Figure 1.
Distinct phenotypes of CD4 T cells activated under Th1 and Th2 polarising conditions.
(A) Intracellular staining of polarised T cells for IFN-γ and IL-4. Bars indicate percentage cytokine expressing CD4 gated T cells after flow cytometric analysis, error bars represent SD. *P<0.05, **P<0.01, ***P<0.001, t-test, n = 8. (B) Illustrative intracellular staining for IFN-γ and IL-4 (quadrants set according to isotype control) from Th1 and Th2 polarised T cells. (C) Surface staining and flow cytometry analysis of cell surface receptors implicated in lymphocyte homing; pooled data for expression of chemokine receptors CXCR3, CCR4, and CD49d. Bars indicate percentage homing receptor expressing CD4 gated T cells after flow cytometric analysis, error bars represent SD. *P<0.05, **P<0.01, t-test, n = 8. (D) Representative staining of chemokine receptors CCR4 and CXCR3 on Th2 and Th1 polarised T cells.
Figure 2.
Adoptively transferred Th1 cells show preferential homing and accumulation in an intracranial tumour compared with Th2 cells.
SMARTA T cells (CD45.1) were labelled with CFSE (Th1) or Violet dye (Th2) and were intravenously transferred (3×106 Th1; 3×106 Th2) into C57BL/6 mice (CD45.2) that had been intracranially implanted with 4×105 MC57-GP tumour cells 4 days previously. After 19 hours (A), or 96 hours (B), BILs were isolated, stained with antibodies for CD4 and CD45.1 and were analysed ex vivo by multicolour flow cytometry. Adoptively transferred T cells were identified as CD45.1+CD4+ cells that were either CFSE+ or Violet dye+ (supporting information Figure S2). Results are expressed as the percentage of Th1 and Th2 cells among the adoptively transferred CD45.1+CD4+ cells in the BILs, each symbol represents an individual mouse (n = 8). ***P<0.001, t-test.
Figure 3.
Enhanced recruitment of cytokine expressing T cells to the brain tumour site by co-transfer of tumour-antigen specific CD4 Th1 T cells.
Th1 or Th2 polarised T cells from SMARTA and OTII mice were labelled with CFSE. Naïve (non-cultured) P14 CD8 T cells were labelled with Violet dye. The different T cell populations were intravenously transferred into C57BL/6 mice (CD45.2) that had been previously intracranially implanted with 4×105 MC57-GP tumour cells. P14 CD8 T cells were transferred 1 day after tumour implantation; SMARTA and OTII CD4 T cells were transferred the next day, in the combinations indicated. The numbers of cells transferred were 3×106 Th1, 3×106 Th2, and 6×106 P14. At day 6 after tumour implantation, BILs were isolated and analysed ex vivo by multicolour flow cytometry. Adoptively transferred T cells were identified as CD45.1+CD4+CFSE+ cells or CD45.1+CD8+ Violet dye+ cells. Each group of mice comprised 3–5 animals, and results from several experiments were pooled to acquire data from 12 mice. (A) Absolute numbers of each population of adoptively transferred T cells per brain. Results are displayed as means+SD. *P<0.05; **P<0.01: Mann-Whitney Rank sum test. (B) Left panel: intracellular staining of BILs gated on CD45.1+CD4+CFSE+ cells. Expression of IFN-γ, IL-4 and TNF-α is shown after ex vivo restimulation with cognate peptides. Results are displayed as means+SD. *P<0.05: t-test. Right panel: representative dot plots of ex vivo restimulated SMARTA CD4 T cells. (C) Total numbers of cytokine expressing adoptively transferred T cells per brain (including both CD4 and CD8 T cells). Results are displayed as means of 3 pools of mice, from 12 mice in total.
Figure 4.
Enhanced survival of brain-tumour bearing mice co-transferred with tumour-antigen specific CD4 and CD8 T cells.
In vitro activated OT-I CD8 T cells and polarised OT-II CD4 T cells were intravenously transferred into C57BL/6 mice that had been intracranially implanted with 5×105 EG-7 tumour cells 6 days previously. Groups were CD8 T cells alone: 12×106 OTI T cells; CD8/CD4 Th2∶7×106 OTI T cells +5×106 OTII; CD8/CD4 Th1∶7×106 OTI T cells +5×106 OTII; CD4 Th1 alone: 12×106; untreated: EG-7 tumour cells alone. Mice were monitored until appearance of terminal symptoms (see Methods), at which point they were euthanised. Survival curves represent accumulated data from 2 experiments with 8–12 mice for all groups except CD4 Th1 alone (6 mice). There was a statistically significant difference between CD4 (Th1 or Th2) co-transferred groups and CD8 T cells alone (P<0.05, t-test) at up to 32 days post-implantation of tumour. At the termination of the experiment at 56 days post-implantation, there was a statistically significant difference for mice adoptively transferred with T cells and tumour alone (CD4 Th1 or Th2: P<0.001; CD8 P<0.05, t-test).