This study was supported by Max McGee National Research Center for Juvenile Diabetesat Medical College of Wisconsin and Children's Research Institute of Wisconsin. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.

1. Before setting up a suppression assay, one needs to coat tosylactivated beads with anti-human CD3 (clone UCHT1, final concentration 1&mu;g/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
<ol>
<li>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. ...

<ol>
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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 1&mu;g/ml (as opposed to commercially availabl...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent or instrument</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>Amersham Pharmacia Biotech</td>
			<td>17-1440-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DPBS-1X</td>
			<td>Gibco </td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Invitrogen</td>
			<td>15250-061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-CD4 microbeads</td>
			<td>Miltenyi</td>
			<td>130-045-101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-separation filters</td>
			<td>Miltenyi</td>
			<td>130-041-407</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>B4287</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD4-APCCy7 (clone RPA-T4)</td>
			<td>BD Pharmingen</td>
			<td>557852</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD25-PE (clone M-A251; IL-2R&alpha;)</td>
			<td>BD Pharmingen</td>
			<td>555432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD8-FITC (clone RPA-T8)</td>
			<td>BD Pharmingen</td>
			<td>555366</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD14-FITC (clone M5E2; LPS receptor)</td>
			<td>BD Pharmingen</td>
			<td>555397</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD32-FITC (clone FLI8.26; Fc&gamma;R-type II)</td>
			<td>BD Pharmingen</td>
			<td>555448</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD116-FITC (clone M5D12; GM-CSFR&alpha; chain)</td>
			<td>BD Pharmingen</td>
			<td>554532</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynalbeads M-450 tosylactivated</td>
			<td>Invitrogen</td>
			<td>140-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-human CD3</td>
			<td>Ancell</td>
			<td>144-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer1</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>0.1M Na2B4O7 pH7.6</td>
		</tr>
		<tr>
			<td>Buffer2</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>PBS/2mM EDTA/ 0.1% BSA pH7.4</td>
		</tr>
		<tr>
			<td>Buffer3</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>0.2M Tris/0.1% BSA pH8.5</td>
		</tr>
		<tr>
			<td>Complete RPMI media</td>
			<td>Homemade</td>
			<td>&nbsp;</td>
			<td>RPMI 1640 media 
2 mM L-glutamine 
5 mM HEPES 
100 U/&mu;g/ml peni/strept 
0.5 mM sodium pyruvate
</td>
		</tr>
		<tr>
			<td>[3H] thymidine</td>
			<td>Perkin Elmer</td>
			<td>NET027Z005MC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human pooled AB serum </td>
			<td>Atlanta Biologicals</td>
			<td>S40110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Multiscreen harvest plate</td>
			<td>Millipore</td>
			<td>MAHFC1H60</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscint 20</td>
			<td>Perkin Elmer</td>
			<td>6013621</td>
			<td>&nbsp;</td>
		</tr>
		
	</tbody>
</table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

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 &beta;-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.

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

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

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.

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

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

human-vitro-suppression-as-screening-tool-for-recognition-an-early

Research

JoVE Journal

Immunology and Infection

14.4K 조회수.  Medical College of Wisconsin. Tregs는 면역 시스템의 강력한 진압하고 있습니다. 그들을 정의하는 독특한 표면 마커의 부족이있다, 그러므로, Tregs의 정의는 주로 기능입니다. 여기서 우리는 최적화된 설명을 체외에서 검정.