Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Summary

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Abstract

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic β-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy¹. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Protocol

1. Before setting up a suppression assay, one needs to coat tosylactivated beads with anti-human CD3 (clone UCHT1, final concentration 1μg/ml) for cell stimulation and afterwards check whether the beads are efficiently coated by setting up an in vitro proliferation assay using human T cells

1. Take 1ml of M-450 tosylactivated beads from original vial, place in magnetic stand and hold until all the beads have adhered to the side of the tube. Remove the buffer while tube is still in the magnetic stand. Take tube out of magnetic stand and add 1ml of buffer1, place the tube in magnetic stand again and remove the buffer1 while tube is in the magnetic stand....

Discussion

As the only unique feature to Tregs, suppressive function should be tested reliably and uniformly between subjects at different phases of disease development within the same and between different studies. We offer details of the suppression assay developed in our laboratory as our contribution to the standardization of this assay. In our extensive optimization study, we have determined that T cell stimulation with anti-human CD3-coated beads (UCHT1 clone, in concentration of 1μg/ml (as opposed to commercially availabl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque PLUS	Amersham	17-1440-03
DPBS-1X	GIBCO, by Life Technologies	14190-144
Trypan Blue	Invitrogen	15250-061
anti-CD4 microbeads	Miltenyi Biotec	130-045-101
Pre-separation filters	Miltenyi Biotec	130-041-407
LS column	Miltenyi Biotec	130-042-401
EDTA	Invitrogen	15575-020
BSA	Sigma-Aldrich	B4287
Anti-human CD4-APCCy7 (clone RPA-T4)	BD Biosciences	557852
Anti-human CD25-PE (clone M-A251; IL-2Rα)	BD Biosciences	555432
Anti-human CD8-FITC (clone RPA-T8)	BD Biosciences	555366
Anti-human CD14-FITC (clone M5E2; LPS receptor)	BD Biosciences	555397
Anti-human CD32-FITC (clone FLI8.26; FcγR-type II)	BD Biosciences	555448
Anti-human CD116-FITC (clone M5D12; GM-CSFRα chain)	BD Biosciences	554532
Dynalbeads M-450 tosylactivated	Invitrogen	140-13
Anti-human CD3	Ancell	144-024
Buffer1	Home made		0.1M Na2B4O7 pH7.6
Buffer2	Home made		PBS/2mM EDTA/ 0.1% BSA pH7.4
Buffer3	Home made		0.2M Tris/0.1% BSA pH8.5
Complete RPMI media	Home made		RPMI 1640 media 2 mM L-glutamine 5 mM HEPES 100 U/μg/ml peni/strept 0.5 mM sodium pyruvate
[3H] thymidine	PerkinElmer, Inc.	NET027Z005MC
human pooled AB serum	Atlanta Biologicals	S40110
Multiscreen harvest plate	EMD Millipore	MAHFC1H60
Microscint 20	PerkinElmer, Inc.	6013621