) as compared to mice which did not receive naïve CD4+ T cells. This effect was abrogated by Reparixin administration (Fig. 4A, 4B). In support of this observation, we incubated primary human ovarian cancer cells with recombinant human CXCL8 for 24 hours and subsequently injected into the NSG mice. CXCL8 treatment resulted in increased tumor weight (Fig. 4C). Thus, naïve T cell-derived CXCL8 is biologically active. CXCL8 is known to promote stemness qualities to cancer cells (30, 31). We performed a sphere assay by culturing primary ovarian cancer cells (OC8) (24, 25) in vitro with recombinant human CXCL8. We found that CXCL8 promoted tumor sphere formation (Fig. 4D). We further tested whether naïve T cells could promote sphere formation in a CXCL8-dependent manner. We observed an increase in tumor sphere numbers in the presence of naïve T cell supernatants. This effect was abolished by Reparixin (Fig. 4E). In addition, we tested the potential effect of naive T cell-derived CXCL8 on neutrophil migration in an in vitro transwell assay. We observed that neutrophils migrated toward naive T cell supernatants. This effect was blocked by Reparixin (Fig. 4F). As confirmation, we detected high levels of CXCL8 in umbilical cord blood naive CD4+ T cells (Fig. 4G). Thus, naive T cell-derived CXCL8 may be functionally important in vivo.

Figure 4.

Figure 4

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Naive T cell-derived CXCL8 promotes tumor growth and neutrophil migration. (A–B) Human primary cells (OC8) were mixed with cord blood naïve T cells and subcutaneously injected into NSG mice. Mice received daily intraperitoneal PBS or Raparixin (5mg/kg) injection. Following two-weeks, tumors were recovered and weighted. Representative tumors are presented in (A) and graphical summary of tumor size (B). n = 5/group. *, P <0.05. (C) OC8 tumor cells were pre-incubated with recombinant human CXCL8 in vitro for 24 hours and subcutaneously injected into NSG mice and tumor growth at endpoint was measured. n = 10/group. *, P < 0.05. (D) Effect of CXCL8 on ovarian cancer cell sphere formation. OC8 cells were cultured in vitro with recombinant human CXCL8 for 5 days and absolute number of spheres formed was reported. n = 3 in duplicates, P < 0.05. (E) Effect of UCB naïve T cell conditioned medium on ovarian cancer cell sphere formation. OC8 cells were cultured in vitro with UCB TN cell conditioned medium for 3 days in the presence or absence of Reparixin. n = 3 donors in duplicates, P < 0.05. (F) Neutrophil migration assay was carried out on cord blood naïve T cell-derived supernatants with increasing concentrations of Reparixin. Migrated neutrophil numbers were counted. n = 5, *, P <0.05. (G) CXCL8 secretion by naïve T cells. Cord blood naïve CD4+ T cells were cultured with anti-CD3 and anti-CD28 for 3 days. CXCL8 was detected by ELISA in the supernatants. n = 3.

Discussion

Naïve T cells are thought to require activation to acquire effector functions. Recent reports suggest that naive T cells carry out active functions prior to their activation such as maintaining lymph node integrity (1). Previous studies have shown that human umbilical cord blood CD4+ naïve T cells express CXCL8 and naive T cell CXCL8 expression is lost in adulthood (7, 8). Here we show that human CD4+ naive T cells spontaneously express high levels of CXCL8 and poised to express CXCL8 upon activation. Furthermore, adult human tonsil naïve T cells express CXCL8 spontaneously and this expression may be a functional feature of these cells. Typical T helper effector cytokines including IFN-γ, IL-4, IL-17 and IL-22 are not co-expressed with CXCL8 in naïve CD4+ T cells. Phenotypic experiments reveal that these cells are enriched in CD4+CD45RA+RO−CD62L+CCR7+CD31+ population. These CXCL8 producers show no signs of prior activation or differentiation, indicating that they are naïve T cells.

Interestingly, although naïve T cells in both fresh umbilical cord blood and adult peripheral blood express high levels of CXCL8, the percentage of CXCL8+ T cells are substantially higher in umbilical cord blood than that in adult peripheral blood. Naïve T cells from human tonsil also expressed CXCL8 under homeostatic conditions. In addition, ex vivo memory T cells express high levels of other effector cytokines including IFN-γ, rather than CXCL8. The data suggest that spontaneous CXCL8 expression may be a functional feature for naïve T cells. In support of this, we have found that naïve T cell-derived CXCL8 mediates neutrophil migration in vitro and promotes primary ovarian cancer growth in the NSG model. CXCL8 expression in naïve T cells may allow humans be prepared against pathogens encountered in early in life. Thus, CXCL8+ naive T cells may regulate innate cell trafficking and shape early immune responses. Given the well-defined role of CXCL8 in angiogenesis, CXCL8+ naïve T cells may support vascularization in newborn tissues. Of course, these cells may represent a remnant of the fetal immune system and their potential role in fetus development has yet to be determined (32). Moreover, the pro-tumor role of CXCL8 has been well-defined in many types of tumor (33, 34). As the CXCL8, CXCR1 and CXCR2 signaling is involved in the regulation of cancer stem cells (30) and tumor often metastasizes into lymph nodes, it is speculated that CXCL8+ naïve T cells may support tumor lymphoid metastasis via CXCL8 production. Altogether, naïve T cell-derived CXCL8 may play an important role in physiologic homeostasis and specific pathological conditions.

Acknowledgments

We thank Melissa Cantrell and Amy Drosd at Mott's Children's Hospital for helping in the delivery of cord blood to the Zou laboratory.

This work was supported in part by research grants from the NIH/NCI R01 grants (W.Z) (CA217510, CA123088, CA099985, CA193136 and CA152470) and the NIH through the University of Michigan’s Cancer Center Support Grant (CA46592).

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