Syngeneic natural killer cell therapy activates dendritic and T cells in metastatic lungs and effectively treat low-burden metastases

Reviewer #1 (Public Review):

Summary:

This is a very nice paper in which the authors addressed the potential for NK cell cellular therapy to treat and potentially eliminate previously established metastases after surgical resections, which are a major cause of death in human cancer patients. To do so they developed a model using the EO771 breast cancer cell line, in which they establish and then resect tumors and the draining lymph node, after which the majority of mice eventually succumb to metastatic disease. They found that when the initiating tumors were resected when still relatively small, adoptive transfers of IL-15/12-conditioned NK cells substantially enhanced the survival of tumor-bearing animals. They then delved into the cellular mechanisms involved. Interestingly and somewhat unexpectedly, the therapeutic effect of the transferred NK cells was dependent on the host's CD8+ T cells. Accordingly, the NK cell therapy contributed to the formation of tumor-specific CD8+ T cells, which protected the recipient animals against tumor re-challenge and were effective in protecting mice from tumor formation when transferred to naive mice. Mechanistically, they used Ifng knockout NK cells to provide evidence that IFNgamma produced by the transferred NK cells was crucial for the accumulation and activation of DCs in the metastatic lung, including expression of CD86, CD40, and MHC genes. In turn, IFNgamma production by NK cells was essential for the induced accumulation of activated CD8 effector T cells and stem cell-like CD8 T cells in the metastatic lung. The authors then expanded their findings from the mouse model to a small clinical trial. They found that inoculations of IL-15/12-conditioned autologous NK cells in patients with various malignancies after resection were safe and showed signs of efficacy.

Strengths:

- Monitoring of long-term metastatic disease and survival after resection used in this paper is a physiological model that closely resembles clinical scenarios more than the animal models usually used, a great strength of the approach.

- Previous literature focused on the notion that NK cells clear metastatic lesions directly, within a short period. The authors' use of a more relevant model and time frame revealed the previously unexplored T cell-dependent mechanism of action of infused NK cells for long-term control of metastatic diseases.

- Also important, the paper provides solid evidence for the contribution of IFNgamma produced by NK cells for activation of dendritic cells and T cells. This is an interesting finding that provokes additional questions concerning the action of the interferon-gamma in this context.

- The results from the clinical trial in cancer patients based on the same type of IL-15/12-conditioned NK cell infusions, were encouraging with respect to safety and showed signals of efficacy, which support the translatability of the author's findings.

Weaknesses:

- Having demonstrated that NK cell IFNgamma is important for recruiting and activating DCs and T cells in their model, one is left to wonder whether it is important for the therapeutic effect, which was not tested.

- Relatedly, previous studies, cited by the authors, reported that NK cells promote T cell activation by producing the chemokines CCL5 and XCL1, and FLT3 ligand, which respectively recruit and activate dendritic cells that can subsequently mobilize a T cell response. The present study demonstrates an important role for NK cell-produced IFNgamma in these processes. One is left wondering whether the model used by the authors is also dependent on CCL5, XCL1, and FLT3 production by NK cells, and if so whether IFNgamma plays a role in that or acts in parallel. The issue could be discussed by the authors, even if they cannot easily resolve it.

- The authors do not address whether the IL-12 in their cocktail is essential for the effects they see. Relatedly, it was of interest that despite the effectiveness of the transferred IL-15/IL-12 cultured NK cells, the cells failed to persist very long after transfer. Published studies have reported that so-called memory-like NK cells, which are pre-activated with a cocktail of IL-12, IL-18 and IL-15, persist much longer in lympho-depleted mice and patients than IL-2 cultured NK cells. It would be illuminating to compare these two types of NK cell products in the author's model system, and with, or without, lymphodepletion, to identify the critical parameters. If greater persistence occurred with the memory-like NK cell product, it is possible that the NK cells might provide greater benefit, including by directly targeting the tumor.

- It was somewhat difficult to gauge the clinical trial results because the trial was early stage and therefore not controlled. Evaluation of the results therefore relies on historical comparisons. To evaluate how encouraging the results are, it would be valuable for the authors to provide some context on the prognoses and likely disease progression of these patients at the time of treatment.

Reviewer #2 (Public Review):

Summary:

The authors show convincing data that increasing NK cell function/frequency can reduce the development and progression of metastatic disease after primary tumor resection.

Strengths:

The inclusion of a first-in-human trial highlighting some partial responses of metastatic patients treated with in vitro expanded NK cells is tantalising. It is difficult to perform trials in preventing further metastasis since the timelines are very protracted. However, more data like these that highlight the role of NK cells in improving local cDC1/T cells anti-tumor immunity will encourage deeper thinking around therapeutic approaches to target endogenous NK cells to achieve the same.

Weaknesses:

As always, more patient data would help increase confidence in the human relevance of the approach.