between viruses and the immune system are replicated during
immune responses against established tumors. Here, CTLs
that recognize mutational tumor neoantigens also exert varying
levels of immune control but, similar to CTLs in chronic viral
infection, adopt a hypofunctional state.
Recent studies have revealed the heterogeneity and dynamics
of the hypofunctional CTL populations observed in chronic viral
infection and cancer (He et al., 2016; Im et al., 2016; Leong
et al., 2016; Sade-Feldman et al., 2018; Snell et al., 2018;
Utzschneider et al., 2016; Wu et al., 2016). These include
stem-like CTLs that express the HMG box transcription factor
TCF-1 and the SLAM family member Slamf6/Ly108, possess
the capacity for self-renewal, and are found primarily in lymphoid
tissues but in smaller numbers also at immunological effector
sites such as tumors (Miller et al., 2019; Siddiqui et al., 2019).
TCF-1
pos
CTLs continually give rise to TCF-1
neg
effector-like
cells that acquire cytotoxic function, but also upregulate inhibi-
tory receptors, such as TIM-3, predicted to attenuate their
effector activity. TCF-1
neg
CTLs include cells with a continuum
of differentiation states ranging from highly proliferative and
functional to irreversibly hypofunctional. Highly proliferative
TCF-1
neg
CTLs referred to as transitory CTLs express the che-
mokine receptor CX3CR1 and mediate antiviral control during
chronic viral infection. Terminally differentiated TCF-1
neg
CTLs,
on the other hand, are characterized by expression of CD101
and stable epigenetic repression of effector genes ( Hudson
et al., 2019; Li et al., 2019; Philip et al., 2017; Zander et al., 2019).
DCs not only initiate anti-tumor responses in tumor-draining
lymph nodes (tdLNs) but also support and regulate T cell func-
tions in the tumor microenvironment (TME) (Gerhard et al.,
2021; Wculek et al., 2020). Developmental studies have identi-
fied two subsets of conventional DCs named cDC1s and
cDC2s as well as plasmacytoid DCs (pDCs) as lineages distinct
from monocytes, monocyte-derived DCs, and macrophages
(Murphy et al., 2016). cDC1s are more efficient at cross-present-
ing tumor cell-derived antigen to CTLs (Broz et al., 2014),
whereas cDC2s may be more relevant for CD4
+
T cell activation
(Binnewies et al., 2019).
We recently identified an intratumoral DC state characterized
by co-expression of IL12b, Fascin1, and the chemokine receptor
gene CCR7, which we initially classified as cDC1s (Garris et al.,
2018). Reanalysis of these and additional mouse and human
data led us to re-classify these cells as a discrete DC state we
named DC3 (Gerhard et al., 2021; Zilionis et al., 2019). Others
then reported on similar cell states in mouse and human tumors,
to which they referred as LAMP3
+
DC (Zhang et al., 2019),
mregDC (Maier et al., 2020), or Ccl22
+
cDC1s (Zhang et al.,
2020). The respective roles of cDC1s, cDC2s and DC3s in intra-
tumoral CTL activation and specifically how these cells support
the differentiation of stem- to effector-like and to terminally
differentiated CTLs requires further study.
An unanswered question is how CTLs at various stages of dif-
ferentiation navigate the TME in order to orchestrate their cross-
talk with different DC subsets and ultimately to engage with their
malignant target cells. Considering their well-established roles in
lymphoid tissues, chemokines and their receptors are likely cen-
tral orchestrators of this process. Inflammatory chemokine re-
ceptors such as CXCR3, CCR5, and CCR4 are generally
assumed to be important for the recruitment of blood-borne
T cells to tumor tissue, although this has only in some cases
been directly demonstrated, e.g., for CXCR3 (Mikucki et al.,
2015). In addition, CXCR3 guides the local positioning of
T cells in both lymphoid and non-lymphoid tissues (Ariotti
et al., 2015; Groom et al., 2012). Expression of the CXCR3 ligand
CXCL9 specifically by cDC is required for the efficacy of anti-PD-
1 cancer immune checkpoint therapy through mechanisms unre-
lated to T cell trafficking from tdLNs to tumor tissue, hinting at a
role for organizing local cDC interactions with tumor-infiltrating
CXCR3
+
T cells (Chow et al., 2019). However, the full spectrum
of chemokine receptors and their ligands expressed in the
TME by both immune and non-immune cells, but in particular
by CTL subsets, has not been systematically explored.
Here, we generated a comprehensive account of all chemo-
kine and chemokine receptor genes expressed by all cells of
the TME in mouse models of immunogenic cancer in order to
provide a road map for the systematic exploration of their roles
in organizing cellular interactions. We identified CXCR6 as the
most highly expressed chemokine receptor in tumor-infiltrating
CTLs and DC3s as the cell state most highly expressing its ligand
CXCL16. Using multiphoton intravital microscopy (MP-IVM), we
found that CXCR6 optimizes the positioning of TCF-1
neg
CTLs in
perivascular clusters of DC3s in the tumor stroma and uncovered
its critical role in rescuing the proliferative transitory CTL subset
from activation-induced cell death (AICD) through exposure to
trans-presented interleukin-15 (IL-15) cytokine, which was crit-
ical to sustain their population size and anti-tumor function.
RESULTS
CXCR6 is critical for CTL-mediated tumor control
In order to explore chemokine receptors expressed by tumor-
infiltrating CTLs, we used the immunogenic mouse melanoma
model D4M.3A-pOVA (Di Pilato et al., 2019). Tumor single-cell
suspensions were enriched for immune cells and all single-cell
transcriptomes annotated to cell states (see STAR Methods).
We detected three main cell clusters containing T and natural
killer (NK) cell, myeloid cell, and non-immune cell states, as
well as three minor clusters classified as pDC, B cell, and mast
cell states (Figures 1A and S1A). Comparisons to published sin-
gle-cell RNA sequencing (scRNA-seq) datasets revealed that T
and NK cell states resembled those in MC38 mouse colorectal
tumors, and CD8 T cell states resembled those in ovalbumin
(OVA)-expressing B16.F10 mouse melanoma and in spleens of
lymphocytic choriomeningitis virus (LCMV)-infected mice
(Figure S1B) (Miller et al., 2019; Zhang et al., 2020). DC states
resembled those in KP1.9 mouse lung and in MC38 tumors (Fig-
ure S1C), mirroring DC state conservation observed across hu-
man solid cancers (Gerhard et al., 2021; Maier et al., 2020; Zhang
et al., 2020; Zilionis et al., 2019). Cell state annotation was further
validated by marker genes (Figure S1D) and distinct cell state-
enriched gene expression revealing known marker genes
(Figure S1E; Table S1A).
The T/NK cell cluster contained an NK cell state (NK), a CD4
+
T cell state containing both regulatory and helper T cells (CD4 T
R/H), as well as two CD8
+
T cell states annotated as effector-like
(CD8 T E) and memory-like (CD8 T M). CD8 T E expressed the
ll
Cell 184, 4512–4530, August 19, 2021 4513
Article
