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.
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 allotransplants1-6. 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.
Peripheral αβTCR+CD3+CD4–CD8–double-negative (DN) T cells are divided into various subsets that possess distinct phenotypes and functions1-4. DN T cells are poorly understood but increasingly being implicated in pathophysiological immune responses in different disease models4-6.
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 (spleen and lymph nodes). However, they are major residents of the normal kidney and gut epithelium10-12. Very little is known about their function7 in the kidney in the steady state and under pathological conditions such as acute kidney injury (AKI) associated with kidney transplant.
Because of the intricate roles of different immune cells in regulating immune responses including alloresponses, defining the role of each player is critical in understanding alloresponses and designing new therapeutics. Given the significant numbers of DN T cells present in the kidney under physiologic and different disease conditions, DN T cells are likely to play a critical role in regulating immune and autoimmune responses in mice and humans, and alloresponses in transplant recipients. Accumulating though still scattered evidence implicates DN T cells in both pathogenic and immunosuppressive functions but it is poorly understood why and how they exhibit a specific harmful or suppressive function and how the environment influences them.
Due to their low abundance in the kidney, improved methods of isolation are necessary.
There is currently no standard protocol for isolation of DN T cells from the non-lymphoid tissues. Our protocol describes a novel method for isolation of DN T cells from the kidney; however, the method can also be used for various non-lymphoid tissues.
1. Preparation of Instruments, Culture Media and Reagents
2. Preparation of Kidney Digestion
3. Preparation of Kidney Mononuclear Cells (KMNC) from the Kidney Digestion
4. Isolation of Hematopoietic (CD45+) Cells from KMNC (Step I)
5. Isolation of DN T Cell from CD45+ Preparation by Negative Selection (Step II)
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 subjected to positive selection using CD45+ magnetic microbeads as described in section 4 (STEP I). The column bound cells were then eluted and analyzed for CD45+ by FACS. This results in enrichment of CD45+ cells to about 80 to 95% as analyzed by flow cytometry (Figure 2, middle panel). Among enriched hematopoietic CD45+ cells, about 40-60% was αβ TCR+ (data not shown), about 30-60% were DN T cells (Figure 2, right panel).
The final step involved subjecting enriched CD45+ cells to negative selection to label and remove CD4+, CD8+, MHC class II+ and CD16/32+ cells using biotin specific mAbs and anti-biotin microbeads and magnetic columns. This result in highly purified (90-95%) DN T cells (Figure 3). Following this protocol it was possible to obtain up to 0.5 x 106 hematopoietic CD45+ magnetically labeled cells per mouse (two kidneys).
Figure 1. Preparation of the kidney for digestion with 5% collagenase solution. The mouse kidney is cut into small pieces of 1-2 mm and placed in collagenase D 5% solution for 30 min for digestion. Please click here to view a larger version of this figure.
Figure 2. Strategy for enrichment of CD45+ hematopoietic cells from the Kidney. Left: CD45 staining of kidney MNC before enrichment using CD45+ kit by positive selection. Center: CD45 staining of kidney MNC after enrichment using CD45+ kit by positive selection. Right: CD4 & CD8 profile of αβTCR+ cells among gated CD45+ cells different subsets of T cells. Please click here to view a larger version of this figure.
Figure 3. Purity of isolated DN T cells. Left: CD45 staining of isolated kidney DN T cells by negative selection. Center: TCRαβ staining of isolated kidney DN T cells by negative selection. Right: CD4 & CD8 staining among isolated DN T cells by negative selection. Please click here to view a larger version of this figure.
Figure 4. Lymphocytes in the kidney before purification. Left: total cells in kidney tissue before CD45 cells purification. Right: CD45 staining in kidney tissue before purification. Please click here to view a larger version of this figure.
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), glomerulonephritis8,13. 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 major reason is paucity of DN T cells in secondary lymphoid organs and hence difficulty in obtaining sufficient cells for in vitro analysis and adoptive transfer experiments. DN T cells are primarily located in non-lymphoid tissues such as the kidney8 but the lack of reliable methods for their isolation from solid organ is impeding the effort to investigate their function. Therefore, our novel protocol for isolation of DN T cells from the kidney is expected to expedite research into the function of DN T cells. DN T cells accumulate in lymph nodes in large numbers in Fas deficient (lpr) or FasL deficient (gld) mice and they can be easily isolated from lymphoid organs 9,14,15, but it still remains a challenge to isolate these cells from other tissues.
According to this protocol, kidneys from two mice yield approximately 0.5 x 106 hematopoietic cells (CD45+) and around 0.2 x 106 DN T cells. This number is sufficient to perform in vitro culture, functional assays and/or FACs analysis. The purity of the DN T cell population must be confirmed by flow cytometry. This protocol provides a population of DN T cells that can be as pure as 98%. While this protocol is designed for isolation of DN T cells from the kidney, it can be modified for isolation of T cells from other non-lymphoid organs such as heart, thyroid etc.
Since DN T cells represent a small population, it is sometimes necessary to increase yield by performing additional rounds of purification. However if a higher number of DN T cells is required (higher than 0.5 x 106 cells) we recommend performing sorting separation in order to reduce time and reagent cost.
In summary, this protocol describes a novel method for isolation of DN T cells from the kidney. It is composed of a preparation (via digestion and enrichment of KMNC) step followed by positive selection and then negative selection for depletion of non-desired cell populations. Therefore, there is minimal direct manipulation of DN T cells during the isolation process. It is noteworthy that there are existing protocols for isolation of DN T cells from peripheral blood in mice and humans and from secondary lymphoid organs, similar to those used for isolation of conventional T cells.
Because it has been shown that some DN T cells express FcR-γ, an alternative option is not to include CD16/32+ in the cocktail 16,17. Successful isolation of DN T cells requires great attention to detail. Particularly crucial steps include: tissue digestion, cutting of the renal tissue into small pieces, and correctly identifying and collecting the light layer in the density gradient. Overall, several rounds of practice and some manual dexterity are required to achieve the desired result.
The authors have nothing to disclose.
This work is supported by NIH PBS (R21 AI095484). We thank NHI tetramer core facility for CD1d tetramer.
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) | |
Microscope | Olympus | Or equivalent equipment | |
Analytical flow cytometer | LSR II | ||
Name of the reagent | Company | Catalog Number | Comments |
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-Fc receptor (CD16/32) biotinylated | BD | 553143 | |
anti-MHC class II (I-A d) biotinylated | BD | 553609 | |
anti-CD1d PBS-57 tetramer | NHI tetramer | lote # 15621-PBS57 | |
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 | |
70mc 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 |
Petri Dish | Fisher Scientific | S33580 | |
Plate 24 wells | BD Biosciences | 354723 |