Michele W. L. Teng

CD4+CD25+ T Regulatory Cells Suppress NK Cell-Mediated Immunotherapy of Cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-β-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.

Immune-mediated dormancy: an equilibrium with cancer

This brief review discusses the role of the immune system in tumor development, covering a history of cancer immunity and a summary of the concept of cancer immunoediting, including its three phases: elimination, equilibrium, and escape. The latter half of this review then focuses specifically on the equilibrium phase, making note of previous work, suggesting that immunity might maintain cancer in a dormant state, and concluding with a description of a tractable mouse model unequivocally demonstrating that immunity can indeed hold preformed cancer in check. These findings form a framework for future studies aimed at validating immune‐mediated cancer dormancy in humans with the hopes of devising new, immunotherapeutic strategies to treat established cancer.

Cancer immunoediting by the innate immune system in the absence of adaptive immunity

In the absence of adaptive immunity, NK cells polarize M1 macrophages to facilitate cancer immunoediting.

Supernatural T cells: genetic modification of T cells for cancer therapy

Immunotherapy is receiving much attention as a means of treating cancer, but complete, durable responses remain rare for most malignancies. The natural immune system seems to have limitations and deficiencies that might affect its ability to control malignant disease. An alternative to relying on endogenous components in the immune repertoire is to generate lymphocytes with abilities that are greater than those of natural T cells, through genetic modification to produce supernatural T cells. This Review describes how such T cells can circumvent many of the barriers that are inherent in the tumour microenvironment while optimizing T-cell specificity, activation, homing and antitumour function.

TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer

T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer.

Combination Therapy of Established Tumors by Antibodies Targeting Immune Activating and Suppressing Molecules

The blockade of immune suppression against antitumor responses is a particularly attractive strategy when combined with agents that promote tumor-specific CTLs. In this study, we have attempted to further improve the CTL induction and potent antitumor efficacy of a combination mAb-based therapy (termed “trimAb therapy”) that comprises tumor cell death-inducing anti-death receptor 5 mAb and immune activating anti-CD40 and anti-CD137 mAbs. Among trimAb-treated tumors, the infiltration of CD4+ Foxp3+ cells was greater in progressing tumors compared with stable tumors. Blockade of CTLA-4 (CD152)-mediated signals by an antagonistic mAb substantially increased the tumor rejection rate of trimAb therapy, although the immune responses of draining lymph node cells were not augmented. Interestingly, by comparison, additional treatment with agonistic anti-glucocorticoid-induced TNF receptor mAb, antagonistic anti-programmed death-1 (CD279) mAb, or agonistic anti-OX40 (CD134) mAb significantly augmented immune responses of draining lymph node cells, but did not augment the therapeutic effect of trimAb. CD4 T cell depletion reduced the antitumor effect of anti–CTLA-4 mAb treatment alone, but did not reduce the tumor rejection rate of trimAb in conjunction with anti–CTLA-4 mAb. Thus, the blockade of the CTLA-4–mediated inhibitory signal in tumor infiltrating CTL may be the most effective strategy to augment the effect of immune therapies that generate tumor-specific CTL.

Multiple Antitumor Mechanisms Downstream of Prophylactic Regulatory T-Cell Depletion

Several reports have shown that prophylactic depletion of regulatory T cells (Treg) using various monoclonal antibodies (mAb) in mice can stimulate potent antitumor immune responses and prevent tumor development. These same depletion methods do not significantly suppress tumor growth in a therapeutic setting. Although different strategies to deplete FoxP3(+) Treg have been used, no study has systematically compared these qualitatively for the effector mechanisms they each liberate. Herein, using prophylactic depletion of FoxP3(+) Tregs with either anti-CD4, anti-CD25, or anti-FR4 mAbs, we have compared the cellular and effector requirements for elimination of the renal carcinoma RENCA and prevention of methylcholanthrene-induced fibrosarcoma. Collectively from these two models, it was clear that CD8(+) T cells and natural killer cells played an important role downstream of Treg depletion. However, whereas all three mAbs quantitatively depleted FoxP3(+) T cells to a similar extent, subtle differences in the downstream mechanisms of tumor control existed for all three approaches. In general, neutralization of any lymphocyte subset or effector mechanism was insufficient to alter tumor suppression initiated by Treg depletion, and in some settings, the neutralization of multiple effector mechanisms failed to prevent tumor rejection. These studies reveal that Tregs control multiple redundant elements of the immune effector response capable of inhibiting tumor initiation and underscore the importance of effectively targeting these cells in any cancer immunotherapy.

Combined Natural Killer T-Cell–Based Immunotherapy Eradicates Established Tumors in Mice

A rational monoclonal antibody (mAb)-based antitumor therapy approach has previously been shown to eradicate various established experimental and carcinogen-induced tumors in a majority of mice. This therapy comprised an agonistic mAb reactive with tumor necrosis factor-related apoptosis-inducing ligand receptor (DR5), expressed by tumor cells, an agonistic anti-CD40 mAb to mature dendritic cells, and an agonistic anti-4-1BB mAb to costimulate CD8(+) T cells. Because agonists of CD40 have been toxic in patients, we were interested in substituting anti-CD40 mAb with other dendritic cell-maturing agents, such as glycolipid ligands recognized by invariant natural killer T (iNKT) cells. Here, we show that CD1d-restricted glycolipid ligands for iNKT cells effectively substitute for anti-CD40 mAb and reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs (termed NKTMab therapy). NKTMab therapy-induced tumor rejection was dependent on CD4(+) and CD8(+) T cells, NKT cells, and the cytokine IFN-gamma. NKTMab therapy containing either alpha-galactosylceramide (alpha-GC) or alpha-C-galactosylceramide (alpha-c-GC) at high concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of alpha-GC (>250 ng/injection), limiting the use of this glycolipid. By contrast, even very low doses of alpha-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, alpha-c-GC showed a considerably greater therapeutic index. In summary, sequential tumor cell apoptosis and amplification of dendritic cell function by NKT cell agonists represents an exciting and novel approach for cancer treatment.

Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.

The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.

Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-γ in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later. Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.