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. 2004 Sep 20;200(6):771-82.
doi: 10.1084/jem.20041130.

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Mary Jo Turk  1 , José A Guevara-Patiño, Gabrielle A Rizzuto, Manuel E Engelhorn, Shimon Sakaguchi, Alan N Houghton
Affiliations

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Mary Jo Turk et al. J Exp Med. .

Erratum in

  • J Exp Med. 2005 Jan 3;201(1):159

Abstract

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.

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Figures

Figure 1.
Figure 1.
Concomitant immunity to B16 melanoma can be induced by GM-CSF expression in tumor cells and/or by CD4+ T cell depletion. C57BL/6 mice (10–15/group) were inoculated with B16 cells (A and C) or B16-GMCSF cells (B and D) in the right flank. 6 d later, mice were challenged with B16 cells in the left flank, and growth of primary and challenge tumors was monitored. In each panel, the left graph represents growth of primary tumors, the middle graph depicts growth of challenge tumors, and the right graph shows growth of the challenge tumor inoculum given in naive mice. Mice were either left untreated (A and B) or treated (C and D) with GK1.5 CD4 depleting antibody on days 4, 10, and 17 relative to primary tumor inoculation (arrows). Significance for each group was determined by log rank analysis comparing growth of challenge tumors in tumor-bearing mice versus naive mice. (A) P = 0.145; (B) P = 0.014; (C) P = 0.001; (D) P = 0.0002. Results are shown for representative experiments.
Figure 2.
Figure 2.
Concomitant tumor immunity is dependent on CD8+ cells. C57BL/6 mice (5–15/group) were inoculated with B16 cells (A) or B16-GMCSF cells (B) in the right flank, followed 6 d later by B16 cells in the left flank. In addition, all mice received CD4-depleting antibody on days 4 and 10 to induce concomitant immunity. In each panel, the left graph depicts growth of primary tumors and the right graph depicts growth of challenge tumors in the same mice. The two bottom panels show tumor outgrowth in mice coinjected with CD8-depleting antibody (arrows). Significance for each group was determined by log rank analysis comparing growth of challenge tumors in CD8-depleted versus nondepleted mice. (A) P = 0.0892; (B) P = 0.0022.
Figure 3.
Figure 3.
Priming of concomitant immunity is less effective in mice lacking CD4+ T cell help. Mice (15/group) were inoculated with B16 cells in the right flank, followed 6 d later by an identical inoculum in the left flank. Mice received intraperitoneal injections of CD4-depleting antibody on days −2, 4, 10, and 17 (A, arrows) or days 4, 10, and 17 (B). In each panel, the left graph represents growth of primary tumors, the middle graph represents growth of challenge tumors, and the right graph represents growth of the challenge tumor inoculum given in naive mice.
Figure 4.
Figure 4.
Concomitant immunity to B16 is shared with JBRH melanoma, but not with Lewis lung carcinoma or LiHa fibrosarcoma. Mice were inoculated (left flank) on day 0 with either Lewis lung carcinoma (A), LiHa fibrosarcoma (B), or JBRH melanoma (C). Mice were either untreated; depleted of CD4+ T cells on days −2, 4, and 11; or depleted of CD4+ T cells on days −2, 4, and 11 and inoculated with B16 tumors (right flank) on day −6 to induce concomitant immunity. Tumor diameter represents the mean of the greatest diameters (± SEM) for 15 mice/group. For JBRH tumors, the difference between mean sizes in naive versus B16 tumor-bearing mice was statistically significant (P < 0.05; by two-tailed Student's t test) at all time points between days 12 and 20.
Figure 5.
Figure 5.
CD8+ T cells from concomitantly immune mice recognize epitopes from DCT and gp100 melanocyte differentiation antigens. Mice received (A) inoculation of B16 cells in the right flank followed 6 d later by an identical inoculum in the left flank, (B) inoculation of B16 cells in the right flank combined with CD4 depletion on days 4 and 10, or (C) inoculation of B16 cells in the right flank followed 6 d later by an identical inoculum in the left flank and CD4 depletion on days 4 and 10. 12 d after primary tumor inoculation, CD8+ T cells from spleen and inguinal lymph nodes were tested by IFN-γ ELISPOT analysis using as targets either B16 cells or peptide-pulsed EL4 lymphoma cells. Lymph node and spleen cells were pooled from groups of 5–10 mice. Values represent the mean number of spots (n = 3–4 replicates/point) ± SD. Asterisks depict statistically significant differences (P < 0.05; by two-tailed Student's t test) between T cell responses to target EL4 cells pulsed with relevant versus irrelevant (Irr) peptide; or, for target B16 cells, differences between responses from naive versus tumor-bearing mice.
Figure 6.
Figure 6.
CD8+ T cells from concomitantly immune donors adoptively transfer immunity to T cell–deficient hosts. Donor C57BL/6 mice were made concomitantly immune by inoculation of B16 cells in the right flank followed 6 d later by a secondary inoculation in the left flank and CD4 depletion on days 4 and 10. 12 d after the primary tumor inoculation, CD8+ T cells were harvested from spleens and tumor-draining lymph nodes of concomitantly immune mice (CD8 immune), or from spleens of naive C57BL/6 mice (CD8 naive). Cells were purified and adoptively transferred into RAG1 −/− recipients, which were challenged with an inoculum of B16 cells on the following day. Tumor growth was monitored in adoptively transferred recipients for 60 d. The difference between tumor incidence in mice receiving naive versus immune CD8+ T cells was statistically significant (P = 0.013), as determined by log rank analysis.
Figure 7.
Figure 7.
GITR stimulation induces potent concomitant immunity. Mice (10–15/group) were inoculated with B16 cells in the right flank followed 6 d later with an identical inoculum in the left flank. In each panel, the left graph represents growth of primary tumors, the middle graph depicts growth of challenge tumors, and the right graph shows growth of the challenge tumor inoculum given to naive mice. 1 mg anti-GITR antibody (clone DTA-1) was administered intraperitoneally on days 1 and 7 (A) or days 0 and 7 (B) relative to primary tumor inoculation. (C) Mice received injections of 1 mg rat IgG isotype control antibody. Arrows indicate time of antibody administration. The difference between challenge tumor incidence in mice receiving rat control IgG versus DTA-1 was statistically significant for both treatment schedules as determined by log rank analysis. (A) P = 0.0002; (B) P = 0.0050.
Figure 8.
Figure 8.
Single-dose cyclophosphamide induces concomitant immunity when administered 4 d before primary tumor inoculation. (A) Mice (10/group) were treated with cyclophosphamide on day −4, inoculated with B16 cells in the right flank on day 0, and reinoculated with B16 in the left flank on day 6. The left graph represents growth of primary tumors, the middle graph depicts growth of challenge tumors, and the right graph shows growth of the challenge tumor inoculum in naive mice that were treated with cyclophosphamide according to the same schedule. (B) Incidence of B16 secondary tumors (left flank) was measured in mice bearing day-6 primary tumors and receiving the following: no treatment or cyclophosphamide treatment on days −4, −2, or 0 relative to primary tumor inoculation. The difference between challenge tumor incidence in untreated mice and those that received cyclophosphamide on day −4 was statistically significant (P = 0.002) as determined by log rank analysis.
Figure 9.
Figure 9.
CD4+CD25+ T cells suppress priming of concomitant immunity. RAG1 −/− mice were adoptively transferred with various populations of naive T cells from C57BL/6J donor mice as follows: (A) no cells, (B) CD8+ and CD4+ T cells, (C) CD8+ and CD4+CD25− T cells, (D) CD8+ and CD4+CD25+ T cells, or (E) CD8+ T cells alone. Adoptively transferred mice were inoculated with primary B16 tumors on day 1, followed by challenge tumors on day 7. In each panel, the left graph depicts growth of primary tumors and the right graph depicts growth of challenge tumors in the same mice. In D, the experiment was terminated 8 d after tumor challenge because growth of primary tumors was too rapid. Five out of nine tumors had already appeared by day 9.
Figure 10.
Figure 10.
CD4+CD25+ T cells from naive mice suppress preactivated effectors of concomitant immunity. CD8 immune T cells were generated as described in Fig. 6. (A) Incidence of B16 tumors in RAG1 −/− mice that had been previously (day −1) reconstituted with CD8 immune T cells or CD8 immune T cells mixed with various potential suppressor populations as follows: naive CD4+CD25+ T cells, CD4+CD25+ T cells from day-12 tumor-bearing mice, or CD4+CD25− T cells from day-12 tumor-bearing mice. As determined by log rank analysis, the difference between tumor incidence in mice receiving immune CD8 cells alone and those receiving immune CD8 cells combined with naive CD4+CD25+ T cells was statistically significant (P = 0.0026). However, there was no statistical difference for mice cotransferred with tumor-bearing CD4+ CD25− T cells (P = 0.992) or tumor-bearing CD4+CD25+ T cells (P = 0.677). Naive versus tumor-bearing CD4+CD25+ T cells did not reach statistical significance (P = 0.0729). (B) Tumor growth curves depicting growth rates of individual tumors in each group of adoptively transferred mice.
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