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In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

Yamaguchi Y, Kudoh J, Yoshida T, Shimizu N - Biol Open (2014)

Bottom Line: Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state.Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs.Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk".

View Article: PubMed Central - PubMed

Affiliation: Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.

No MeSH data available.


Related in: MedlinePlus

Detection of endogenous Aire protein and observation of in vitro some aspect for negative selection of thymocytes in vivo by three lines of Aire+ cells.(A) Detection of mouse endogenous Aire proteins with anti-Aire antibody. Aire+ cell lysates were electrophoresed on 5–20% gradient polyacrylamide gel and processed for Western blotting with mouse polyclonal antibody (anti-Aire-pAb). Arrow indicates the location of Aire-protein (Mr = 62 kDa) along protein markers. Asterisk in Control indicates the FLAG-Aire fusion protein that was overexpressed in the transformed Aire+DC. In A9 cell line used as negative control, target size band was not detected. The anti-Aire-pAb pre-absorbed with synthetic mouse Aire peptides and pre-immune serum were used to confirm immune specificity. (B) Microscopic observation of co-cultures and separate cultures. Fresh thymocytes (7.0×106) and Aire+ cells (7.5×104 for Aire+TEC1 and TEC2, 3.0×105 for Aire+DC) were grown for 24 hrs as separate cultures or co-culture. Almost equal numbers of living Aire+ cells were counted in the separate culture and co-culture (59 and 61 cells in a visual field). Scale bar, 100 µm. (C) Estimation of viable cell number. Aire+ cells and the equivalent numbers of thymocytes were inoculated and cultured for 24 hrs, then living cells were stained with MTT and absorbance at 570 nm (A570) was measured. The cells used were: TEC1 and TEC2 (2.76×104 and 2.88×104), DC (9.60×104) and thymocytes (2.58×106). Average of four separate experiments was shown with standard deviation (bar). The reduction of absorbance value directly indicates the number of killed thymocytes. Note: significant reduction of thymocytes (dashed line regions) in contrast to thymocytes only. The number of eliminated thymocytes was estimated using A570 and Aire+ cell number. It was calculated that within 24 hrs single Aire+ cell appeared able to kill ∼30 thymocytes in vitro. (D) Apoptosis and non-apoptosis of fresh thymocytes induced by Aire+ cells: Fresh thymocytes (7.0×106) were added into the culture of Aire+TEC1 (7.5×104) and incubated for 16 h and 24 h. DNA fragmentation as an indication of apoptosis was detected with FITC (green) by TUNEL method and nuclear DNA was stained with PI (red). The wavy broken lines indicate the outline of Aire+TEC1 detected under DIC. Scale bar, 50 µm. [16 h] White arrows indicate the clusters of apoptotic thymocytes adhering on Aire+TEC1 (stained with both FITC and PI). [24 h] Open arrowheads indicate the clusters of apoptotic thymocytes (stained with both FITC and PI), under which smashed thymocytes on membrane of Aire+TEC1 are also stained with FITC. A white arrowhead indicates a cluster of thymocytes that seem alive (stained with PI but not with FITC) while tightly adhering on a living Aire+TEC1. NIH3T3 and A9 cells were used as control, and no adherence with thymocytes was observed.
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f01: Detection of endogenous Aire protein and observation of in vitro some aspect for negative selection of thymocytes in vivo by three lines of Aire+ cells.(A) Detection of mouse endogenous Aire proteins with anti-Aire antibody. Aire+ cell lysates were electrophoresed on 5–20% gradient polyacrylamide gel and processed for Western blotting with mouse polyclonal antibody (anti-Aire-pAb). Arrow indicates the location of Aire-protein (Mr = 62 kDa) along protein markers. Asterisk in Control indicates the FLAG-Aire fusion protein that was overexpressed in the transformed Aire+DC. In A9 cell line used as negative control, target size band was not detected. The anti-Aire-pAb pre-absorbed with synthetic mouse Aire peptides and pre-immune serum were used to confirm immune specificity. (B) Microscopic observation of co-cultures and separate cultures. Fresh thymocytes (7.0×106) and Aire+ cells (7.5×104 for Aire+TEC1 and TEC2, 3.0×105 for Aire+DC) were grown for 24 hrs as separate cultures or co-culture. Almost equal numbers of living Aire+ cells were counted in the separate culture and co-culture (59 and 61 cells in a visual field). Scale bar, 100 µm. (C) Estimation of viable cell number. Aire+ cells and the equivalent numbers of thymocytes were inoculated and cultured for 24 hrs, then living cells were stained with MTT and absorbance at 570 nm (A570) was measured. The cells used were: TEC1 and TEC2 (2.76×104 and 2.88×104), DC (9.60×104) and thymocytes (2.58×106). Average of four separate experiments was shown with standard deviation (bar). The reduction of absorbance value directly indicates the number of killed thymocytes. Note: significant reduction of thymocytes (dashed line regions) in contrast to thymocytes only. The number of eliminated thymocytes was estimated using A570 and Aire+ cell number. It was calculated that within 24 hrs single Aire+ cell appeared able to kill ∼30 thymocytes in vitro. (D) Apoptosis and non-apoptosis of fresh thymocytes induced by Aire+ cells: Fresh thymocytes (7.0×106) were added into the culture of Aire+TEC1 (7.5×104) and incubated for 16 h and 24 h. DNA fragmentation as an indication of apoptosis was detected with FITC (green) by TUNEL method and nuclear DNA was stained with PI (red). The wavy broken lines indicate the outline of Aire+TEC1 detected under DIC. Scale bar, 50 µm. [16 h] White arrows indicate the clusters of apoptotic thymocytes adhering on Aire+TEC1 (stained with both FITC and PI). [24 h] Open arrowheads indicate the clusters of apoptotic thymocytes (stained with both FITC and PI), under which smashed thymocytes on membrane of Aire+TEC1 are also stained with FITC. A white arrowhead indicates a cluster of thymocytes that seem alive (stained with PI but not with FITC) while tightly adhering on a living Aire+TEC1. NIH3T3 and A9 cells were used as control, and no adherence with thymocytes was observed.

Mentions: In our initial characterization study (Yamaguchi et al., 2011), expression of Aire mRNA was clearly detected in all three Aire+ cell lines (Aire+TEC1, TEC2 and DC) but endogenous Aire protein could not be detected, perhaps due to poor quality of polyclonal antibodies used. In the present study, we succeeded to produce a new polyclonal anti-Aire antibody (pAb) by immunizing mice with a mixture of synthetic peptides, which correspond to two peptide fragments (126–140 aa and 541–552 aa) of mouse Aire protein. The resulting pAb clearly detected endogenous mouse Aire proteins of Mr = 62 kDa on Western blots in all three Aire+ cell lines (Aire+TEC1, Aire+TEC2 and Aire+DC). Also, FLAG-tagged Aire protein was detected (see arrowed three bands and an asterisked band in Fig. 1A, leftside panel). Pre-immune serum or antibodies pre-absorbed with synthetic peptides detected no protein band of Mr = 62 kDa (Fig. 1A, middle and right-side panel). Thus, the newly made anti-Aire pAb had proper immune specificity. Moreover, the protein band of Mr = 62 kDa was not detected in mouse A9 skin fibroblasts. Thus, endogenous Aire protein was detected in all three Aire+ cell lines.


In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

Yamaguchi Y, Kudoh J, Yoshida T, Shimizu N - Biol Open (2014)

Detection of endogenous Aire protein and observation of in vitro some aspect for negative selection of thymocytes in vivo by three lines of Aire+ cells.(A) Detection of mouse endogenous Aire proteins with anti-Aire antibody. Aire+ cell lysates were electrophoresed on 5–20% gradient polyacrylamide gel and processed for Western blotting with mouse polyclonal antibody (anti-Aire-pAb). Arrow indicates the location of Aire-protein (Mr = 62 kDa) along protein markers. Asterisk in Control indicates the FLAG-Aire fusion protein that was overexpressed in the transformed Aire+DC. In A9 cell line used as negative control, target size band was not detected. The anti-Aire-pAb pre-absorbed with synthetic mouse Aire peptides and pre-immune serum were used to confirm immune specificity. (B) Microscopic observation of co-cultures and separate cultures. Fresh thymocytes (7.0×106) and Aire+ cells (7.5×104 for Aire+TEC1 and TEC2, 3.0×105 for Aire+DC) were grown for 24 hrs as separate cultures or co-culture. Almost equal numbers of living Aire+ cells were counted in the separate culture and co-culture (59 and 61 cells in a visual field). Scale bar, 100 µm. (C) Estimation of viable cell number. Aire+ cells and the equivalent numbers of thymocytes were inoculated and cultured for 24 hrs, then living cells were stained with MTT and absorbance at 570 nm (A570) was measured. The cells used were: TEC1 and TEC2 (2.76×104 and 2.88×104), DC (9.60×104) and thymocytes (2.58×106). Average of four separate experiments was shown with standard deviation (bar). The reduction of absorbance value directly indicates the number of killed thymocytes. Note: significant reduction of thymocytes (dashed line regions) in contrast to thymocytes only. The number of eliminated thymocytes was estimated using A570 and Aire+ cell number. It was calculated that within 24 hrs single Aire+ cell appeared able to kill ∼30 thymocytes in vitro. (D) Apoptosis and non-apoptosis of fresh thymocytes induced by Aire+ cells: Fresh thymocytes (7.0×106) were added into the culture of Aire+TEC1 (7.5×104) and incubated for 16 h and 24 h. DNA fragmentation as an indication of apoptosis was detected with FITC (green) by TUNEL method and nuclear DNA was stained with PI (red). The wavy broken lines indicate the outline of Aire+TEC1 detected under DIC. Scale bar, 50 µm. [16 h] White arrows indicate the clusters of apoptotic thymocytes adhering on Aire+TEC1 (stained with both FITC and PI). [24 h] Open arrowheads indicate the clusters of apoptotic thymocytes (stained with both FITC and PI), under which smashed thymocytes on membrane of Aire+TEC1 are also stained with FITC. A white arrowhead indicates a cluster of thymocytes that seem alive (stained with PI but not with FITC) while tightly adhering on a living Aire+TEC1. NIH3T3 and A9 cells were used as control, and no adherence with thymocytes was observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f01: Detection of endogenous Aire protein and observation of in vitro some aspect for negative selection of thymocytes in vivo by three lines of Aire+ cells.(A) Detection of mouse endogenous Aire proteins with anti-Aire antibody. Aire+ cell lysates were electrophoresed on 5–20% gradient polyacrylamide gel and processed for Western blotting with mouse polyclonal antibody (anti-Aire-pAb). Arrow indicates the location of Aire-protein (Mr = 62 kDa) along protein markers. Asterisk in Control indicates the FLAG-Aire fusion protein that was overexpressed in the transformed Aire+DC. In A9 cell line used as negative control, target size band was not detected. The anti-Aire-pAb pre-absorbed with synthetic mouse Aire peptides and pre-immune serum were used to confirm immune specificity. (B) Microscopic observation of co-cultures and separate cultures. Fresh thymocytes (7.0×106) and Aire+ cells (7.5×104 for Aire+TEC1 and TEC2, 3.0×105 for Aire+DC) were grown for 24 hrs as separate cultures or co-culture. Almost equal numbers of living Aire+ cells were counted in the separate culture and co-culture (59 and 61 cells in a visual field). Scale bar, 100 µm. (C) Estimation of viable cell number. Aire+ cells and the equivalent numbers of thymocytes were inoculated and cultured for 24 hrs, then living cells were stained with MTT and absorbance at 570 nm (A570) was measured. The cells used were: TEC1 and TEC2 (2.76×104 and 2.88×104), DC (9.60×104) and thymocytes (2.58×106). Average of four separate experiments was shown with standard deviation (bar). The reduction of absorbance value directly indicates the number of killed thymocytes. Note: significant reduction of thymocytes (dashed line regions) in contrast to thymocytes only. The number of eliminated thymocytes was estimated using A570 and Aire+ cell number. It was calculated that within 24 hrs single Aire+ cell appeared able to kill ∼30 thymocytes in vitro. (D) Apoptosis and non-apoptosis of fresh thymocytes induced by Aire+ cells: Fresh thymocytes (7.0×106) were added into the culture of Aire+TEC1 (7.5×104) and incubated for 16 h and 24 h. DNA fragmentation as an indication of apoptosis was detected with FITC (green) by TUNEL method and nuclear DNA was stained with PI (red). The wavy broken lines indicate the outline of Aire+TEC1 detected under DIC. Scale bar, 50 µm. [16 h] White arrows indicate the clusters of apoptotic thymocytes adhering on Aire+TEC1 (stained with both FITC and PI). [24 h] Open arrowheads indicate the clusters of apoptotic thymocytes (stained with both FITC and PI), under which smashed thymocytes on membrane of Aire+TEC1 are also stained with FITC. A white arrowhead indicates a cluster of thymocytes that seem alive (stained with PI but not with FITC) while tightly adhering on a living Aire+TEC1. NIH3T3 and A9 cells were used as control, and no adherence with thymocytes was observed.
Mentions: In our initial characterization study (Yamaguchi et al., 2011), expression of Aire mRNA was clearly detected in all three Aire+ cell lines (Aire+TEC1, TEC2 and DC) but endogenous Aire protein could not be detected, perhaps due to poor quality of polyclonal antibodies used. In the present study, we succeeded to produce a new polyclonal anti-Aire antibody (pAb) by immunizing mice with a mixture of synthetic peptides, which correspond to two peptide fragments (126–140 aa and 541–552 aa) of mouse Aire protein. The resulting pAb clearly detected endogenous mouse Aire proteins of Mr = 62 kDa on Western blots in all three Aire+ cell lines (Aire+TEC1, Aire+TEC2 and Aire+DC). Also, FLAG-tagged Aire protein was detected (see arrowed three bands and an asterisked band in Fig. 1A, leftside panel). Pre-immune serum or antibodies pre-absorbed with synthetic peptides detected no protein band of Mr = 62 kDa (Fig. 1A, middle and right-side panel). Thus, the newly made anti-Aire pAb had proper immune specificity. Moreover, the protein band of Mr = 62 kDa was not detected in mouse A9 skin fibroblasts. Thus, endogenous Aire protein was detected in all three Aire+ cell lines.

Bottom Line: Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state.Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs.Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk".

View Article: PubMed Central - PubMed

Affiliation: Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.

No MeSH data available.


Related in: MedlinePlus