Identification of a Common Developmental Pathway for Thymic Natural Killer Cells and Dendritic Cells by Carlos Márquez, César Trigueros, Jaime M. Franco, Almudena R. Ramiro, Yolanda R. Carrasco, Miguel López-Botet, and Marı́a L. Toribio Blood Volume 91(8):2760-2771 April 15, 1998 ©1998 by American Society of Hematology

Three-color flow cytometry analysis of postnatal CD34+ CD1− thymocytes. Three-color flow cytometry analysis of postnatal CD34+ CD1− thymocytes. CD34+CD1− thymocytes isolated as described in the Materials and Methods were analyzed by flow cytometry for the correlated expression of CD44, CD34, and one of the indicated MoAbs. Electronic gates were set as shown in the upper biparametric plot to analyze the expression of CD2, CD5, CD38, CD7, CD33, CD13, HLA-DR, and CD45RA antigens on CD44brightCD34bright(unshaded areas) and CD44intCD34int (shaded areas) thymocytes. Background values (dashed histograms) were determined with isotype-matched irrelevant antibodies. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

CD34+CD33lo and CD34+CD33− postnatal thymocytes cultured with multiple cytokines give rise to phenotypically distinct cell progenies. CD34+CD33lo and CD34+CD33− postnatal thymocytes cultured with multiple cytokines give rise to phenotypically distinct cell progenies. (A) Sorted CD34+CD33lo and CD34+CD33− postnatal thymocytes were reanalyzed for the expression of CD34 and CD33. (B) The correlated expression of CD44 and CD5 was independently analyzed on sorted CD34+CD33lo (left panels) and CD34+CD33− (right panels) cells either before (day 0) or after 3 and 5 days of culture with a mixture of IL-7, IL-1α, IL-6, SCF, and GM-CSF. (C) The correlated expression of CD34 and CD33 was analyzed on electronically gated CD44bright(left panel) and CD44lo (right panel) cell progenies recovered at day 3 from the multicytokine-supported cultures of CD34+CD33lo thymocytes. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

CD34+CD33lo thymocytes develop efficiently into DCs in multicytokine-supported cultures. CD34+CD33lo thymocytes develop efficiently into DCs in multicytokine-supported cultures. CD34+CD33lo thymocytes were cultured for 10 days with the cytokine mixture described in Fig 2. Biparametric histograms on the left show the correlated expression of CD44 and CD33, CD44 and CD4, CD44 and CD1, CD40 and CD33, and CD7 and CD13 on the cultured cells. Background fluorescence values were set by use of FITC- and PE-conjugated isotype-matched irrelevant antibodies. Monoparametric histograms on the right show the expression of HLA-DR, -DP, and -DQ, CD11c, CD80, and CD86 (shaded histograms) on the same cultured cells. Background fluorescence (unshaded histograms) was determined by staining with isotype-matched irrelevant MoAbs plus PE-conjugated goat antimouse Igs. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

CD34+CD33lo thymocytes develop simultaneously into NK Cells and DCs in multicytokine-supported cultures containing IL-2. CD34+CD33lo thymocytes develop simultaneously into NK Cells and DCs in multicytokine-supported cultures containing IL-2. CD34+ CD33lothymocytes were cultured with the mixture of IL-7, IL-1α, IL-6, SCF, and GM-CSF described in Fig 2, plus IL-2. (A) Depicts the correlated expression of CD44 versus CD5, CD44 versus CD34, and CD34 versus CD33 at day 4. CD34+CD44brightCD5lo/− cells represent 90% of total cells. High surface CD33 expression was observed on 75% of total cells. (B) Shows the phenotype of the cellular progeny at day 8. Cells were analyzed for the correlated expression of CD7, CD13, and either CD1, CD4, or CD56. Monoparametric histograms show the expression of CD1, CD4, and CD56 (shaded areas) on the CD7+CD13lo/− and CD7lo/−CD13+ cell subsets electronically gated as shown in the biparametric plot. Background fluorescence (unshaded histograms) was determined by staining with isotype-matched irrelevant MoAbs plus PE-Cy5-conjugated goat antimouse Igs. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

Growth and differentiation kinetics of NK cells and DCs derived from CD34+CD33lo thymocytes. Growth and differentiation kinetics of NK cells and DCs derived from CD34+CD33lo thymocytes. (A) CD34+CD33lo thymocytes (105/well) were cultured during 9 days in 0.2 mL of medium supplemented with the mixture of IL-7, IL-1α, IL-6, SCF, and GM-CSF either without IL-2 (•) or with IL-2 (▪). An additional culture was set up without IL-2, and IL-2 was added at day 4 (▴). The number of total viable cells recovered at the indicated days was determined by trypan blue dye exclusion. (B) CD34+CD33lo thymocyte cultures set up as described in (A) were analyzed by flow cytometry for the correlated expression of either CD7, CD13, and CD56, or CD7, CD13, and CD1, at the indicated days. The data show the absolute numbers of CD7+CD13lo/−CD56+ NK cells (open symbols) and CD7lo/−CD13+CD56− DCs (solid symbols) recovered from cultures lacking IL-2 (•, ○) or supplemented with IL-2 either from day 0 (▪, □) or from day 4 (▴, ▵). Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

NK cells and DCs develop simultaneously through a common CD44bright intermediate stage. NK cells and DCs develop simultaneously through a common CD44bright intermediate stage. Cells derived from CD34+CD33lo thymoctes cultured with IL-7, IL-1α, IL-6, SCF, and GM-CSF were recovered at day 2 and cultured for 5 additional days with the same cytokine mixture either with or without IL-2. (A) Shows CD44 expression versus cell size (FS, forward scatter; left plot) and the correlated expression of CD7 and CD13 on electronically gated CD44bright large (forward scatter >350) cells (right plot) at day 2. (B) Shows the kinetics of CD7 and CD13 expression on electronically gated CD44bright large cells recovered at the time points indicated after reculture with the cytokine mixture either with or without IL-2. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

Induction of NKR-P1A expression on cultured CD34+ CD33lo thymocytes. Induction of NKR-P1A expression on cultured CD34+ CD33lo thymocytes. Cultures of CD34+ CD33lo thymocyes were set up as described in Fig 6. The correlated expression of NKR-P1A and CD13 was analyzed by flow cytometry on electronically gated CD44bright large cells at the indicated time points. Percentages of NKR-P1A+ CD13+ cells recovered at days 3, 4, 5, and 7 were 27%, 22%, 7%, and 5%, respectively, in cultures lacking IL-2; and 29%, 40%, 30%, and 40%, respectively, in cultures containing IL-2. The correlated expression of NKR-P1A and CD56 was analyzed on electronically gated CD44bright large cells recovered under both culture conditions at day 7. Background fluorescence was determined by sequential staining with isotype-matched (IgG1) irrelevant MoAbs, FITC-conjugated goat antimouse IgG1, and PE-conjugated isotype-matched irrelevant MoAbs. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

Induction of CD25 (IL-2Rα) and CD122 (IL-2Rβγ) expression on cultured CD34+CD33lothymocytes. Induction of CD25 (IL-2Rα) and CD122 (IL-2Rβγ) expression on cultured CD34+CD33lothymocytes. CD34+CD33lo thymocytes cultured for 1 or 3 days with IL-7, IL-1α, IL-6, SCF, and GM-CSF were analyzed for the correlated expression of CD34 and CD25 or CD34 and CD122. Background fluorescence was determined by sequential staining with isotype-matched irrelevant MoAbs, FITC-conjugated goat antimouse Igs, and PE-Cy5-conjugated isotype-matched irrelevant MoAbs. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology

Identification of CD34+CD44brightCD5lo/−CD33−intermediate thymocytes in vivo. Identification of CD34+CD44brightCD5lo/−CD33−intermediate thymocytes in vivo. Lin−thymocytes depleted of the most immature CD34int-bright precursors, as described in the Materials and Methods, were analyzed by flow cytometry for the correlated expression of CD44, CD34, and one of the indicated MoAbs. Electronic gates were set as shown in the upper biparametric plot to analyze either the cell size (mean forward scatter, FSC) or the expression of CD5, CD13, and CD33 antigens (shaded histograms) on CD34+CD44bright (gate I) and CD34+CD44lo/− (gate II) thymocytes. Background staining values (unshaded histograms) were determined with isotype-matched irrelevant antibodies. Carlos Márquez et al. Blood 1998;91:2760-2771 ©1998 by American Society of Hematology