Tissue-resident NK cells support survival in pancreatic cancer through promotion of cDC1-CD8T activity

Reviewer #1 (Public Review):

Summary:

The authors demonstrate that the immunosuppressive environment in pancreatic ductal adenocarcinoma (PDAC) can be mitigated by a combination of ionizing radiation (IR), CCR5 inhibition, and PD1 blockade. This combination therapy increases tissue-resident natural killer (trNK) cells that facilitate CD8 T cell activity, resulting in a reduction of E-cadherin positive tumor cells. They identify a specific "hypofunctional" NK cell population in both mouse and human PDAC that supports CD8 T cell involvement. A trNK signature is found to be associated with better survival outcomes in PDAC and other solid tumors.

Strengths:

Overall, I think this is an interesting study that combines testing of therapeutic concepts in mice with bioinformatics analysis of single-cell transcriptome data in primary tumors and exploration of clinical outcomes using signature genes in TCGA data. The key finding is that immunoregulatory properties of tumor-infiltrating/resident CD56-bright NK cells (assumed to be non-cytotoxic) are beneficial for outcome through cross-talk with DC and recruitment of CD8 T cells. The latter is specifically induced by irradiation combined with CCR5i and PD1 blockade.

"These results collectively support the notion that IR/CCR5i/αPD1 combination treatment alters immune infiltration by reducing Tregs and increasing NK and CD8 T cells, thereby resulting in greater local tumor control." I agree with this conclusion.

Weaknesses:

There are a few points to discuss and that the authors may want to address.

1. "Notably, CCR5i significantly reduced Treg infiltration but had no effect on the infiltration of other immune cells, indicating the active recruitment of CCR5+ Tregs in PDAC (Figure 2B)."
   CCR5i treatment seems to inhibit infiltration of CD8 T cells and NK cells to a greater extent, in relative terms, compared to Treg, albeit it is not statistically significant. If this visual inspection of the graph does not reflect reality, additional experiments may be needed to verify the selective targeting of Tregs or confirm the fact that also CD8 T cells and NK cells are affected by single agent CCR5i. The reduced recruitment of Treg, NK cells, and CD8T cells was completely reversed when combined with irradiation. In the data shown in Figure 3E it seems as if CCR5i induced infiltration of Tregs along with other immune cells. However, this said, I agree with the conclusion of the authors that this combined treatment leads to an altered immune composition and ratio between Tregs and effector cells (CD8T cells and NK cells). Could this altered composition be displayed more clearly?

2. The definition of active and hypofunctional NK cells based on solely NKG2D expression alone seems like an oversimplification. I realize it is not trivial to test tumor-infiltrating NK cells from these tumors functionally but perhaps scRNAseq of the tumors would allow for characterization of cytotoxicity scores using KEGG or GO analysis or reversed gene set enrichment in responders/non-responders. It seems as if the abstract refers to this phenotype incorrectly since the "hyporesponsive" subset is described as NKG2C-negative.

3. "The NK_C1 cluster correlates best with the hypofunction NK phenotype observed in mice as similarly displayed reduced activation (reduced NKG7, NKp80, GZMA, and PRF1) with additional expression of tissue residency markers CD103, CD49a and, surprisingly, the adaptive activating receptor NKG2C (KLRC2) (Figure 5B, C)."

There is no doubt that NK_C1 represents tumor-infiltrating NK cells with a CD56bright gene signature with a strong tissue resident score. However, the transcriptional expression of KLRC2 on these is not surprising! It is well established that KLRC2 transcripts (but not protein) are highly expressed on conventional CD56bright NK cells. There are several published sources where the authors can find such data for confirmation. Thus, this is not to be confused with adaptive NK cells having an entirely different transcriptional signature and expressing high levels of NKG2C at the cell surface. I strongly recommend re-interpreting the results based on the fact that KLRC2 is expressed at high levels in conventional CD56bright NK cells. If not, it would be important to verify that these tissue-resident NK cells express NKG2C and not NKG2A at the cell surface.

1. NCAM1 transcript alone is not sufficient to deconvolute CD56bright NK cells in TCGA data (Figure 7A). As a single marker, it likely reflects NK cell infiltration without providing further evidence on the contribution of the bright/dim components. Therefore, the use of the bright Tr NK signature described in Table 1 is very important (Figure 7B). Table 1 is not provided. Nor Supplementary Table 1. There is only one supplementary figure in the ppt attached.

Reviewer #2 (Public Review):

Summary:

This work elaborates on a combined therapeutic approach comprising ionizing radiation and CCR5i/αPD1 immunotherapy as a promising strategy in pancreatic cancer. Previous research has established that NK cell-derived CCL5 and XCL1 play a crucial role in recruiting cDC1 cells to the tumor microenvironment, contributing to tumor control. In this study, by using a murine pancreatic cancer model, the authors propose that the addition of radiation therapy to CCR5i and αPD1 immunotherapy could upregulate CD8+ T cells and a subgroup of NK cells within the tumor and result in better tumor control. They further analyzed human single-cell sequencing data from pancreatic cancer patients and identified one subgroup of NK cells (NK C1) with tissue-resident features. Subsequent cell-cell contact analysis reveals the NK-cDC1-CD8 cell axis in pancreatic cancer. By analyzing TCGA data, they found that high NK C1 signature levels were associated with better survival in pancreatic cancer patients. Thus, radiotherapy could benefit the outcome of patients bearing low NK C1 signatures. Importantly, the positive correlation between NK C1 score with survival extends beyond pancreatic cancer, showing potential applicability across various solid cancers.

Strengths:

This study could add new insight into the clinical practice by introducing such novel combined therapy and shed light on the underlying immune cell dynamics. These findings hold potential for more effective and targeted treatment in the future. Mouse experiments nicely confirmed that such combined therapy could significantly reduce tumor volume. The elegant use of single-cell sequencing analysis and human database examination enriches the narrative and strengthens the study's foundation. Additionally, the notion that NK C1 signature correlates with patient survival in various solid cancers is of high interest and relevance.

Weaknesses:

1. The role of CCR5i requires further clarification. While the authors demonstrated its capacity to reduce Treg in murine tumors, its impact on other cell populations, including NK cells and CD8+ T cells, was not observed. Nevertheless, the effect of CCR5i on tumor growth in Figure 2B should be shown. If the combination of radiotherapy and αPD1 already can achieve good outcomes as shown in Figure 3A, the necessity to include CCR5i is questioned. Overall, a more comprehensive elucidation of the roles of CCL5 and CCR5i in this context would be good.

2. In line with this, spatial plots in Figure 4 did not include the group with only radiotherapy and αPD1. This inclusion would facilitate a clearer comparison and better highlight the essential role of CCR5i.

3. NK C1 cells should be also analyzed in the mouse model. The authors suggest that NKNKG2D-ve could be the cell population. Staining of inhibitory markers should be considered, for example, TIGIT and TIM3 as presented in Figure 5B.

4. While the cell-cell contact analysis generated from single-cell sequencing data is insightful, extending this analysis to the mouse model under therapy would be highly informative. NK and CD8 cells in the tumor increased upon the combined therapy. However, cDC1 was not characterized. Analysis regarding cDC1 would provide more information on the NK/cDC1/CD8 axis.

5. Human database analysis showed a positive correlation between NK C1 score and CCL5 in pancreatic cancer. Furthermore, radiotherapy could benefit the outcome of patients bearing low NK C1 scores. It would be interesting to test if radiotherapy could also benefit patients with low CCL5 levels in this cohort.

Reviewer #3 (Public Review):

Summary:

In the submitted manuscript by Go et al, the authors evaluated the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and made a number of interesting observations, including the following: 1) CCL5 expression within the tumor microenvironment negatively correlated with clinical outcomes in human patients with PDAC; 2) there were both positive and negative correlations between CCL5 expression and the expression of specific genes (e.g. those encoding CD56 and CD16, respectively) included among gene signature lists for Treg, MDSC, TAM, and NK cells; 3) CCR5 inhibition with the inhibitor, maraviroc, reduced Treg infiltration but not that of other immune cell types in an orthotopic murine model of PDAC; 4) CCR5 inhibition augmented anti-PD1 immunotherapy when combined with ionizing radiation (IR) therapy in the murine model; 5) the above therapy resulted in increased infiltration of CD8+ cytotoxic T cells as well as of a subset of NKG2D-negative, tissue-residency (tr) marker expressing NK cells (deemed Cluster 1 NK in their data sets) that inversely correlated with the number of E-cadherin+ cells (i.e. tumor cells) and showed predicted interactions with cDC1 dendritic cells (including XCL1/XCL2 expressed by the NK and XCR1 expressed by the cDC1); 6) the authors identified a number of putative signals stemming from the trNK (e.g. IL-16, TNFSF14, FASLG, CSF, MIF) as well as incoming from cDC1s to NK (e.g. BAG6-NKp30); 7) these trNK cells positively correlated with good outcomes and with CD8+ T cell infiltrations in human PDAC as well as in many other solid tumor types; and 8) importantly, the benefit of IR therapy was specific to the subset of PDAC patients (represented in the TCGA dataset) that were predicted to have low amounts of trNK cells. The authors used murine experimental models, multiplexed imaging analyses, and a number of publicly available sequencing data sets from human tumor samples to perform their investigations. Based on their findings, the authors proposed that combining IR with CCR5 inhibition and anti-PD1 immunotherapy is a promising strategy to treat solid cancers.

Strengths:

Overall, the collective analyses and conclusions appear to be novel and could be of high and rapid impact on the field, particularly in terms of directing clinical trials to incorporate IR with CCR5 inhibition and immunotherapy. The manuscript is well written; the figures are for the most part clear; and the Discussion is very thoughtful.

Weaknesses:

There were a number of minor typographical errors, missing references, or minor issues with the figures. In general, while many of the observations provided strong suggestive evidence of relationships, phenotypes, and functions, the authors often used language to indicate that such things were confirmed, validated, or proven. In fact, there was a paucity of such functional/confirmatory experiments. This does not necessarily detract from the overall significance, excitement for, and potential impact of the study; but the language could likely be adjusted to be more in keeping with the true nature of the findings. The main title and running title are a bit different; consider making them more similar.