Deciphering Immune Regulatory Mechanism for the Immune Control of Human Viral Infections

1Minh Thu Nguyen, Harry T .Wynn-Williams1, Fabio E. Leal1, Lana Garimine2, Lishomwa C. Ndhlovu1, Department of Tropical Medicine, John A. Burns School of Medicine,

University of Hawaii, Manoa, HI, 2University of Hawaii Cancer Center, HI

The human immune system maintains a constant balance between tolerance and inflammatory responses though the use of a diverse set of mechanisms including T regulatory cells (Tregs). Tregs have traditionally been described as having the phenotype: CD4+CD25+FOXP3+CD127lo and while these cells constitute a relatively small percentage of CD4 cells, they play a critical role in maintaining tolerance by suppressing effector T-cell responses (Fig. 1A).

Introduction

Increased CD161 Surface Expression on CD39+CD25- T cells. CD161 was initially discovered on rodent Natural killer (NK) cells and has been reportedly expressed on IL-17 producing inflammatory CD4+ T-cell subsets in humans. From the flow cytometric analysis we observed a statistically significant increased frequency of CD161+ CD39+CD25- (Tind) CD4+ T cells compared with the CD161+ CD39+CD25- (Treg) CD4+ T cells in the peripheral blood mononuclear cells from five healthy donors (p = 0.0079) (Fig 4A,B). The increased CD161 in Tind cell expression compared to Treg cell populations correlated with the elevated gene expression of the KLRB1 gene analysis between the Tind and Treg cell populations in Fig 3 and validated our microarray result.

Results Conclusions

Subjects: Freshly isolated blood was obtained from buffy coats derived from six healthy blood donor from Stanford Blood Bank, CA. Flow Cytometry Cell Sorting Isolation and RNA Extraction: CD4+ T cell subsets were cell sorted into 4 populations according to expression of CD25 and CD39 to a purity >95%. RNA extraction was performed using Trizol RNA extraction. Total RNA was eluted from the columns, quantitated on a Nanodrop spectrophotometer, and aliquoted for further applications. In addition, the quality of the total RNA (~250 ng) was assessed on an Agilent 2100 Bioanalyzer Nanochip for subsequent steps and determined to be pure and of high-quality.Microarray: An Affymetrix GeneChip microarray using the Gene 1.0ST whole transcript array covering 36,079 refernence sequences. Moderated t statistics were used to examine differential expression based on expression of CD39+CD25+ and CD39+CD25- CD4+ T cells and a heat map was generated representing those genes with large variations in expression. Background research was conducted on those genes highly expressed in the CD39+ CD25- T cell population.Flow Cytometry Analysis: Cryopreserved PBMC’s from five healthy donors were rapidly thawed in RPMI 1640 with 2% FBS and washed in PBS buffer. Cells were counted using Guava staining procedure and resuspended for final concentrations of 106 cells/well. Stimulated cells were incubated with PMA/Ionomycin and BFA/Monensin for 4 hours at 37°C. For surface staining, conjugated antibodies to CD3, CD4, CD25, CD39 and CD161 were used. Intracellular staining was carried out using BD FACS Lysing Solution with 10 minutes of RT incubation. Samples were washed and resuspended with FACS Perm 1X with another round of RT incubation. The intracellular antibody mix consisted of conjugated antibodies for IFN-ɣ and IL-17. Samples were resuspended in 1% PFA prior to flow cytometric analysis using a BD Fortessa cytometer. Data was analyzed using Flowjo Software.

Differential Gene expression profile of CD39 Expressing CD4 T cells

AcknowledgementsThis project was supported in part by grants from the National Center for Research Resources (5P20RR016467-11) and the National Institute of General Medical Sciences (8 P20 GM 103466-11) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Heat map showing differential gene expression profile between CD39+CD25+ versus CD39+CD25- CD4+ T-cell populations. Bright red indicates high expression and bright green indicates low expression. (Fig 3). CD25-CD39+ vs CD25+CD39+ paired analysis was performed. Differences in expression values between these two groups were derived within each donor. We observed 96 differentially expressed genes containing coding for proteins associated with all types of processes, from anti-inflammatory to neurological functioning.

We observed that CD39+CD25+ T cells have an increased gene expressions of previously known genes expressed on suppressive T regs (CTLA-4, FOXP3 and CD25 (IL-2RA) and low expression of CD127 ( IL-7R). These specific gene transcripts were reversed on the CD39+CD25- CD4 T cells. Several novel genes were identified exclusive to CD39+CD25- CD4 T cells. One particular gene of interest was the KLRB1 gene encoding the CD161 immunoreceptor.

Objective

The mechanism by which these CD4+ (CD39+CD25-Foxp3-) T” inducer” cells function is still unclear and a distinct phenotypic profile is still uncertain.

In this study we performed a gene chip microarray of the two CD39 expressing CD4 T cell subsets to analyze their differential gene expression profile and identify a gene signature that could inform on their function properties. We also tested the enzymatic activity of CD39+ cells using a luciferase-based assay to detect ATP hydrolysis.

CD39-CD25- CD39+CD25+ CD39+CD25-

Measurement of CD161 Protein Expression CD4 T cell subsets by Flow CytometryAfter sorting leukocytes based on surface protein expression we see that:• CD39 expressing CD4 T cells are represented by two distinct phenotypic subsets and by using gene chip microarray we confirm that over 96 genes are differential expressed between these two immunoregulatory CD39+ CD4+ T cell subsets.• Killer cell lectin-like receptor subfamily B, member 1, also known as KLRB1, NKR-P1A or CD161 immunreceptor, was one of several genes that was differentially expressed at a higher level on CD39+ CD25- T cells. By flow cytometry assessment we confirmed our gene expression data and reveal that CD161 protein expression is markedly upregulated on the CD39+ CD25- Tind cells compared to CD39+CD25+ Treg cells.• Further characterization of the phenotype and function of these distinct CD39 ATP-hydrolyzing, adenosine- producing cells is fundamental in enabling us to understand their role in immunoregulation within human diseases and further by the microenvironment in which they reside or to which they are recruited.

Methods

The CD39 ectoenzyme has been shown to be exclusively expressed on Treg cells in mice and humans. However, CD39 by releasing adenosine through its enzymatic activity, can mediate inhibitory as well as immunostimulatory effects (Fig. 1B). We observed that a novel subset of CD39+ CD4+ T cells that lacks Foxp3 or CD25 have no suppressive function on the proliferation of responder T cells as opposed to CD39+CD25+ cells that retain suppressive characteristics (Fig. 2A, 2B). This novel subset, designated T "Inducer” cells is expanded during viral infections (Ndhlovu L,Leal F. et al, Euro J. Immunol.2010; Leal F,Ndhlovu L,Wynn-Williams H. et al PloSNTD.2013) in cancer and autoimmunity.

CD4+ T regulatory cells (TReg)

Express: CD25 (IL-2RA), FOXP3, CTLA-4,

Lack expression: CD127 (IL-7R)

Metabolic disruption: CD39Mechanism of TReg Suppression: Through CD39 ectoenzymatic activity

(A)

(B)

Figure 1

(B) TReg Suppression Assay: CFSE labeledCD39-CD25- CD4+T cells were co –cultured withthe unlabeled indicated subsets above for 4 days with anti-CD3;anti CD28 stimulation Figure 2

CD39+CD25- CD39+CD25+

Figure 3

Figure 4

(A) Flow cytometry Gating Strategy

Figure 4

Figure 4CD39

CD161

IFN-g

CD25

(B) CD4+ CD161 expression

CD161

IFN-g

KLRB1/CD161

(A) Two distinct CD39 expressing CD4 T cell subsets:

-CD39+CD25+Foxp3+-CD39+CD25-Foxp3-

CD39+CD25+

CD39+CD25-