1. Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy
by Julia K. Tietze, Danice E. C. Wilkins, Gail D. Sckisel, Myriam N. Bouchlaka, Kory L. Alderson, Jonathan M. Weiss, Erik Ames, Kevin W. Bruhn, Noah Craft, Robert H. Wiltrout, Dan L. Longo, Lewis L. Lanier, Bruce R. Blazar, Doug Redelman, and William J. Murphy
Blood
Volume 119(13):
March 29, 2012
©2012 by American Society of Hematology

2. Effects of immunotherapy regimens on memory CD8 T cell expansion and function in vivo.
Effects of immunotherapy regimens on memory CD8 T cell expansion and function in vivo. (A) Survival after anti-CD40 and IL-2 treatment of subcutaneously implanted 3LL (left), subcutaneously implanted B16 (middle), or intravenously injected Renca (right) tumor models. (B) Total numbers of splenic CD44highCD8+ T cells on day 11 of anti-CD40 and IL-2 (left) and 5 days after IL-2 and IL-12 (right) immunotherapy regimens in naive mice. (C) BrdU incorporation of CD44highCD8+ T cells in response to immunotherapy. Numbers in dot plots represent percentages of CD8+ T cells. (D) Percentage of naive CD8+ T cells in wildtype and thymectomized mice 11 days after anti-CD40 and IL-2 therapy. (E) Percentage of memory CD8+ T cells in WT and thymectomized mice on day 11 of anti-CD40 and IL-2 treatment. (F) Expression of CD122 on CD44high (dashed line) compared with CD44low CD8+ T cells (shaded) from resting C57BL/6 mice. Data are representative of at least 3 independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology

3. Phenotype of cytokine induced CD8+ T cells in vivo and in vitro.
Phenotype of cytokine induced CD8+ T cells in vivo and in vitro. (A) Gating schema and expression of NKG2D and CD25 on CD44highCD8+ T cells. (B) Percentages of CD25 and/or NKG2D expressing CD44highCD8+ populations in the spleen (left) and lymph node (right) on day 12 of anti-CD40 and IL-2 treatment. (C) Percentage of PD-1+ CD8+ T cells 12 days after anti-CD40 and IL-2 treatment in the spleen and lymph node. (D) Histograms depicting expression of NKG2D+ (left), PD-1+ (middle), and CD25+ (right) CD8+ T cells after anti-CD3 and anti-CD28 (solid line) or IL-2 (dashed line) stimulation compared with media alone (shaded). Data are representative of at least independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology

4. Characterization of functional ability and tumor efficacy of immunotherapy expanded CD8+ T cells.
Characterization of functional ability and tumor efficacy of immunotherapy expanded CD8+ T cells. (A) Granzyme B expression in CD44highCD8+ T cells on day 12 of anti-CD40 and IL-2 treatment. (B) Lytic ability of whole splenocytes (left) or sorted CD44high and CD44low CD8+ T cells (right) from anti-CD40 and IL-2 or control treated animals redirected against anti-CD3 labeled P815 targets. (C) NKG2D expression and (D) quantification of CD25 (pos) and (neg) CD8+ T cells from mice bearing orthotopic Renca tumors. (E) Tumor growth after immunotherapy concurrent with blockade of NKG2D in sc NKG2D ligand expressing Renca tumors. Data are representative of at least 3 independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology

5. Phenotype of OT-I CD8+ T cells after cytokine immunotherapy versus immunization.
Phenotype of OT-I CD8+ T cells after cytokine immunotherapy versus immunization. (A) Expression of Vα2 and Vβ5.1/5.2 on peripheral blood CD8+ T cells from WT (left) and OT-I (right) mice. Numbers in the quadrants denote the percentages of CD8+ T cells expressing each marker. (B) Percentage and (C) numbers of CD44high expressing OT-I cells 11 days after anti-CD40 and IL-2 immunotherapy or OVA vaccination. (D) Dot plots depicting BrdU incorporation gated by CD44high expressing population in adoptively transferred OT-I cells after immunotherapy. Percentages of (E) CD25− and (F) PD-1 expression on CD44highOT-I cells from anti-CD40/IL-2 or OVA-vaccinated mice. (G) Percentage (left) and total numbers (right) of NKG2D+CD25− of CD44highOT-I cells on day 12 of anti-CD40 and IL-2 treatment. (H) Frequencies of NKG2D (left), PD-1 (middle), and CD25 (right) on OT-I cells after in vitro anti-CD3 and anti-CD28 (solid line) or IL-2 (dashed line) stimulations compared with media alone (shaded). Data are representative of at least 2 independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology

6. Functional analysis of OT-I cells after anti-CD40 and IL-2 treatment.
Functional analysis of OT-I cells after anti-CD40 and IL-2 treatment. (A) Granzyme B expression by OT-I T cells on day 12 day of anti-CD40 and IL-2 treatment. (B) Lytic ability (presented as percentage specific lysis at a 50:1 E/T ratio) of OT-I and WT mice on day 12 of anti-CD40 and IL-2 treatment. (C) Specific lysis of OVA-expressing EG7 and OVA-negative EL4 tumor lines after anti-CD40 and IL-2. Presented as percentage specific lysis at a 25:1 E/T ratio. (D) Growth of subcutaneous 3LL tumors in control and anti-CD40/IL-2-treated OT-I and WT mice compared. Data are representative of at least 2 independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology

7. Human CD8 T cells exhibit similar phenotypic characteristics to mice after in vivo and in vitro antigen nonspecific stimulation.
Human CD8 T cells exhibit similar phenotypic characteristics to mice after in vivo and in vitro antigen nonspecific stimulation. CD8 (purple) and CD25 (brown) expression in melanoma lesional biopsies after application of (A) vehicle or (B) 5% imiquimod cream daily for 14 days (40× magnification). (C) Dot plots depict gating schema and expression of HLA-DR and CD25 expression on human CD8+ T cells after 3 days of stimulation with PHA/IL-2 (TCR mimicking) or IL-2 alone in vitro. (D) Percentages of (TCR-dependent) CD25+HLA-DR+ (left) or PD-1+ (right) expressing CD45RO+CD8+ human T cells. (E) Percentage (left) and numbers (right) of CD25−HLA-DR+ (TCR-independently activated) human CD45RO+CD8+ T cells. (F) Percentages of CD25+ (left) and CD25− (right) human CD45RO+CD8+ T cells after 14 days of indicated stimulation. Data are representative of at least 2 independent experiments (*P < .05, **P < .01, ***P < .001).
Julia K. Tietze et al. Blood 2012;119:
©2012 by American Society of Hematology