Patients with advanced melanoma have expanded circulating CD8⁺ CD161^hi T cell populations.

<p>(<b>A</b>) The frequency of CD161^hi lymphocytes within the CD3⁺ γδ TCR⁻ population. (<b>B</b>) The CD4/CD8 profile of the CD161^hi T cells as identified in (A). (<b>C</b>) The frequency of CD161^hi T cells within the CD3⁺CD8⁺ γδ TCR⁻ lymphocyte gate. (<b>D</b>) CD161^hiCD8⁺ T cells from patients with advanced melanoma retain PLZF expression, as compared to conventional CD8 T cells. PBMC's from ten patients with metastatic melanoma were isolated via leukapheresis. The cells were stained and analyzed by FACS as described above. A representative control and two patient samples are shown for each analysis. Data from the control and patient groups are summarized in scatter plots. P values are shown in figures (n.s.  =  not significant).</p

Human γδ T cells express PLZF.

<p>(<b>A</b>) The MFI of PLZF expression in γδ T cells as compared to CD3⁺CD56⁻CD161⁻ T cells (P value is shown). Histogram shows PLZF expression in γδ T cells collected from two healthy donors in comparison to γδ T cells from the PLZF-deficient donor. (<b>B</b>) The frequency of γδ T cells from a healthy control and the PLZF deficient donor is shown in the panels at the top. Although the frequency of γδ T cell in PLZF deficient sample appears higher than the controls, the percentage is actually consistent with a broader analysis (data not shown) and with published reports. The bottom panels show CD161 and CD8 expression on γδ T cells from the healthy control and the PLZF deficient donor. γδ T cells were identified as shown in the top panels. Data from PLZF deficient donor were consistent from three independent blood samples. Numbers indicate the frequency of cells within each panel.</p

PBMCs from a healthy control and the PLZF-deficient donor were analyzed by FACS.

<p>(<b>A</b>) Histogram shows PLZF levels in permeabilized CD3⁻CD56⁺ NK cells. (<b>B</b>) CD56 levels on CD3⁻ PBMCs from a healthy control or the PLZF-deficient donor. CD16 and CD161 levels on CD3⁻CD56⁺ NK cells (solid line  =  PLZF-deficient donor; dotted line  =  healthy control). (<b>C</b>) CD3⁻CD56⁺ NK cells were sorted and then activated with beads coated with anti-NKp46 and anti-CD2. Supernatants were collected after 72 hours and analyzed by cytokine bead array. Control-1 was obtained from a healthy donor the day of the sort; control-2 was obtained from a healthy donor and the sample was shipped with the PLZF-deficient sample. FACS analysis was consistent for eight healthy controls. Samples from the PLZF-deficient donor were collected three times and were analyzed in three independent FACS experiments. Cells for the cytokine release experiment were collected from one sample. Sorted cells were split into multiple wells (between 3 and 8 wells) and treated as independent samples for statistical purposes. (** P value  = 0.0080; *** P value <0.0001).</p

Patients with systemic lupus erythematosus show decreased circulating have altered frequencies of PLZF-expressing T cells.

<p>(<b>A</b>) The frequency of CD161^hi lymphocytes within the CD3⁺ γδ TCR⁻ population. (<b>B</b>) The CD4/CD8 profile of the CD161^hi T cells as identified in (A). (<b>C</b>) The frequency of CD161^hi T cells within the CD3⁺CD8⁺ γδ TCR⁻ lymphocyte gate. (<b>D</b>) The remaining CD161^hiCD8⁺ T cells in SLE patients express low levels of PLZF. PBMC's were collected from six patients with SLE and stained as previously described. A representative control and two patient samples are shown for each analysis. Data from the control and patient groups are summarized in scatter plots. P values are shown in figures (n.s.  =  not significant).</p

Expression of PLZF in peripheral T cell subsets.

<p>(<b>A</b>) Intranuclear FACS analysis of PLZF expression in total CD3⁺ T cells; (<b>B</b>) CD161 and CD56 expression on CD3⁺, NKT cell⁻, γδ TCR⁻ lymphocytes; (<b>C</b>) PLZF expression in the indicated cell populations. Expression is compared to that of PLZF negative CD3⁺CD161⁻CD56⁻ conventional T cells (black line). (<b>D</b>) CD4 and CD8 expression on electronically gated CD3⁺CD161^hi T cells. (<b>E</b>) CD161 expression on electronically gated CD3⁺CD8⁺ T cells. (<b>F</b>) PLZF expression in the indicated cell populations. (<b>G</b>) Mean fluorescence intensity (MFI) of PLZF expression in indicated cells (N = 8). P values are indicated (n.s.  =  not significant); (<b>H</b>) cDNA made from RNA collected from the indicated populations was analyzed by real-time quantitative rtPCR. Units are relative to the expression of GAPDH. Data are the average of two independent experiments with an N = 4. (<b>I</b>) Healthy donor lymphocytes were sorted into four T cell subsets: CD161⁻CD4⁺, CD161⁻CD8⁺, CD161^hiCD4⁺ and CD161^hiCD8⁺. Representative data for CD161 expression on the four populations analyzed immediately post-sort is shown (black line  =  CD161⁻ cells; red line  =  CD161^hi cells). (<b>J</b>) CD161 levels of the indicated sorted cells, analyzed three days after activation with anti-CD3/CD28 coated beads, is shown (black line  =  CD161⁻ cells; red line  =  CD161^hi cells). (<b>K</b>) IFN- γ expression by peripheral blood T cells 6 hours after activation with PMA/Ionomycin or anti-CD3/C28 coated beads. Numbers indicate percentage of cells within the indicated gate. Data in A–G are representative of eight donors, each analyzed at least two times. Data in I and J are representative of two individuals done in two independent experiments. Data shown in K are from two different individuals and were two independent experiments.</p

Radiation followed by lymphocyte infusion leads to improved persistence of responses to tumor vaccination and to cure of some mice with established tumors.

<p>A) Mice were treated with radiation and lymphocyte infusion, and were then immunized with VP22-Opt-TRP1 DNA vaccine starting 1 day afterwards for 3 immunizations. Either 5, 12, or 19 days after the last immunization, splenocytes were restimulated with TRP1_455-463 peptide and IFNγ production from CD8⁺ T cells was quantified by flow cytometry. n=3/group, results shown from one of two experiments with similar results. B) Mice were treated as in A with hTRP2 or VP22-Opt-TRP1 DNA vaccine starting 1 day after radiation and lymphocyte infusion. Either 5, 12, or 19 days after the last immunization, mice were challenged intradermally with B16 melanoma. Mice were then monitored for development of palpable tumors. n=10-15/group, results shown from one of two experiments with similar results. C) Mice were inoculated intradermally with B16 melanoma. Three days later, some mice received radiation and lymphocyte infusion, followed 1 day later by immunizations with VP22-Opt-TRP1 DNA vaccine every 5 days. Mice were harvested on day 21 after irradiation and splenic and tumor-infiltrating lymphocytes were evaluated by flow cytometry. Additional mice were treated similarly for a total of 8 immunizations, n=10/group, and followed for overall survival, with results shown from one of three experiments with similar results. D) Tg(Grm1)EPv-transgenic mice began treatment at 8-12 weeks of age with 5 weekly vaccinations of VP22-Opt-TRP1 DNA or an empty control plasmid; some mice were pre-treated with irradiation and lymphocyte infusion one day prior. Mice were evaluated weekly for development of tail and ear melanomas. n=4-5/group, with combined results from 2 experiments.</p

Irradiation followed by lymphocyte infusion leads to marked T cell populations and increased frequency of IFNy+ tumor-antigen specific CD8+ T cells.

<p>A) CD3, CD4 and CD8-expressing splenocytes were quantified by flow cytometry at the indicated time points following irradiation and lymphocyte infusion. Typical numbers from normal mice are indicated by the dotted line. Results shown from one of two experiments with similar results. B) Regulatory T cell splenocytes (CD3⁺CD4⁺Foxp3⁺) and the CD8⁺ T cell to regulatory T cell ratio were quantified by flow cytometry. Expression of IL-15Rα and CD122 on tetramer⁺ CD8⁺ T cells was also quantified by flow cytometry. Results shown from one of two experiments with similar results. C) Mice were irradiated, received an infusion of 30×10⁶ splenocytes from naïve Pmel mice, which express a CD8⁺ TCR transgene recognizing the melanoma antigen gp100, and immunized weekly with hgp100 DNA plasmid vaccine for 3 doses. Naive Pmel splenocytes, and splenocytes isolated 20 days after infusion into irradiated animals +/- vaccination were restimulated with gp100_25-33 peptide (1μg/ml) and evaluated for staining with Pmel-specific tetramer and expression of IFNγ by flow cytometry.</p

Irradiation results in a short-lived enhancement of dendritic cell numbers and phenotype, corresponding to a similarly short-lived enhanced response to tumor vaccination.

<p>A) Mice were treated with irradiation and lymphocyte infusion, and splenic dendritic cell (DC) (CD11c⁺MHCII⁺) percentages and CD86 expression were quantified by flow cytometry. B) Examination of inguinal lymph nodes (LN) for the percentages and relative ratios of skin-derived DCs (CD11c⁺MHCII^high) and LN-resident DCs (CD11c⁺MHCII^intermediate) was also evaluated by flow cytometry. Results shown from one of two experiments with similar results. C) Mice were treated with radiation and lymphocyte infusion, and were then immunized with hTRP2 DNA vaccine starting 1, 3, or 7 days afterwards. After 3 immunizations, splenocytes were restimulated with TRP2_181-188 peptide or irradiated B16 cells, and IFNγ production from CD8⁺ T cells was quantified by flow cytometry. n=3/group, results shown from one of two experiments with similar results. D) Mice were treated as in A with varying days of initial hTRP2 DNA immunizations, and then were challenged intradermally with B16 melanoma. Mice were then monitored for development of palpable tumors. n=7-12/group, results shown from one of two experiments with similar results.</p

Deletion size and deletion separation can distinguish common fragile sites from tumor suppressor genes.

<p>(A) Box and whisker plots for “Deletion Size,” which measures the median size of deletions within a 2-Mb window centered on the gene, common fragile site genes (orange) and tumor suppressor genes (blue) (B) Box and whisker plots for “Deletion Separation,” which measures the separation (non-overlapping) of deletions within a 2-Mb window centered on the gene, (C) The co-deletion tendency of common fragile site genes (orange) and tumor suppressor genes (blue) relative to the co-deletion of genes of different classes (gray).</p

Deletion properties that distinguish common fragile site genes from tumor suppressor genes.

<p>Scatter dot plots of values for statistics that distinguish focal deletions affecting common fragile site (CFS) genes (orange) from tumor suppressor genes (blue). (A) “RNA/DNA Correlation,” which corresponds to the Pearson’s correlation coefficient of log2-transformed DNA copy number values and relative RNA expression values for the 115 tumor samples with both array CGH and RNA expression profiling data, (B) DNA Copy Number is the median value of segmented DNA copy number values. (C) Cell Line Proportion” which provides a relative measure of how frequently deletions for a given gene are found in cell lines rather than primary tumors. Panels (D) and (E) show the number of deletions found in each gene within a 10 Mb region centered on the <i>MACROD2</i> locus (D) and TP53 and <i>MAP2K4</i> loci (E). (F) Box and whisker plots of “Deletion Isolation,” which measures how frequently neighboring genes are deleted relative to the featured gene.</p