PHF6 在調控免疫反應中的潛在作用

自我更新和分化之間的平衡調節 HSC 和前驅細胞的數量。這種平衡由處於靜止狀態的 HSC 與循環的相對數量控制，並且在循環的 HSC 中，由對稱自我更新、不對稱和對稱分化 分裂的相對數量控制。這些過程受細胞內在和外在過程的調節。干擾素信號通路整合外部線 索來調節 HSC 行為以響應高水平的干擾素，這可能是病原體誘導的。當免疫系統被激活時，

HSC 和前驅細胞對乾擾素的反應增加的循環被認為使乾細胞和祖細胞能夠補充造血系統。

本論文發現 Phf6 cKO 小鼠的總白細胞計數(WBC)高於其野生型同窩小鼠。具體來說，

Phf6 cKO 小鼠的 B220⁺ B 細胞和單核細胞的絕對計數顯著升高，並且在 8 至 12 週時外週 血中 CD4⁺和 CD8⁺ T 細胞的計數顯著降低。將來，可能會對該結果進行研究，這可以透過在 Phf6 在 T 細胞內特異性缺失的模型中，例如通過使用 Lck-cre 轉基因，來測試 Phf6 在 T 細 胞分化與功能上的角色。

5.4 PHF6 缺失引起的造血變化如何導致白血病易感性

隨著小鼠年齡的增長，HSC 內的 DNA 損傷會增加，複製壓力會加劇這種情況。因此，

可以想像，Phf6 缺失的 HSC 循環增加可能導致其他突變的可能性增加。在這篇論文中，本 論文已經證明 Phf6 缺失增加了 HSCs 和前驅細胞的再增殖活性，並導致持續的自我更新。

以類似的方式，造血腫瘤中的 PHF6 突變可能有助於促進白血病細胞或白血病幹細胞的增殖 和持續自我更新。

雖然一般而言，分化後代的輸出受阻而不是增強與白血病有關，但 Phf6 缺失系統中分化 後代的輸出增強發生在成人造血發育的最早階段，導致前驅細胞數量增加。因此，偏向淋巴 樣的前驅細胞群的擴大可能是處於惡性轉化風險的人群。

此外，在 T 細胞發育的 DN2 階段，年齡依賴性 PHF6 缺失誘導的部分阻滯可能導致惡 性腫瘤。缺乏 PHF6 的細胞可能採用補償機制來彌補使它們易發生惡性轉化的發育缺陷，例 如在缺乏 TCF1 的小鼠中，由於補償性 Wnt 通路轉錄因子 LEF-1 的上調以克服發育阻滯而發 展為 T 細胞白血病。或者，Phf6 缺失小鼠中 DN2 細胞的減少可能導致對 DN3 生態位的競 爭減少，因此 DN3 階段的細胞將暴露於增殖壓力，類似於移植到祖細胞剝奪宿主中的胸腺移 植物中觀察到的情況，這導致獲得自我更新屬性。

有趣的是，5%的 AML 具有 DN2 T 細胞特徵，並且預計來自 T 細胞前驅細胞，這表明 DN2 發育階段的部分阻斷，可能不僅與理解 PHF6 在 T 細胞白血病，也可以部分解釋急性骨 髓性白血病中的作用。

5.5 PHF6 突變在腫瘤維持中的作用

本論文中提出的結果，解決了 PHF6 對造血腫瘤起始的影響。這對於了解 T-ALL 中第五 種最常見突變的分子基礎非常重要，並且在了解男性和女性 BFLS 患者和女性 PHF6 突變攜 帶者的造血系統惡性腫瘤風險方面具有臨床意義。

未來，一個需要解決的重要問題是 PHF6 在白血病進展中的作用。到目前為止，僅通過 在 B-ALL 細胞系中缺失 Phf6 來評估 PHF6 對腫瘤生長的影響，其中 PHF6 的缺失導致腫瘤 生長延遲。尚無 Phf6（或 PHF6）缺失在白血病進展中賦予生長優勢的例子。一旦腫瘤已經 建立，就白血病細胞的生長而言，具有和不具有 Phf6 突變的腫瘤之間的差異未明。然而，一 個懸而未決的問題是 PHF6 表達的喪失，對於那些具有 Phf6 突變的腫瘤的生長是否重要。

PHF6 對腫瘤維持的具體影響可以首先通過在具有 Phf6 突變的白血病細胞中重新表達 PHF6 來評估對生長的影響。PHF6 的缺失是否有助於持續的腫瘤維持對於了解 PHF6 突變是否具 有治療相關性很重要。雖然一般來說，腫瘤抑制基因的突變很難靶向，但受 PHF6 缺失影響 的下游途徑可能是一種潛在的策略，值得在該領域進一步研究。

圖 1：PHF6 基因和蛋白質結構域結構

Figure 1 PHF6 gene and protein domain structures. (A) The PHF6 gene contains 11 exons and is located on the X chromosome in human and mice. Source: UCSC Genome Browser.(Kent, Sugnet et al. 2002) (B) The gene encodes a protein of 365 amino acids with two ZaP (zinc knuckle, atypical PHD) domains and localization signals for the nucleus (*) and nucleolus (••).(Todd, Ivanochko et al. 2015)

圖 2：使用 CRISPR/Cas9 基因組編輯策略，培育 Phf6 floxed 小鼠 (A)

(B)

(C)

(D)

Figure 2 The CRISPR/Cas9 genome editing strategy used in the generation of Phf6 floxed mice. (A) Schematic of the Cas9/sgRNA/ODNs targeting sites in Phf6 between exon1 and 2 (5’ loxP site), and the intron after exon 11(3’ loxP site). A single-strand guiding RNA (sgRNA) was designed corresponding to the Cas9-targeting sequence

located at the 5’ loxP site and 3’ loxP site. (B) Two donor oligonucleotides (ODNs) carrying the 5’ and 3’-LoxP sequences were also designed for the targeted insertion. The Cas9 expression vector, sgRNA and the loxP donor strands were co-delivered into the zygotes.

(C) Different PCR products in the 10 offspring mice generated from targeted male mouse and two female wild type C57BL/6J mice using the primer Phf6 5VF1 and Phf6 5VR1 over the 5’ loxp site, and Phf6 3VF1 and Phf6 3VR1 over the 3’ loxp site. Mice containing the loxP sequence in 5’ and 3’ loxP sites are marked by stars. (D) The restriction fragment length polymorphism (RFLP) analysis of the 3 offspring mice obtained both loxP

sequences with the HaeII enzyme activity. (HaeII : Haemophilus aegyptius, number II, a restriction endonucleases; ODN: donor oligonucleotides)

圖 3：西方墨點法(Western blotting)顯示 Phf6^KO小鼠的骨髓、胸腺和脾臟中幾乎完全缺失 Phf6 蛋白

Figure 3 The western blot showed nearly complete absence of Phf6 protein in the bone marrow, thymus and spleen of Phf6 KO mice. (1 and 4: wild type mice; 2 and 3: Phf6 KO mice)

圖 4：Phf6 條件性剔除(KO)導致 8 至 10 週小鼠的淋巴生成異常

Figure . Phf6 conditional knockout (cKO) leads to bias in lymphogenesis. (A) Western hybridization of bone marrows harvested from 3 wild type mice (left 3 lane) or 3 Phf6 cKO mice (right 3 lane), Phf6 (~41kDa) were absent in bone marrows of Phf6 cKO. (B) Phf6 cKO mice have higher monocyte, WBC and lymphoid counts than wild type mice. (C) Phf6 cKO have higher B220⁺B cell (left panel) counts, lower CD4⁺ T cell (middle panel) counts and lower CD8⁺T cell (right panel) counts in their peripheral blood. (D) Wild type and Phf6 cKO have similar spleen weight (left panel) and thymus weight (right panel).

PHF6 (~41kDa) Tubulin (55kDa) Wild type Phf6 KO

C B C c o u n t

103/mm3

WBC

NEUT

LYMPH

MONO

EOS

BASO 0

5 1 0

1 5 W ild - ty p e

P h f6 ^/

-*

*

*

A B

B 2 2 0⁺

103/L

Wild-ty pe Ph f6 KO 0

2 4 6

8 *

C D 4⁺

103/L

Wild-ty pe Ph f6 KO 0 . 0

0 . 5 1 . 0 1 . 5 2 . 0

***

C D 8⁺

103/L

Wild-ty pe Ph f6 KO 0 .0

0 .5 1 .0 1 .5

* ***

C

S p le e n w e ig h t (g )

Wild ty pe Ph f6K

O 0 .0 6

0 .0 7 0 .0 8 0 .0 9 0 .1 0 0 .1 1 0 .1 2

T h y m u s w e ig h t (g )

Wild ty pe Ph f6K

O 0 .0 0

0 .0 2 0 .0 4 0 .0 6 0 .0 8 0 .1 0

D

Figure 4 Phf6 conditional knockout (cKO) leads to bias in lymphogenesis in 8- to

10-week mice. (A) Western hybridization of bone marrows harvested from 3 wild type mice (left 3 lane) or 3 Phf6 cKO mice (right 3 lane). Phf6 (~41kDa) were significantly abolished in bone marrow cells of Phf6 cKO. (B) Phf6 cKO mice have higher monocyte, WBC, and lymphocyte counts than wild type mice. (C) Phf6 cKO have higher B220⁺ B cell (left panel) counts, lower CD4⁺ T cell (middle panel) counts and lower CD8⁺ T cell (right panel) counts in their peripheral blood. (D) Wild type and Phf6 cKO have similar spleen weight (left panel) and thymus weight (right panel). (* <0.05, **<0.01, ***<0.001, ****<0.0001)

圖 5：在基礎狀態下，8-10 週小鼠造血幹細胞和前驅細胞的組成

Figure . Composition of hematopoietic stem and progenitor cells under steady state. (A) Phf6 cKO mice have higher proportion of LSK bone marrow cells compared to wild-type mice. (B) The proportion of ST-HSC was higher in Phf6 cKO. (C) Loss of Phf6 did not altered the proportion of LT-ST-HSC. (D) Phf6 knockout mice have less GMP. (E) Tissue sections showed that Phf6 knockout and wild-type have similar cellularity and architecture in their bone marrow.

A B C D

40X

400X

200µm 20µm

E

L S K

% in total BMCs

Wild-ty pe Ph f6 KO 0 .0

0 .2 0 .4 0 .6 0 .8

1 .0 *

S T - H S C

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0 0

0 .0 5 0 .1 0 0 .1 5

**

L T -H S C

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0 0

0 .0 5 0 .1 0 0 .1 5

G M P

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0

0 .5 1 .0 1 .5 2 .0

*

Wild-type Phf6 KO

Figure . Composition of hematopoietic stem and progenitor cells under steady state. (A) Phf6 cKO mice have higher proportion of LSK bone marrow cells compared to wild-type mice. (B) The proportion of ST-HSC was higher in Phf6 cKO. (C) Loss of Phf6 did not altered the proportion of LT-ST-HSC. (D) Phf6

knockout mice have less GMP. (E) Tissue sections showed that Phf6 knockout and wild-type have similar cellularity and architecture in their bone marrow.

A B C D

40X

400X

200µm 20µm

E

L S K

% in total BMCs

Wild -ty pe

Ph f6 KO 0 .0

0 .2 0 .4 0 .6 0 .8

1 .0 *

S T - H S C

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0 0

0 .0 5 0 .1 0 0 .1 5

**

L T -H S C

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0 0

0 .0 5 0 .1 0 0 .1 5

G M P

% in total BMCs

Wild -ty p

e Ph f6 KO 0 .0

0 .5 1 .0 1 .5 2 .0

*

Wild-type Phf6 KO

Figure 5 Composition of hematopoietic stem and progenitor cells under the steady state in 8- to 10-week mice. (A) Phf6 cKO mice have higher proportion of LSK bone marrow cells compared to wild-type mice. (B) The proportion of ST-HSC was higher in the marrow cells of Phf6 cKO mice. (C) The proportion of LT-HSC in the marrow is not different

between wild-type and Phf6 cKO mice. (D) Phf6 knockout mice have less GMP in the marrow. (E) Tissue sections show that Phf6 knockout and wild-type mice have similar cellularity and architecture in their bone marrow. (* <0.05, **<0.01)

圖 6：Phf6 剔除導致淋巴球比例異常 (A) CBC.

(B) Circulating lymphocyte % in peripheral blood.

(C) Circulating lymphocyte counts in peripheral blood.

(D) Lymphocyte % in the spleen.

Figure 6 Phf6 KO leads to bias in lymphogenesis. (A) Phf6 KO mice had similar WBC, Hgb, and PLT with wild type mice in the peripheral blood. (B)Phf6 KO mice had lower CD4⁺ T cell and CD8⁺ T cell percentages and higher B220⁺ B cell percentage in their peripheral blood at 8 weeks and 12 weeks. (C) Phf6 KO mice had lower CD4⁺ T cell and CD8⁺ T cell counts and higher B220⁺ B cell counts in their peripheral blood at 8 weeks. At 12 weeks, only CD8⁺ T cell counts and B220⁺ B cell counts were significantly different in the same way with that at 8 weeks. (D) Phf6 KO mice also had a higher proportion of B cells (left panel) and a lower proportion of CD8⁺ T cells (right panel) than the wild type mice in the spleen at 8 weeks. There was a trend of lower splenic CD4⁺ T cell percentage in Phf6 KO mice (middle panel) (Data were collected in two independent analysis). (*

<0.05, **<0.01, ***<0.001)

圖 7：Phf6^KO小鼠胸腺中 CD4 和 CD8 雙陰性(DN)細胞的比例

DN

% in bulk thymocytes

Wild-type

Phf6KO 0

1 2 3

4 *

DP

% in bulk thymocytes

Wild-type

Phf6KO 75

80 85 90 95

CD4 SP

% in bulk thymocytes

Wild-type

Phf6KO 7

8 9 10 11

CD8 SP

% in bulk thymocytes

Wild-type

Phf6KO 2.5

3.0 3.5 4.0 4.5

Figure 7 Phf6 KO mice had lower proportion of CD4 and CD8 double negative (DN) cells in their thymus. On the other hand, CD4 and CD8 double positive (DP), CD4 single positive (CD4 SP) and CD8 single positive (CD8 SP) were unaltered.

圖 8：骨髓移植測定(Bone marrow transplantation assay)結果

Figure 8 Bone marrow transplantation assay of Phf6 cKO and wild-type bone marrow.

The recipient mice of Phf6 cKO BMCs have (A) higher total WBC counts, (B) higher total neutrophil counts, and (C) lower total lymphocyte counts than recipients of wild-type BMCs.

(D) Within donor-derived blood cells (CD45.2), Phf6 cKO BMCs produced lower proportion of CD4⁺ and CD8⁺ peripheral blood cells in recipient mice compared to wild-type BMCs.

On the other hand, Phf6 cKO BMCs produced higher proportion of CD11b⁺Gr1^- blood cells in recipient mice. (* <0.05, **<0.01, ***<0.001, ****<0.0001)

圖 9：老年的的 Phf6^KO和野生型小鼠血像圖

W B C #

103/L

Wild-ty p e

Ph f6 KO 0

5 1 0 1 5 2 0 2 5

N E U T #

103/L

Wild -ty p

e Ph f6 KO 0

5 1 0 1 5 2 0

L Y M P H #

103/L

Wild -ty p

e Ph f6 KO 0

5 1 0 1 5 2 0

*

M O N O #

103/L

Wild -ty p

e Ph f6 KO 0 .0

0 .2 0 .4 0 .6

H G B

g/dL

Wild -ty p

e Ph f6 KO 1 0

1 2 1 4 1 6 1 8

P L T #

103/L

Wild -ty pe

Ph f6 KO 0

5 0 0 1 0 0 0 1 5 0 0 2 0 0 0

2 5 0 0 ***

A B C D E F

C o m p o s it io n

% in bone marrow

B2 20 +IgM+ B2 20 +IgM

-CD4 + CD8 +

CD1 1b+ G r1+

CD1 1b+ G r1

-Te r1 19 + 0

2 0 4 0 6 0 8 0

W il d - t y p e P h f6 K O

*

G H

Phf6 cKO Wild-type

s p le e n w e ig h t (g )

Wild type Phf6 cKO 0 .0

0 .1 0 .2 0 .3

s p le e n w e ig h t (g )

Wild typ e P hf6 cKO 0 .0

0 .1 0 .2 0 .3

I

P=0.064

*

*

***

Figure 9 Hemogram of 18-month age wild-type and Phf6 cKO mice. (A) Both mice have similar WBC counts in blood. (B) Both mice have similar neutrophil counts in blood.

(C) Phf6 knockout mice have lower lymphocyte counts in blood. (D) Both mice have similar monocyte counts in blood. (E) Both mice have similar hemoglobin levels in blood.

(F) Old Phf6 cKO mice have significantly lower platelet counts than wild-type mice in blood.

(G) Old Phf6 knockout mice have higher proportion of CD4⁺ cells in their bone marrow. (H) Representative pictures of spleen of wild type and Phf6 cKO mice. (I) The spleen weights of wild type and Phf6 cKO mice at 18-month age. (* <0.05, **<0.01, ***<0.001)

圖 10：老年的 Phf6^KO和野生型小鼠病理分析

Figure 10 Analysis of old wild-type and Phf6 knockout mice. f. Tissue sections showed increased extramedullary hematopiesis in the red pulp of Phf6 knockout mice’s spleens (inside 20x, outside 100x). g. Bone marrow sections showed increased

megakaryocytes number, decreased cell size and nuclear lobation of megakaryocytes (indicated by white arrows) in Phf6 knockout mice (inside 100x, outside 400x). h.

Megakaryocytes of old Phf6 knockout mice have decreased cell size (p=0.0007; both N=3)). i. Old Phf6 knockout mice have increased megakaryocytes number (p=0.0004; both N=3). j. Old Phf6 knockout mice had higher percentages of CD4+ cells (left) and lower percentages of CD8+ cells (right) in the spleens (p=0.0105 and 0.0247 respectively;

wild-type N=5, Phf6 knockout N=9). k. In the fraction of CD4+ splenic T cells, Phf6 knockout mice had higher percentages of naïve regulatory T cells (left panel) and lower percentages of effector T cells (right panel) (p=0.018 and 0.0205 respectively; wild-type N=5, Phf6 knockout N=9). l. In the bone marrow, old Phf6 knockout mice have decreased LT-HSC counts (p=0.0424; wild-type N=7, Phf6 knockout N=10).

圖 11：Phf6 缺失和 ICN1 過表達的協同作用

Figure 11 The synergistic effects of Phf6 deletion on ICN1 overexpression. a. Six weeks after transplantation, the recipients of Phf6 KO+ICN1 cells had lower hemoglobin levels, and trends of higher counts in WBC and neutrophils, lower counts in platelets, and higher LDH levels, suggesting higher tumor burden. b. The recipients of Phf6 KO+ICN1 cells had larger spleens. c. The recipients of Phf6 KO+ICN1 cells had and a shorter overall survival (p<0.0001). d. The survival disadvantages of Phf6 KO+ICN1 mice held true with different cell doses. e. There were more LICs (CD4-CD8-CD25+CD127+) in Phf6 KO+ICN1 mice marrow. f. Phf6 KO+ICN1 mice had more LIC (p<0.0001) by limiting

dilution assays.

圖 12：Phf6 缺失增強了 HSPC 的分化和細胞週期相關功能

Figure 12 Phf6 deletion enriched differentiation and cell cycle-associated

functions in HSPCs. a. Enrichment map showing the significantly perturbed functions in Phf6-null compared with wild-type HSPCs Nodes are GO gene sets and edges indicate shared genes. b. GSEA plot for representative gene sets enriched in Phf6 knockout versus wide-type cells. c. Heatmap representation of oncogenic signatures significantly enriched or depleted (p < 0.05) in Phf6 knockout HSPC. d. GSEA plot for representative oncogenic signatures enriched in Phf6 knockout versus wide-type cells. E2F1, MYC and mTOR signatures were positively enriched in Phf6 knockout LT-HSC, MPP2 and MPP3.

圖 13：培育 Phf6^KOIdh2^R172K小鼠和基因分型

Idh2^r172ktm2/+; Phf6^f/Y

Idh2^r172k; Phf6^f Idh2^r172k; Phf6^Y +; Phf6^f +; Phf6^Y Vav1-cre/+ ;

Phf6^f/f

Vav1cre;Phf6^f Vav1-cre;

Idh2^r172k;Phf6^f/f

Vav1-cre;

Idh2^r172k;Phf6^f/Y

Vav1-cre;

Phf6^f/f

Vav1-cre;

Phf6^f/Y +; Phf6^f Idh2^r172k; Phf6^f/f Idh2^r172k; Phf6^f/Y Phf6^f/f Phf6^f/Y

• Experiment

• Vav1-icre;Idh2^R172k;Phf6^f/Y

• Control

• Vav1-icre;Phf6^f/Y

• Idh2^R172k;Phf6^f/Y

• Phf6^f/Y

IDH2 ^R172K KI m ice cross Phf6 cKO m ice

+ +

-Phf6 5V Phf6 3V

Vav1Cre 44~47 IDH2

44~47

44~47 44~47

IDH2 X Phf6Cre 44-47 A

B

Figure 13 Animal mattings and genotyping mice. (A) Transgenic mice with heterozygous Idh2^R172K and floxed Phf6 were crossed with transgenic mice with

heterozygous Vav1-cre and floxed Phf6. (B) A representative gel picture of the strategies of genotyping by PCR on genomic DNA prepared from tail clips of Phf6 floxed mice, Vav1-cre transgenic mice and Idh2^R172K transgenic mice.

圖 14：在 8 週至 12 週齡時，Phf6^KOIdh2^R172K、Phf6^KO、Idh2^R172K和野生型小鼠週邊血中血 像圖和網織紅細胞(reticulocyte)計數

A B C

D E F

P=0.004 P=0.015

P=0.002 P=0.009

P=0.047

P=0.026

Figure 14 The CBC/DC and reticulocyte counts in the peripheral blood of the Phf6KOIdh2^R172K, Phf6KO, Idh2^R172K, and wild type mice at age 8 weeks to 12 weeks.

(A) White blood cell counts, (B) Neutrophil counts, (C) Lymphocytes counts, (D) Hemoglobulin levels, (E) Reticulocyte counts, and (F) Platelet counts of these four genotypes.

Doi:10.6342/NTU202300119

55

圖 15：在 8 週至 12 週齡時，Phf6^KOIdh2^R172K、Phf6^KO、Idh2^R172K和野生型小鼠週邊血中流 式細胞儀分析圖

Vav1icre;

Idh2^R172k; Phf6^f/Y Vav1icre;

Phf6^f/Y Phf6^f/Y

Idh2^R172k; Phf6^f/Y

% in viable cells

B220: 54.2%

CD4: 11.0%

CD8: 8.88%

CD11b+: 4.95%

CD11b+Gr.1+: 18.2%

Others: 2.77%

B220: 62.2%

CD4: 2.66%

CD8: 4.23%

CD11b+: 6.81%

CD11b+Gr.1+: 21.4%

Others: 2.7%

B220: 62.6%

CD4: 4.75%

CD8: 5.90%

CD11b+: 4.36%

CD11b+Gr.1+: 19.3%

Others: 3.09

B220: 23.8%

CD4: 2.41%

CD8: 7.16%

CD11b+: 22.7%

CD11b+Gr.1+: 38.9%

Others: 5.33%

FSC-A, SSC-A subset 2.30%

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

FSC-A, SSC-A subset 4.48%

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

FSC-A, SSC-A subset 5.59%

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

FSC-A, SSC-A subset 1.43%

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

2 103 104 105

Comp-PE-Cy7-A:: CD8

2 103 104 105

Comp-BV421-A:: B220

2 103 104 105

Comp-PE-A:: CD11b

FSC-A

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

Q13 4.95%

Q14 18.2%

Q15 2.60%

Q16 74.3%

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

Q13 6.81%

Q14 21.4%

Q15 1.74%

Q16 70.0%

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

Q13 4.36%

Q14 19.3%

Q15 2.63%

Q16 73.7%

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

Q13 22.7%

Q14 38.9%

Q15 3.90%

Q16 34.6%

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

Q1 8.88%

Q2 0.040%

Q3 11.0%

Q4 80.1%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

Q1 4.23%

Q2 0.035%

Q3 2.68%

Q4 93.1%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

Q1 5.90%

Q2 0.066%

Q3 4.75%

Q4 89.3%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

Q1 7.16%

Q2 0.139%

Q3 1.79%

Q4 90.9%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

CD4

CD8

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

Q5 54.2%

Q6 0.366%

Q7 11.0%

Q8 34.4%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

Q5 62.2%

Q6 0.721%

Q7 2.67%

Q8 34.4%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

Q5 62.6%

Q6 0.987%

Q7 4.67%

Q8 31.8%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-PerCP-Cy5-5-A:: 7AAD

0 50K 100K 150K 200K 250K

FSC-A 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV605-A:: CD45-2 0

50K 100K 150K 200K 250K

SSC-A

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

Q5 23.8%

Q6 0.532%

Q7 2.41%

Q8 73.3%

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 50K 100K 150K 200K 250K

FSC-A 0

102 103 104 105

Comp-FITC-A:: CD3

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV711-A:: CD44 0

102 103 104 105

Comp-BV510-A:: CD4

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-PE-Cy7-A:: CD8

0 102 103

104 105 Comp-BV510-A:: CD4 0

102 103 104 105

Comp-BV421-A:: B220

0 102 103

104 105 Comp-APC-Cy7-A:: Gr1 0

102 103 104 105

Comp-PE-A:: CD11b

CD4

B220

Gr.1

CD11b

Figure 15 Representative FACS profiles of the peripheral blood of Phf6KOIdh2^R172K, Phf6^KO, Idh2^R172K, and wild type mice at age 8 weeks to 12 weeks.