This study analyzed the gene expression profiles of two subsets of CD4+ T cells that express the ectoenzyme CD39: CD39+CD25+ T regulatory cells (Tregs) and CD39+CD25- T "inducer" cells (Tinds). Microarray analysis identified 96 genes differentially expressed between these subsets, including higher expression of KLRB1 (which encodes the CD161 protein) on Tinds. Flow cytometry confirmed higher CD161 protein levels on Tinds compared to Tregs. The findings suggest CD161 may be a marker for the novel Tind subset and provide insight into their immunoregulatory roles and functions.

1. 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
Acknowledgements
This 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 Cytometry
After 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: CD39
Mechanism of TReg Suppression: Through
CD39 ectoenzymatic
activity
(A)
(B)
Figure 1
(B) TReg Suppression Assay: CFSE labeled
CD39-CD25- CD4+T cells were co –cultured with
the 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 4
0 50K 100K 150K 200K 250K
FSC-A
0
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FSC-H
95.3
0 102 10 3 104 105
<YARD-A>: Live
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SSC-A
92.2
0 50K 100K 150K 200K 250K
FSC-A
0
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SSC-A
94.8
0 102 103 104 10 5
<Ax700-A>: CD4
0
10
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<V500-A>:CD3
49.4
0 102
103
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<PE-A>: CD39
0
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105
<PE-Cy7-A>:CD25
8.24 2.53
5.0984.1
0 10
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<APC-A>: CD161
0
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<V450-A>:IFNg
7.59
0 102 103 104 105
<APC-A>: CD161
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<V450-A>:IFNg
13.2
CD39
CD161
IFN-g
CD25
(B) CD4+ CD161 expression
CD161
IFN-g
KLRB1/C
D161
(A) Two distinct
CD39 expressing CD4 T
cell subsets:
-CD39+CD25+Foxp3+
-CD39+CD25-Foxp3-
CD39+CD25+
CD39+CD25-