CD8+ T cell-mediated control of distant tumours following local photodynamic therapy is independent of CD4+ T cells and dependent on natural killer cells

Abstract

Cancer survival rates decrease in the presence of disseminated disease. However, there are few therapies that are effective at eliminating the primary tumour while providing control of distant stage disease. Photodynamic therapy (PDT) is an FDA-approved modality that rapidly eliminates local tumours, resulting in cure of early disease and palliation of advanced disease. Numerous pre-clinical studies have shown that local PDT treatment of tumours enhances anti-tumour immunity. We hypothesised that enhancement of a systemic anti-tumour immune response might control the growth of tumours present outside the treatment field. To test this hypothesis we delivered PDT to subcutaneous (s.c.) tumours of mice bearing both s.c. and lung tumours and monitored the growth of the untreated lung tumours. Our results demonstrate that PDT of murine tumours provided durable inhibition of the growth of untreated lung tumours. The inhibition of the growth of tumours outside the treatment field was tumour-specific and dependent on the presence of CD8(+) T cells. This inhibition was accompanied by an increase in splenic anti-tumour cytolytic activity and by an increase in CD8(+) T cell infiltration into untreated tumours. Local PDT treatment led to enhanced anti-tumour immune memory that was evident 40 days after tumour treatment and was independent of CD4(+) T cells. CD8(+) T cell control of the growth of lung tumours present outside the treatment field following PDT was dependent upon the presence of natural killer (NK) cells. These results suggest that local PDT treatment of tumours lead to induction of an anti-tumour immune response capable of controlling the growth of tumours outside the treatment field and indicate that this modality has potential in the treatment of distant stage disease.

Figure 1

Control of distant disease by PDT is immune cell mediated. (A) S.c. tumours present on BALB/cJ mice bearing s.c. and lung tumours were either treated with light only (Control, n=5), surgically removed 18–24 h post-Photofrin administration (SR, n=16), or received PDT (n=16). The PDT/skin column (n=6) represents results from mice bearing both s.c. and lung tumours that were treated with Photofrin and had a 1 cm³ area of skin on the non-tumour bearing shoulder illuminated with 630 nm light. The average number of lung tumours was determined as described in Materials and Methods; error bars represent s.e.m. Significance was calculated by Student's t-test. (B) Spleen cells isolated from naïve animals (•) or from mice whose s.c. tumours were surgically removed (▪) were mixed with ⁵¹Cr-labeled EMT6 target cells at various effector-to-target cell ratios. Spleen cells from mice whose tumours were PDT treated were mixed with either ⁵¹Cr-labeled EMT6 (□) or Colon 26 (▴) target cells. Effector and target cells were incubated for 4 h and the percent specific lysis was determined as described in Materials and Methods. Spleen cells from a minimum of six mice were used for each point; error bars represent s.e.m.

Figure 2

Control of distant tumours by PDT is dependent upon the presence of adaptive immune cells. S.c. tumours present on BALB/cJ or SCID mice bearing s.c. and lung tumours were treated with PDT (BALB/cJ, n=16; SCID, n=18). The average number of lung tumours was determined as described in Materials and Methods; error bars represent s.e.m. Significance was calculated by Student's t-test.

Figure 3

Control of distant disease by PDT is mediated by CD8⁺ T cells. (A) BALB/cJ mice were treated with isotype control, anti-CD4 or anti-CD8 monoclonal antibodies. Antibody treated animals were bled and the extent of the depletion was confirmed by flow cytometry. Representative dot plots are shown of samples collected on day 3 and day 10. (B) BALB/cJ mice were treated with isotype control (n=11), anti-CD4 (n=11), anti-CD8 (n=10), or a combination of anti-CD4 and anti-CD8 antibodies (n=11) before inoculation with EMT6 tumour cells. S.c. EMT6 tumours were treated with Photofrin-PDT (135 J cm⁻² given at 75 mW cm⁻²) and 10 days post-PDT the average number of lung tumours was determined. (C) Splenocytes were harvested from mice depleted of immune cells as in (B) 3 days after PDT treatment of s.c. tumours and adoptively transferred into SCID mice (10⁷ cells mouse⁻¹). Recipient animals were challenged with 10⁴ EMT6 tumour cells 2 days post-transfer; the presence of lung tumours was assessed 10 days after tumour challenge. (D) S.c. EMT6 tumours of BALB/cJ (filled bars) or CD40^-/- (open bars) mice bearing s.c. and lung tumours were treated with Photofrin-PDT. Ten days post-PDT the average number of lung tumours was determined as described in Materials and Methods. Each group contains a minimum of five mice. Error bars represent s.e.m. in all figures; significance was calculated by Student's t-test.

Figure 4

Increased CD8⁺ T-cell infiltrate into distant EMT6 tumours following PDT. The left tumour of mice bearing a tumour on each shoulder was treated with PDT or removed surgically (SR); at various time post-treatment the right untreated tumours were removed and examined by flow cytometry for the presence of CD45⁺, CD3⁺, CD4⁺ cells (circles) or CD45⁺, CD3⁺, CD8⁺ cells (squares) as described previously (Gollnick et al, 1997). Results are presented as the number of cells per mg of tumour tissue. Open symbols represent the infiltrate present in untreated tumours when the left tumour was removed surgically; closed symbols represent the infiltrate present in right tumours following treatment of the left tumour with PDT. Tumours from a minimum of three animals were examined from each group. Error bars represent s.e.m.; significance was calculated by Student's t-test.

Figure 5

Enhancement of memory CD8⁺ T cells by PDT is independent of CD4⁺ T cells. Spleen cells were isolated from naïve BALB/cJ mice and CD8⁺ or CD 4⁺ T cells were enriched by negative selection as described in Materials and Methods. (A) An aliquot of the enriched population was subjected to flow cytometry to assess purity. A representative dot plot from the starting population (Control), the enriched CD8⁺ T-cell population and a recipient mouse after tumour challenge (day 40) is shown. (B) The enriched CD8⁺ T cell or combined CD8⁺ and CD4⁺ T cell populations was adoptively transferred into SCID mice (10⁷ mouse⁻¹). Recipient mice were inoculated with EMT6 cells and the resulting tumours were treated with Photofrin-PDT. Mice cured of tumours were challenged >40 days post-treatment by i.v. injection of EMT6 cells; 10 days following challenge lungs were examined for the presence of tumours The average number of lung tumours was determined as described in Materials and Methods; error bars represent s.e.m. Significance was calculated by Student's t-test.

Figure 6

NK cells contribute to control of distant tumours by local PDT. (A) SCID mice were treated with TMβ1 to deplete NK cells as described in Materials and Methods. Five days after TMβ1 administration, NK-depleted and isotype-treated mice were injected intravenously with PBS or CD8⁺ T cells purified from naïve BALB/cJ mice, followed 2 days later by inoculation of EMT6 tumour cells as described in Materials and Methods. S.c. EMT6 tumours were treated with PDT (135 J cm⁻² given at 75 mW cm⁻²) and the average number of lung tumours per mouse was determined as described; error bars represent s.e.m. Open bars represent animals treated with TMβ1; filled bars represent animals treated with isotype control antibodies. Each group contained a minimum of five mice per group. Significance was calculated by Student's t-test. ^*P<0.0007, when the number of lung tumours per mouse treated with TMβ1 was compared to the number of lung tumours present per mouse treated with isotype control antibody. ^#P<0.0001, when the number of lung tumours per mouse with CD8⁺ T cells and treated with isotype control antibodies was compared to the number of lung tumours per mouse treated with isotype control antibodies. (B) Spleen cells were harvested from CD8⁺ recipient mice treated with either TMβ1 or isotype control antibodies at the time of lung tumour assessment. Cells were stained with antibodies specific for CD3, CD8 and CD49; flow cytometry analysis was performed and cells were gated based on CD3 expression. Representative single colour histograms are shown for cells that were CD3⁺ (left panel) or CD3^- (right panel) and expressed either CD8 or CD49 respectively. Filled histograms indicate cells isolated from non-depleted mice stained with isotype control antibodies for anti-CD3 or anti-CD49; open histograms with dashed lines indicate cells isolated from NK depleted mice; open histograms with solid lines indicate cells isolated from non-depleted, isotype treated mice. (C) SCID mice were treated with anti-asialo-GM1 to deplete NK cells as described. Depleted and control mice were inoculated with EMT6 tumours as described in Materials and Methods. S.c. EMT6 tumours were treated with PDT (135 J cm⁻² given at 75 mW cm⁻²) and the average number of lung tumours per mouse was determined as described; error bars represent s.e.m. Open bars represent animals treated with anti-asialo-GM1; filled bars represent animals treated with isotype control antibodies. Each group contained a minimum of five mice per group. Significance was calculated by Student's t-test. ^*P<0.032, when the number of lung tumours per mouse treated with anti-asialo-GM1 was compared to the number of lung tumours present per mouse treated with isotype control antibody. ^#P<0.0001, when the number of lung tumours per mouse with CD8⁺ T cells and treated with isotype control antibodies was compared to the number of lung tumours per mouse treated with isotype control antibodies.