Blood and tissue samples - MATERIALS AND METHODS

2. MATERIALS AND METHODS

2.5. Blood and tissue samples

Samples representing cell populations were obtained from our collaborators:

Natural Immunity Group (University Clinic of Düsseldorf, Germany) and Faculty of Medicine (University of Southampton, UK).

Taking blood and tissue samples with subsequent cell isolation was conducted with the approval of ethical authorities. Sample collection in the University Clinic of Düsseldorf was approved by the ethical committee of the Heinrich Heine University Düsseldorf (study numbers 3756, 3240, 3880, and 4723); blood and bone marrow samples were obtained from healthy donors, while tonsils were resected due to medical recommendations. Liver sample collection was approved by the National Research Ethics Service (reference number 13/WA/0329).

In all cases cells were isolated by density gradient centrifugation using Ficoll medium and specific lymphocyte subpopulations were selected by fluorescence-activated cell sorting (FACS) based on known surface markers. Details about origin and sorting strategy of cell populations are summarized in Table 1.

The analyzed sample set included human NK cells (n=57), T lymphocytes (n=17), B lymphocytes (n=2), monocytes (n=2), and CD34⁺ lymphocyte precursors isolated from peripheral blood, umbilical cord blood, bone marrow, tonsils, and liver. Besides this, NK cells from two independent in vitro differentiation assays were analyzed (n=9). These NK cells were generated from bone marrow-derived CD34⁺ progenitors by induction with the combination of cytokines including IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, and trombopoetin. The differentiation assays were performed by the Natural Immunity Group (University Clinic of Düsseldorf).

Table 1 Characteristic of studied lymphocyte populations

Name Source Cell

Type Phenotype Specific

characteristic

pbNKst4_1 Peripheral blood NK CD56^brightCD16^- Stage 4

pbNKst4_2 Peripheral blood NK CD56^brightCD16⁺ Stage 4

pbNKst5_1 Peripheral blood NK CD56^dimKIR2DL3⁺ Stage 5

cbNKst4 Cord blood NK CD56^bright Stage 4

pbNKst5_2 Peripheral blood NK KIR^-NKG2A^-CD56⁺CD3^- Stage 5

pbNKst5_3 Peripheral blood NK KIR^-NKG2A^-CD56⁺CD3^- Stage 5

pbNKst5lic_1 Peripheral blood NK KIR3DL1^-NKG2A^-KIR2DL1 -KIR2DL3⁺CD56⁺CD3^-

Stage 5 licensed pbNKst5lic_2 Peripheral blood NK KIR3DL1^-NKG2A

-KIR2DL1⁺KIR2DL3^-CD56⁺CD3^-

Stage 5 licensed

cbNKst5 Cord blood NK CD56^dimKIR2DL3⁺ Stage 5

cbCD34pos_1 Cord blood CD34 CD34⁺CD38⁺ Lymphocyte

precursors

cbCD34pos_2 Cord blood CD34 CD34⁺CD38⁺ Lymphocyte

precursors

cbTCD4pos_1 Cord blood T CD4⁺ T lymphocytes

cbTCD4pos_2 Cord blood T CD4⁺ T lymphocytes

cbTCD8pos_1 Cord blood T CD8⁺ T lymphocytes

cbTCD8pos_2 Cord blood T CD8⁺ T lymphocytes

pbNKst5lic_3 Peripheral blood NK KIR2DL3⁺KIR2DL1⁺ KIR3DL1⁺

Stage 5 licensed

cbCD34_1 Cord blood CD34 CD34⁺ Cultured without

feeder layer

cbCD34_2 Cord blood CD34 CD34⁺ Cultured with

feeder layer

pbNKst5lic_4 Peripheral blood NK KIR2DL1⁺ Stage 5 licensed

pbNKst5lic_5 Peripheral blood NK KIR2DL3⁺ Stage 5 licensed

pbNKst5lic_6 Peripheral blood NK KIR2DL3⁺ Stage 5 licensed

pbNKst5lic_7 Peripheral blood NK KIR2DL1⁺ Stage 5

daNKst2_1 Differentiation

assay

NK CD34⁺CD117⁺CD56^-CD94^- Stage 2

daNKst3_1 Differentiation

assay

NK CD34⁺CD117⁺CD56⁺CD94^- Stage 3

daNKst4_1 Differentiation

assay

NK CD34^-CD117

-CD56⁺CD94⁺CD62L⁺/ CD117^brightCD56⁺CD94^-KIR

-Stage 4

daNKst5_1 Differentiation

assay

NK CD117^dimCD56⁺CD94⁺ KIR⁺

Stage 5

pbTKIRpos_1 Peripheral blood T CD4⁺KIR2DL2⁺ KIR-positive

pbTKIRpos_2 Peripheral blood T CD4⁺KIR3DL2⁺ KIR-positive

pbTKIRneg_1 Peripheral blood T CD4⁺KIR^- KIR-negative

daNKst4_2 Differentiation

assay

NK CD117^brightCD56⁺CD94⁺ CD62L⁺/CD117^brightCD56⁺ CD94^-KIR

-Stage 4

daNKst2_2 Differentiation

assay

NK CD34⁺CD3^-CD94^-CD117⁺CD56^- Stage 2 day 7

daNKst3_2 Differentiation

assay

NK CD34^-CD117⁺CD56⁺CD94 -NKG2A^dim

Stage 3 day 11

daNKst3_3 Differentiation

assay

NK CD94^-CD117⁺CD56⁺NKG2A -KIR

-Stage 3 day 25

daNKst4_3 Differentiation

assay

NK CD94⁺CD117 -CD56⁺NKG2A⁺KIR

-Stage 4 day 25

daNKst5_2 Differentiation

assay

NK CD94⁺CD117 -CD56⁺NKG2A⁺KIR⁺

Stage 5 day 25

pbTCKIRneg_2 Peripheral blood T CD4⁺KIR^- KIR-negative

pbTKIRpos_4 Peripheral blood T CD4^-KIR3DL2⁺ KIR-positive

pbTKIRpos_5 Peripheral blood T CD4⁺KIR3DL2⁺ KIR-positive

stimulated pbTKIRpos_6 Peripheral blood T CD4⁺KIR3DL2⁺ CD3/CD28 KIR-positive

stimulated

pbTKIRneg_3 Peripheral blood T CD8⁺KIR^- KIR-negative

pbTKIRpos_7 Peripheral blood T CD8⁺KIR3DL2⁺ KIR-positiv

pbNKst5mem_1 Peripheral blood NK CD56^dimNKG2A^-CD57 -KIR2DL1⁺NKG2C⁺CD3^-

Stage 5 memory-like

pbNKst5mem_2 Peripheral blood NK CD56^dimNKG2A

+/-CD57⁺KIR2DL1⁺NKG2C^-CD3^-

Stage 5 memory-like

pbNKst5mem_3 Peripheral blood NK CD56^dimNKG2A

-CD57⁺KIR2DL1⁺NKG2C⁺CD3^-

Stage 5 memory-like

pbNKst5lic_8 Peripheral blood NK CD56^dimNKG2A^-CD57 -KIR2DL1⁺NKG2C^-CD3^-

Stage 5 licensed pbNKst5lic_9 Peripheral blood NK CD56^dimNKG2A⁺KIR2DL1

-KIR2DL3^-KIR3DL1^-CD3^-

Stage 5 licensed pbNKst5nonl_2 Peripheral blood NK CD56^dimNKG2A

-KIR2DL1⁺KIR2DL3^-KIR3DL1 -CD3^-

Stage 5 nonlicensed

pbNKst5lic_10 Peripheral blood NK CD56^dimNKG2A^- KIR2DL1

-KIR2DL3⁺KIR3DL1^-CD3

-Stage 5 licensed

pbNKst4_3 Peripheral blood NK CD56^bright Stage 4

pbNKst5nonl_3 Peripheral blood NK CD56^dimNKG2A^-KIR2DL1 -KIR2DL3^-KIR3DL1^-CD3^-

Stage 5 nonlicensed

cbB Cord blood B CD19⁺ B lymphocytes

pbTKIRneg_4 Peripheral blood T CD4⁺KIR⁺ Stimulated

pbTKIRneg_5 Peripheral blood T CD4⁺KIR⁺ CD3/CD28 KIR-positive

pbNKst5mem_4 Peripheral blood NK CD56^dimCD3

-CD57⁺KIR2DL3⁺NKG2C -NKG2A^-KIR2DL1^-

Stage 5 memory-like pbNKst5mem_5 Peripheral blood NK CD56^dimCD3^-CD57

-KIR2DL3⁺NKG2C⁺NKG2A -KIR2DL1

-Stage 5 memory-like pbNKst5lic_12 Peripheral blood NK CD56^dimCD3^-CD57

-KIR2DL3⁺NKG2C^-NKG2A -KIR2DL1^-

Stage 5 licensed

pbNKst5mem_6 Peripheral blood NK CD56^dimCD3

-CD57⁺KIR2DL3⁺NKG2C⁺NKG2 A^-KIR2DL1^-

Stage 5 memory-like pbNKst5nonl_4 Peripheral blood NK CD56^dimCD3^-CD57^-KIR2DL3

-NKG2C^-NKG2A^-KIR2DL1⁺

Stage 5 nonlicensed bmNKst1_1 Bone marrow NK CD34⁺CD117^-CD56^-CD94^-CD3

-CD14^-CD19

-Stage 1

bmNKst2_1 Bone marrow NK CD34⁺CD117⁺CD56^-CD94^-CD3

-CD14^-CD19

-Stage 2

bmNKst3_1 Bone marrow NK CD34⁺CD117⁺CD56^-CD94^-CD3

-CD14^-CD19

-Stage 3

bmNKst4_1 Bone marrow NK CD117^-CD34

-CD56^brightCD94⁺CD3^-CD14 -CD19

-Stage 4

bmNKst5_1 Bone marrow NK CD117^-CD34

-CD56^dimCD94⁺CD3^-CD14^-CD19

-Stage 5

cbNKst2 Cord blood NK CD3^-CD14^-CD19^-CD117⁺CD34⁺ Stage 2

cbNKst3 Cord blood NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^-CD94

-Stage 3

cbNKst4_2 Cord blood NK CD3^-CD14^-CD19

-CD56^brightCD94⁺

Stage 4

cbNKst5_2 Cord blood NK CD3^-CD14^-CD19^-CD117^-CD34

-CD56^dimCD94⁺

Stage 5

toNKst3_1 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^+/-CD94

-Stage 3

toNKst4_1 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^brightCD94⁺

Stage 4

liNKCXCR6n_1 Liver NK CD56^dimCXCR6^- CXCR-negative

liNKCXCR6n_2 Liver NK CD56^dimCXCR6^- CXCR-negative

liNKCXCR6p Liver NK CD56^dimCXCR6⁺ CXCR-positive

bmNKst1_2 Bone marrow NK CD3^-CD14^-CD19^-CD117^-CD34⁺ Stage 1 bmNKst2_3 Bone marrow NK CD3^-CD14^-CD19^-CD117⁺CD34⁺ Stage 2 bmNKst3_2 Bone marrow NK CD3^-CD14^-CD19^-CD117⁺CD34⁺ Stage 3

bmNKst4_2 Bone marrow NK CD3^-CD14^-CD19

-CD56^brightCD94⁺

Stage 4 bmNKst5_2 Bone marrow NK CD3^-CD14^-CD19^-CD56^dimCD94⁺ Stage 5

toNKst2 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34⁺ Stage 2

toNKst3_2 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^+/-CD94

-Stage 3

toNKst4_2 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^brightCD94⁺

Stage 4

toNKst5 Tonsils NK CD3^-CD14^-CD19^-CD117^-CD34

-CD56^dimCD94⁺

Stage 5

toNKst3a Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^-CD94

-Stage 3a

toNKst3b Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56⁺CD94

-Stage 3b

toNKst3_4 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^-CD94^-NKp44⁺

Stage 3

NKp44-positive

toNKst3_5 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56⁺CD94^-NKp44⁺

Stage 3

NKp44-positive

toNKst3_6 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56⁺CD94^-NKp44

-Stage 3

toNKst3_7 Tonsils NK CD3^-CD14^-CD19^-CD117⁺CD34

-CD56^-CD94^-NKp44

-Stage 4

pbB Peripheral blood B CD19⁺ B lymphocytes

cbMono Cord blood MC CD14⁺ Monocytes

pbMono Peripheral blood MC CD14⁺ Monocytes

pbNKst5lic_11 Peripheral blood NKs NKG2A⁺CD56^dim Stage 5

licensed

Im Dokument Analysis of the transcriptome of human NK lymphocytes (Seite 30-33)