Karin Schreiber

Bystander killing of cancer requires the cooperation of CD4+ and CD8+ T cells during the effector phase

Killing of nonmalignant stroma requires cooperation between CD4+ and CD8+ T cells during the effector phase in the tumor microenvironment.

Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Strong synergy between mutant ras and HPV16 E6/E7 in the development of primary tumors

E6/E7 oncogenes of high-risk human papilloma virus (HPV) subtypes are essential for the development of certain types of cancers. However, these oncogenes are insufficient to transform normal cells into an immortalized or malignant state. Mutant Ha-ras cooperates with E6/E7 of HPV subtype 16 in transformation of cells in vitro and may contribute to some HPV-associated cancers in humans. This study investigates whether HPV16 E6/E7 and v-Ha-ras synergize in vivo. FVB/n mice transgenic for v-Ha-ras gene (R+) were crossed with transgenic C57BL/6 mice that harbor E6/E7 of HPV16 (E+). Beginning at about 3 months of age, the bitransgenic E+R+(C57BL/6 × FVB/n) F1 mice developed mouth, eye and ear tumors. By 6 months, the prevalence of these types of mouth, eye and ear tumors was 100, 71 and 79% respectively in the E+R+ mice. Most tumors grew progressively until the mice had to be killed. The median times for the appearance of the first mouth, eye and ear tumor were 3.6, 4.3 and 4.2 months, respectively. For the two singly transgenic groups of mice, the prevalence of mouth, eye and ear tumors was 0, 0 and 6% (E−R+) and 0, 0 and 0% (E+R−), respectively, and the median time to first tumor was greater than 12 months for singly transgenic mice (E−R+, E+R−). Thus, a remarkable synergy occurred between the v-Ha-ras and HPV16 E6/E7 oncogenes in the development of primary tumors in mice.

Long-Term Suppression of Tumor Growth by TNF Requires a Stat1- and IFN Regulatory Factor 1-Dependent IFN-γ Pathway but Not IL-12 or IL-18

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-γ pathway consisting minimally of IFN-γ, IFN-γ receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-γ-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-γ nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-γ was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-γ pathway that without measurable IFN-γ production or activity affects the ability of TNF to suppress tumor development.

Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

Purpose: Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. Experimental Design: By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. Results: When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Conclusions: Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734–43. ©2015 AACR. See related commentary by Liu, p. 2602

Spleen cells from young but not old immunized mice eradicate large established cancers.

Purpose: Solid tumors that have grown two weeks or longer in mice and have diameters larger than 1 cm are histologically indistinguishable from autochthonous human cancers. When experimental tumors reach this clinically relevant size, they are usually refractory to most immunotherapies but may be destroyed by adoptive T-cell transfer. However, TCR-transgenic T cells and/or tumor cells overexpressing antigens are frequently used in these experiments. Here we studied the requirements for destroying clinical size, unmanipulated 8101 tumors by adoptive cell therapy. Experimental Design: 8101 arose in an old mouse after chronic exposure to UV light. A cancer line was established, which was never serially transplanted. The immunodominant CD8+ T cell-recognized antigen of this tumor is caused by a somatic tumor–specific mutation in the RNA helicase p68. 8101 tumors were treated with spleen cells from young naive, or young and old immunized mice to ascertain the characteristics of immune cells that lead to rejection. Results: Here we show that the mutant p68 peptide has an exceptionally high affinity to the presenting MHC class I molecule Kb and that spleen cells from immunized young syngeneic mice adoptively transferred to Rag−/− or cancer-suppressed euthymic mice eradicate 8101 tumors larger than 1 cm in average diameter and established for several weeks. Spleen cells from naive young mice or from old and boosted (reimmunized) mice were ineffective. Conclusions: Relapse-free destruction of large and long-established tumors expressing a genuine very high-affinity tumor-specific antigen can be achieved by using adoptive transfer of lymphocytes from immunized young individuals. Clin Cancer Res; 18(9); 2526–33. ©2012 AACR.

Adoptively Transferred Immune T Cells Eradicate Established Tumors despite Cancer-Induced Immune Suppression

Myeloid-derived CD11b+Gr1+ suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that adoptively transferred immune T cells eradicate well-established tumors in the presence of MDSCs and TAMs, which are strongly immunosuppressive ex vivo. These MDSCs and TAMs were comparable in numbers and immunosuppressive capacity among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer, tumor vasculature and cancer cells disappeared simultaneously. During T cell–mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAMs) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor burden, supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses.

Radiation can inhibit tumor growth indirectly while depleting circulating leukocytes

The growth of certain UV-radiation-induced tumors is suppressed by T lymphocytes, but it has been proposed that non-T lymphocytes stimulate the growth of several of these tumors. In this study, the indirect effects of X irradiation on the growth of one such tumor in T-cell-deficient nude mice was tested. Even when the site of tumor cell injection was shielded, whole-body irradiation with 6 Gy before tumor challenge inhibited subsequent tumor growth significantly. The interval during which this indirect inhibition was observed correlated with the depletion of circulating leukocytes, which did not return to normal levels until 12-21 days after irradiation. These data are consistent with earlier results using an antibody to deplete Gr-1+ leukocytes and indicate that radiation can inhibit the growth of certain tumors indirectly without direct effects on the tumor cells or the tumor bed.

Transfer of Allogeneic CD4+ T Cells Rescues CD8+ T Cells in Anti-PD-L1–Resistant Tumors Leading to Tumor Eradication

Tumor-specific CD8+ T cells temporarily control tumor growth, but eventually tumors can escape. Adoptive transfer of tumor-recognizing CD4+ T cells rescued the function of exhausted CD8+ T cells through co-cross-presentation by stroma, which led to complete tumor eradication. Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127–36. ©2017 AACR.