Isolation of Double Negative αβ T Cells from the Kidney

Summary

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Abstract

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants^1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function⁷ due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney⁸ in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.

Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d α-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Introduction

Peripheral αβTCR⁺CD3⁺CD4^-CD8^-double-negative (DN) T cells are divided into various subsets that possess distinct phenotypes and functions^1-4. DN T cells are poorly understood but increasingly being implicated in pathophysiological immune responses in different disease models^4-6.

DN T cells are a unique immune cell type that is increasingly implicated in the regulation of various immune and autoimmune responses and the modulation of allotransplant tolerance. ^4-6,9 They are rare in the peripheral blood and secondary lymphoid organs (sp....

Protocol

1. Preparation of Instruments, Culture Media and Reagents

1. Instruments: Prepare the reagents under sterile conditions and use under a laminar flow hood.
2. Prepare 1 L of RPMI tissue culture medium, add 5% of fetal bovine serum, 2.1 g of bicarbonate, 10 ml glutamate 100x, 10 mg of HEPES, 1 mg sodium pyruvate and 10 ml of 100x penicillin/streptomycin.
   Note: the recommended tissue culture medium, RPMI, is interchangeable with DMEM.
3. Prepare 5% collagenase “D” solution (5 ml of collagenase “D” to 9 ml of tissue culture medium).
4. Make Percoll solution at three different concentrations: 100%, 80% and 40%. T....

Results

Wild type C57BL/6 (B6) kidney contains approximately 1.5-2.1 x 10⁶ mononuclear cells per kidney. Approximately less than 10% are hematopoietic CD45⁺ cells. For the preparation of kidney mononuclear cells (KMNC), the kidneys are cut into small pieces as shown in Figure 1 followed by digestion using 5% collagenase.

This is followed by performing a density gradient centrifugation to collect KMNC layer (Figure 2, left panel). KMNC were then s.......

Discussion

There is increasing interest in DN T cells since they are being implicated in different pathologic conditions such as autoimmune disorders, cancer, graft tolerance, and primary diseases of the kidney including acute kidney injury (AKI), glomerulonephritis^8,13. Therefore, there is need to better understand and characterize the pathophysiologic functions of DN T cells. However, currently there is a lack in understanding of these cells’ function as compared to CD4 and CD8 T cells. A ma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Laminar flow hood	Baker Company, Inc		Or equivalent equipment
Centrifuge	Beckman Coulter		Or equivalent equipment
Hemocytometer	Electron Microscopy Sciences	63514-11
Atmosphere-controlled incubator	Fisher Scientific		(37 °C with 5% CO2)
Analytical flow cytometer	LSR II
RPMI 1640	Media Tech	10-040-CV
Collagenase D	Roche	11088858001
Percoll	GE Healthcare	17-0891-01
Fetal bovine serum	Corning Cellgro	35-011-CV
0.1% sodium azide (NaN3)	Sigma-Aldrich	S2002
Buffer phosphate buffered saline (PBS)	Corning Cellgro	21-040-CV
PBS 10x	Mediatech	46-013-CM
EDTA buffer	Sigma-Aldrich	E1161
LS columns	MiltenyiBiotec	130-042-401
CD45+ microbeads	MiltenyiBiotec	6.78E-51
Biotin microbeads	MiltenyiBiotec	130-090-485
anti-CD45 (clone: 30-F11)  PerCP	eBioscience	G1397
anti-CD45 (clone: 30-F11)  APC-Cy7	Biolegend	103116
anti-TCR Pacific Blue  (ab-chain, clone: H57-597)	Invitrogen	HM3628
anti-CD4 PE	eBioscience	12-0043
anti-CD8 FITC	eBioscience	8011-0087
anti-CD4 biotinylated (GK1.5)	BD	553728
anti-CD8 biotinylated  (clone 53-6.7)	BD	553029
anti-MHC class II (I-A b) biotinylated	BD	553609
AutoMACS Running Buffer - MACS Separation Buffer	MIlteny I Biotec	130-091-221
C57BL/6J mice	Jackson Labs	000664	Kidneys - Lymph Nodes
Petri dish	BD Biosciences	356517
70 μm filter	Fisher Scientific	22363548
Plunger			Or equivalent equipment
GeneMate Tubes 50 ml	Bioexpress	C-3394-3	Or equivalent equipment
GeneMate Tubes 15 ml	Bioexpress	C-3394-1	Or equivalent equipment
CD1d α-galcer tetramer	NHI