Tumor-infiltrating regulatory T cells inhibit endogenous cytotoxic T cell responses to lung adenocarcinoma

Abstract

Immune cells comprise a substantial proportion of the tumor mass in human nonsmall cell lung cancers (NSCLC), but the precise composition and significance of this infiltration are unclear. In this study, we examined immune complexity of human NSCLC as well as NSCLC developing in CC10-TAg transgenic mice, and revealed that CD4(+) T lymphocytes represent the dominant population of CD45(+) immune cells, and, relative to normal lung tissue, CD4(+)Foxp3(+) regulatory T cells (Tregs) were significantly increased as a proportion of total CD4(+) cells. To assess the functional significance of increased Tregs, we evaluated CD8(+) T cell-deficient/CC10-TAg mice and revealed that CD8(+) T cells significantly controlled tumor growth with antitumor activity that was partially repressed by Tregs. However, whereas treatment with anti-CD25-depleting mAb as monotherapy preferentially depleted Tregs and improved CD8(+) T cell-mediated control of tumor progression during early tumor development, similar monotherapy was ineffective at later stages. Because mice bearing early NSCLC treated with anti-CD25 mAb exhibited increased tumor cell death associated with infiltration by CD8(+) T cells expressing elevated levels of granzyme A, granzyme B, perforin, and IFN-γ, we therefore evaluated carboplatin combination therapy resulting in a significantly extended survival beyond that observed with chemotherapy alone, indicating that Treg depletion in combination with cytotoxic therapy may be beneficial as a treatment strategy for advanced NSCLC.

Figure 1. Immune complexity of human NSCLC

(A) Hematoxylin and eosin (H&E) staining of human non-small cell lung cancer (NSCLC) and adjacent normal tissue (top panel) with representative images showing staining for CD45 (bottom panel). (B) Numbers of CD45⁺ leukocytes per square millimeter of tissue sections as assessed immunohistochemistry. n=8 samples per group. (C) Flow cytometric analysis of immune cell infiltrates within human NSCLC represented as percentage of total CD45⁺ leukocytes. n=6 samples per group. (D) CD19⁺CD20⁺HLA-DR⁺ B cell and CD3⁺CD4⁺ T cell infiltrate within human NSCLC as assessed by flow cytometry, shown as a percent of total CD45⁺ cells. (E) Percent of CD4⁺ and CD8⁺ T cells staining positive for CD69 as assessed by flow cytometry, with representative histograms of CD69 expression shown to the right. *p<0.05; **p<0.01, ***p<0.001.

Figure 2. Immune complexity of NSCLC in CC10-TAg mice

(A) H&E staining of lungs from negative littermates (-LM) and CC10-TAg mice showing adenomas and adenocarcinoma (top panel), with representative staining for CD45 (bottom panel). (B) Numbers of CD45⁺ leukocytes per square millimeter of tissue as assessed by immunohistochemistry. n=5 mice per group. (C) Flow cytometric analysis of immune cell infiltrates in CC10-TAg lungs assessed at various stages of neoplastic development, namely hyperplasia/dysplasia (4 weeks), adenomas (8 weeks) and adenocarcinomas (16 weeks), represented as percentages of total CD45⁺ leukocytes. (D) CD4⁺ T cell lung infiltrate as assessed by flow cytometry, shown as a percent of total CD45⁺ cells. n=5∓8 mice per group. Significant differences are shown relative to negative littermates. *p<0.05; **p<0.01, ***p<0.001.

Figure 3. CD8⁺ T cells restrain NSCLC growth in CC10-TAg mice

(A) H&E staining of lungs from CC10-TAg mice deficient for selective lymphocyte subsets. (B) Quantification of tumor burden from mice shown in A. n=4∓5 mice per group. (C) Survival of CC10-TAg mice compared with those deficient in CD8⁺ T lymphocytes. p<0.0001; n=30 mice per group. (D) Tumor burden quantified as percentage of lung area in CC10-TAg mice following CD8⁺ T cell depletion from 8 weeks until 12 weeks of age. n=3∓7 mice per group, with one of three representative experiments shown. *p<0.05; **p<0.01, ***p<0.001.

Figure 4. Functional significance of T regulatory cells in NSCLC

(A) Frequency of Foxp3⁺ T_reg cells within the CD4⁺ T cell compartment in human NSCLC assessed by flow cytometry, with representative FoxP3 immunohistochemistry shown on left. n=6 per group. (B) Frequency of Foxp3⁺ Treg cells within the CD4⁺ T cell compartment in CC10-TAg tumors at various ages as assessed by flow cytometry, with representative FoxP3 immunohistochemistry shown on left. n=5∓8 mice per group. (C) Percent of CD3⁺CD4⁺FoxP3⁺ cells expressing CD103⁺ in CC10-TAg tumors. n=5∓8 mice per group. (D) T_reg depletion was assessed in CC10-TAg mice in a prevention trial by IP injections of αCD25 mAb every 5 days from 4 weeks until 8 weeks of age. (E) Frequency of Foxp3⁺ T_regs represented as percentage of CD3⁺CD4⁺ T cells in spleen (left) and lung tumors (right) following treatment with αCD25 mAb. (F) Tumor burden represented as percentage of lung area following αCD25 treatment in CC10-TAg mice. (G) Number of BrdU⁺ tumor cells per square millimeter of lung tumors. (H) Angiogenic vasculature represented as percent positive pixels of CD31 staining by automated quantification of representative stained sections. (I) Number of cleaved caspase 3⁺ tumor cells per square millimeter of lung tumors. (J) Immune cell complexity of lung tumors following T_reg depletion represented as percentage of CD45⁺ leukocytes assessed by flow cytometry. (K) CD8⁺ T cell infiltrate of lung tumors as assessed by flow cytometry, shown as a percent of total CD45⁺ cells. (L) Absolute numbers of CD8⁺ cells per square millimeter of lung tumor with representative immunohistochemistry shown to the right. (E-L) n=12∓13 mice per group with data obtained over 3 independent cohorts of animals. *p<0.05; **p<0.01, ***p<0.001.

Figure 5. CD8⁺ T cells in NSCLC following T_reg depletion

(A) Frequency of BrdU⁺ proliferating CD8⁺ T cells represented as percentages of total tumor infiltrating CD8⁺ T cells. (B) Frequency of CD69-expressing activated CD8⁺ T cells represented as percentages of total tumor infiltrating CD8+ T cells. (A-B) n=7 per group, one of two representative experiments is shown. (C-F) Relative expression of Ifng (C), Gzma (D) Gzmb (E) and Prf1 (F) mRNA in flow-sorted CD8⁺ T cells represented as fold change over Tbp as assessed by qPCR. n=7 per group, with data obtained over 2 independent cohorts of animals. (G) Tumor burden represented as percentage of lung area, following treatment with αCD25 mAb from 4 weeks until 8 weeks of age in CC10-TAg mice deficient in CD8⁺ T cells. n=10 per group, with data obtained over 3 independent cohorts of animals. *p<0.05; **p<0.01, ***p<0.001.

Figure 6. T regulatory cell depletion in combination with chemotherapy extends survival

Percent survival of CC10-TAg mice treated with control IgG or αCD25 mAb as monotherapy, or in combination with 50 mg/kg carboplatin (CBDCA). Dosing strategy is shown above the survival graph. Mice received control IgG or αCD25 mAb from 8 weeks of age until end-stage determined by 15% weight loss. Carboplatin was administered in 3 doses, 5 days apart, commencing at 13 weeks. Over 15 cohorts of mice were treated to obtain 10∓16 mice per group. p<0.05, control versus CBDCA alone; p<0.05, CBDCA alone versus αCD25/CBDCA.