Activation and peripheral expansion of murine T-cell receptor γδ intraepithelial lymphocytes  Sarah R. Guehler, Jeffrey A. Bluestone, Terrence A. Barrett  Gastroenterology  Volume 116, Issue 2, Pages 327-334 (February 1999) DOI: 10.1016/S0016-5085(99)70129-0 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Transgenic IEL subsets in G8 and G8 × β2m° mice. Transgenic IEL, thymus, and splenic populations were analyzed by FCM, gating on lymphocytes by standard forward- and side-scatter values. (A) Dot plots were gated based on Vγ2-positive staining, and anti-CD8α and anti–Thy-1 staining were compared for Tg thymic populations. (B) Histograms show anti–Vγ2-PE staining of Tg IEL TCR levels. (C) Dot plots were gated based on Vγ2-positive staining, and anti-CD8α and anti–Thy-1 staining were compared for Tg IEL populations. (D) Anti-CD69 staining was plotted against anti–Thy-1 staining for Tg IEL populations. (E) Anti-CD69 staining for Tg splenic populations. Quadrants were determined on the basis of control staining; percentages of positively stained cells in each quadrant are shown. Data shown represent 5 experiments. Gastroenterology 1999 116, 327-334DOI: (10.1016/S0016-5085(99)70129-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Yields and apoptosis of IELs in G8 × β2m+ and G8 × β2m° mice. (A) IEL total cell yields per mouse (■) are compared with Tg+ cell yield per mouse (▨) for IELs from G8 × β2m+ and G8 × β2m° mice. *Statistically significant difference between G8 × β2m+ and G8 × β2m° IEL yields (n = 7; P < 0.05). Freshly isolated Tg+ IEL populations were analyzed by FCM, gating on Tg+ lymphocytes based on Vγ2-positive staining. (B) Viable Tg+ cells did not stain for annexin V or PI, and apoptotic cells stained positive for annexin V but not PI. To determine the percentages of viable and apoptotic Tg+ cells, quadrants were drawn based on control staining. Gastroenterology 1999 116, 327-334DOI: (10.1016/S0016-5085(99)70129-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Proliferative responses of G8 × β2m° and G8 × β2m+ IELs. Proliferative responses were measured for equivalent numbers of viable Tg+ IELs in response to irradiated H-2b (Ag+) APCs or 20 mg/mL immobilized anti-Vγ2 compared with irradiated H-2d syngeneic control APCs. Results are shown (A) without and (B) with 50 U/mL IL-2. All measurements were performed in triplicate, and the data are expressed as the mean, with an SE of <15%. Data shown represent 3 experiments. Gastroenterology 1999 116, 327-334DOI: (10.1016/S0016-5085(99)70129-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of class I MHC on IL-2 and IFN-γ production in IELs. Equivalent numbers of viable Tg+ IELs of G8 × β2m+ and G8 × β2m° mice were stimulated with irradiated H-2b APCs (Ag+) or immobilized anti-Vγ2 MAb. Culture supernatants were collected at 48 hours, and levels of (A) IL-2 and (B) IFN-γ (in pg/mL) were measured by ELISA. Data represent 3 experiments. Gastroenterology 1999 116, 327-334DOI: (10.1016/S0016-5085(99)70129-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Constitutive cytolytic activity by fresh Tg+ IELs from G8 × β2m+ and G8 × β2m° mice. Fresh effector (A) G8 × β2m+ and (B) G8 × β2m° IELs were titrated against 51Cr-labeled P815 target cells alone or with anti-CD3 (2C11), anti-TCR γδ (GL3), or control (N418) MAbs. Effector-target ratios are indicated, and results are converted to percent specific lysis of target cells. Data represent 3 experiments. Gastroenterology 1999 116, 327-334DOI: (10.1016/S0016-5085(99)70129-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions