1. Partial trisomy 21 contributes to T-cell malignancies induced by JAK3-activating mutations in murine models
by Paola Rivera-Munoz, Anouchka P. Laurent, Aurelie Siret, Cecile K. Lopez, Cathy Ignacimouttou, Melanie G. Cornejo, Olivia Bawa, Philippe Rameau, Olivier A. Bernard, Philippe Dessen, Gary D. Gilliland, Thomas Mercher, and Sébastien Malinge
BloodAdv
Volume 2(13):
July 10, 2018
© 2018 by The American Society of Hematology

2. Paola Rivera-Munoz et al. Blood Adv 2018;2:1616-1627
© 2018 by The American Society of Hematology

3. Generation of the Jak3 knockin model and hematologic disorders.
Generation of the Jak3 knockin model and hematologic disorders. (A) Proportion of JAK3 activating mutations in different types of human hematologic malignancies. The frequency of the JAK3A572/573V compared with mutations affecting other residues is shown (data obtained from and previously reported studies.9,11,21,22,35,36 (B) Representative western blots assessing the activity of mutant Jak3 and downstream pathways in thymocytes from 6- to 8-week-old Jak3WT/WT, Jak3KI/WT and Jak3KI/KI mice. Representative values of band intensity relative to Jak3WT/WT are indicated. (C) Absolute cell number in the bone marrow (BM), spleen (SP), thymus (Thy), and lymph node (LN) of Jak3WT/WT (WT/WT), Jak3KI/WT (KI/WT), and Jak3KI/KI (KI/KI) mice (5 to 13 mice per group, 6-14 months old). Data represented as the mean ± standard error of the mean (SEM); P values are indicated (Student t test). (D) Paraffin-embedded spleen sections from 10-month-old mice were stained with hematoxylin and eosin (H&E) as well as with anti-CD3, anti-factor VIII (von Willebrand factor [vWF]), and anti-myeloperoxidase (anti-MPO) antibodies (original magnification ×100). (E) Representative fluorescence-activated cell sorting (FACS) plots assessing the proportion of CD3+ cells in the spleen of 10-month-old wild-type (WT) and KI/KI mice. (F) Anti-CD3 immunostaining showing of the skin of 10-month-old WT and KI/KI mice (original magnification ×100). AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; CTCL, cutaneous T-cell lymphoma; DS-AMKL, Down syndrome–associated acute megakaryoblastic leukemia; FSC, forward scatter; JMML, juvenile myelomonocytic leukemia; MDS, myelodysplastic syndrome; NKCL, natural killer T-cell lymphoma nasal-type; T-ALL, T-cell acute lymphoblastic leukemia; T-PLL, T-cell prolymphocytic leukemia.
Paola Rivera-Munoz et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

4. Jak3KI/KI mice develop a CTCL-like CD8+ lymphoproliferative disorder in vivo.
Jak3KI/KImice develop a CTCL-like CD8+lymphoproliferative disorder in vivo. (A) Histogram plots presenting the percent of lymphoid cells populations (B220+/IgM+, CD4+/CD8– and CD8+/CD4–) in the bone marrow, spleen, thymus, lymph node, and peripheral blood (PB) (7 to 13 mice per group; 6- to 14-month-old mice). Double-negative (DN) (CD4–/CD8–) and double-positive (DP) (CD4+/CD8+) thymocytes are also depicted. (B) Histogram plots showing the mean ± SEM of p-Stat5; p-Stat3, and p-Erk in spleen cells (CD8+). P values are indicated (Student t test). (C) Evolution of the CD8+ population in PB, BM, SM, Thy, and LN of Jak3KI/KI over time (4 to 8 mice per group). (D) Representative FACS plots describing the TCRα/β+Gr1+ and CD62L/CCR7 phenotype of the CD8+ population in spleen cells of 8-month-old mice.
Paola Rivera-Munoz et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

5. Trisomy of the DSCR cooperates with Jak3KI/KI to enhance the CTCL-like disorder.
Trisomy of the DSCR cooperates with Jak3KI/KIto enhance the CTCL-like disorder. (A) Survival curves comparing Jak3-mutant mice in a euploid vs trisomic (Ts1Rhr) cellular context (4 to 8 mice per group). P values are indicated (log-rank Mantel-Cox test). (B) Histogram plots representing the average weight of spleens in 8- to 10-month-old mice. Data are shown as the mean ± SEM. P values are shown (Student t test). (C) Representative FACS plots of 10-month-old Jak3KI/WT, Jak3KI/KI, Ts1Rhr-Jak3KI/KI, and Ts1Rhr-Jak3KI/WT mice stained for T-cell markers CD4 and CD8 in PB, BM, SP, Thy, and LN cells. (D) Bar graph comparing the percentage of CD8+ cells in PB, BM, SP, Thy, and LN cells of Jak3KI/KI (black) and Ts1Rhr-Jak3KI/KI (blue) mice (4 to 8 mice per group; 4 independent experiments). Data are the mean ± SEM. P values are shown (Student t test). (E) Representative FACS plots assessing the expression of CD62L and CCR7 in the CD8+Gr1+ population of Jak3KI/KI, Ts1Rhr-Jak3KI/KI, and Ts1Rhr mice.
Paola Rivera-Munoz et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

6. Jak3KI/KI CTCL-like disorder is transplantable and is characterized by heterogeneous cell populations.
Jak3KI/KICTCL-like disorder is transplantable and is characterized by heterogeneous cell populations. (A) Kinetics of the percentage of the CD4+ and CD8+ populations in the peripheral blood of sublethally irradiated recipients transplanted with Jak3KI/KI or Ts1Rhr-Jak3KI/KI splenic cells. Data are shown as the mean ± SEM (3 to 4 mice per group). P values are shown (Student t test). (B) Representative FACS plots of the CD4 and CD8 splenic cells gated on CD3+Gr1+ of recipient animals transplanted with Jak3KI/KI and Ts1Rhr-Jak3KI/KI cells. (C) Bar graphs showing the proportion of the different T cell populations in recipient animals (Gated on CD3+Gr1+). Mean ± SEM and P values are indicated (5 mice per group). (D) FACS plot showing the percentage of CD4+Gr1+ and CD8+Gr1+ in the peripheral blood at 20 weeks of recipient animals transplanted with sorted CD4+ or CD8+ donors. (E) Proportion of CD4+ and CD8+ in the peripheral blood of sublethally irradiated recipients transplanted with CD4+ or CD8+ sorted cells (n = 5 to 9 mice per group). The presence of the Jak3KI/KI allele was verified by genotyping CD4+Gr1–, CD4+Gr1+, CD8+Gr1–, and CD8+Gr1+ sorted cell populations from peripheral blood.
Paola Rivera-Munoz et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

7. Jak3KI/KI T cells are sensitive to the JAK3 inhibitor tofacitinib.
Jak3KI/KIT cells are sensitive to the JAK3 inhibitor tofacitinib. (A) Representative western blot showing the efficacy of a 6-hour treatment with tofacitinib on Jak3KI/KI splenocytes. Representative values of band intensity relative to DMSO are indicated. (B) Dose-response curves of tofacitinib treatment over 3 days for KI/KI cell survival in vitro. Data are mean ± SEM. P values are shown (Student t test). (C) Comparison of the sensitivity of CTCL-like Jak3KI/KI, Ts1Rhr-Jak3KI/KI, CD4+ Ts1Rhr-Jak3KI/KI, and T-ALL Ts1Rhr-Jak3KI/KI to tofacitinib treatment. Mean ± SEM from 2 to 5 mice per group (each in triplicates) is indicated. P values are indicated.
Paola Rivera-Munoz et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology