Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

Background: We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma

Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

Background: We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma. Methodology and Principal Findings: To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-c-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-c, TNF-a, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision. Conclusions and Significance: This work establishes that CD4 T cell help is dispensable for the generation of protectiv

Gene expression profile of peripheral blood lymphocytes from renal cell carcinoma patients treated with IL-2, Interferon-α and dendritic cell vaccine

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e50221, doi:10.1371/journal.pone.0050221.Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and TREG-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of TREG-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.This work was supported by grants from the National Institutes of Health (RO1 CA5648, R21CA112761, P20RR016437, and P30CA023108)

Total CD4 depletion does not affect vitiligo incidence or severity.

<p>Proportions of mice with depigmentation 60 days post-surgery.</p

Tumor-specific CD8 memory T cells do not require CD4 T cell help for efficient production of effector cytokines.

<p>(A) Mice received 10⁴ naïve CD8⁺Thy1.1⁺ (pmel) cells one day prior to treatment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026491#pone-0026491-g004" target="_blank">Figure 4A</a>. Sixty days following surgery, the proportion of Thy1.1⁺CD8⁺ cells in the lymph node able to produce cytokine upon restimulation with peptide was determined by flow cytometry. Symbols represent individual mice, and horizontal lines indicate averages. Representative contour plots are shown on left. (B) Sixty days following surgery, an IFN-γ ELISPOT was performed with CD8 T cells (pooled, 6 mice/group), using peptide-pulsed EL4 cells as targets. All analyses were performed on vitiligo-affected mice. Bar graphs show 4 replicate wells with errors bars depicting ± SD. Statistically significant differences were determined by <i>t</i> test (OVA vs. gp100) or one-way ANOVA with Bonferroni post-test (transient help vs. no help) with * <i>P</i><0.05, ** <i>P</i><0.01, and <i>NS</i> denoting <i>P</i>>0.05. Data are representative of two experiments with similar results.</p

T_reg depletion by anti-CD25 treatment fails to prime a CD8 T cell response to B16 melanoma.

<p>(A) Mice received anti-CD25 (PC61) treatment, and four days later, the proportion of CD25⁺ and FoxP3⁺ cells among total CD4⁺ cells was determined by flow cytometry. Representative dot plots of 2–6 mice/group are shown; +/− standard deviation. (B) Mice received primary B16 tumors on day 0, and either anti-CD25 or anti-CD4 treatment was given as indicated in the figure. Mice were sacrificed on day 13, and IFN-γ ELISPOT was performed on CD8 T cells (pooled, 4–7 mice/group), with the indicated peptide-pulsed EL4 cells as targets. Data represent average ± SD of four replicate wells. (C) Mice received 10⁴ naïve CD8⁺Thy1.1⁺ pmel cells on day −1, B16 tumors on day 0, and either anti-CD25 or anti-CD4 treatment was given as indicated in the figure. On day 13, the proportion of Thy1.1⁺ pmel cells among total CD8⁺ cells was determined by flow cytometry. Symbols represent individual mice and horizontal lines represent averages. Statistically significant differences were assessed by <i>t</i> test, with * <i>P</i><0.05, ** <i>P</i><0.01, *** P<0.001, and <i>NS</i> denoting <i>P</i>>0.05 as compared with irrelevant peptide (OVA)-pulsed EL4 cells, or as indicated by brackets. Data are representative of two experiments with similar results.</p

CD4 T cell help is not required for long-lived protection against B16 tumor challenge.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026491#pone-0026491-g004" target="_blank">Figure 4A</a>. Sixty days post-surgery, mice were stratified based on the development of vitiligo. Vitiligo-affected hosts and unaffected hosts that received either Transient Help or No Help, or naïve mice (No Treatment), were challenged with B16 melanoma cells, and tumor incidence was followed. Comparisons are indicated by brackets, and statistical significance was determined by Log-rank analysis, with *** <i>P</i><0.0002 and <i>NS</i> denoting <i>P</i>>0.05. Data are combined from 3 experiments, with a total of 8–12 mice per group.</p

CD4 T cell help enhances, but is not required, for the maintenance of gp100-specific CD8 memory T cells.

<p>(A) Schematic diagram of treatment schedule. (B & C) Mice received 10⁴ naïve CD8⁺Thy1.1⁺ (pmel) cells one day prior to treatment as in Panel A, and the proportion of Thy1.1⁺ (pmel) cells among live CD8⁺ cells was determined 60 days (B) or 90 days (C), following surgery. All analyses were performed on vitiligo-affected mice; although unaffected mice were used as a negative control on day 60. Dot plots depicting representative mice are shown. Symbols represent individual mice, and horizontal bars represent averages. Statistically significant differences were assessed by <i>t</i> test as indicated by brackets, <i>NS</i> denotes <i>P</i>>0.05. Data represent 3 combined experiments.</p

CD4 T cell help is not required for robust primary CD8 T cell responses to B16 melanoma.

<p>(A) Schematic diagram of treatment schedules providing either Early Help (light gray) or No Help (dark gray). (B) Mice received primary tumors and anti-CD4 (mAb GK1.5) treatment as indicated in the figure. On day 13, IFN-γ ELISPOT was performed with CD8 T cells isolated from pooled mice (n = 4–9 mice/group) using EL4 cells pulsed with either TRP-2 or irrelevant (OVA) peptides as targets. Data represent average ± SD of four replicate wells. (C) Mice received 10⁴ naïve CD8⁺ Thy1.1⁺ pmel cells one day prior to primary tumor inoculation and anti-CD4 treatment as indicated in the figure. The proportion of Thy1.1⁺ (pmel) cells among total CD8⁺ cells was determined by flow cytometry on day 13. Symbols represent individual mice and horizontal lines represent averages. Statistically significant differences were assessed by <i>t</i> test, with * <i>P</i><0.05, *** P<0.0002 and <i>NS</i> denoting <i>P</i>>0.05. Unless indicated by brackets, asterisks directly above error bars represent significant differences compared with irrelevant (OVA) peptide. Data in (B) are representative of two experiments with similar results; data in (C) are combined from two repeat experiments.</p