Abstract

   In non–small cell lung cancer (NSCLC), response to immune checkpoint
   blockade (ICB) is associated with programmed cell death ligand 1
   expression that is induced by interferon-γ–producing,
   tumor-infiltrating CD8^+ T cells. However, not all tumors with a CD8^+
   T cell infiltrate respond to ICB, and little is known about the
   mechanisms governing ICB resistance in T cell–infiltrated NSCLC. We
   used an orthotopic NSCLC mouse model to study ICB-refractory CD8^+ T
   cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors
   revealed that lung cancer–specific tumor-infiltrating CD8^+ T cells
   exhibited clonal expansion but lacked expression of genes associated
   with effector and exhausted T cell responses, indicating that they
   underwent a differentiation program distinct from conventional T cell
   exhaustion. This lung cancer–specific T cell dysfunction program was
   established early during priming in the mediastinal lymph node and was
   characterized by robust proliferation but a failed up-regulation of
   effector and exhausted T cell characteristics. Intriguingly, CD8^+ T
   cells from patients with NSCLC expressed an analogous gene expression
   program, which appeared distinct from conventional T cell exhaustion.
   Administration of recombinant interleukin-2 (IL-2) and IL-12 was
   sufficient to restore effector T cell differentiation and induce
   control of KP lung tumors. These findings imply that a CD8^+ T cell
   differentiation trajectory, activated during T cell priming in the
   mediastinal lymph node, limits the response of CD8^+T cells to ICB and
   thereby may contribute to failure of ICB in a subset T cell–infiltrated
   NSCLC.

INTRODUCTION

   Immune checkpoint blockade (ICB) provides long-term tumor control and
   survival for a fraction of patients with non–small cell lung cancer
   (NSCLC) ([40]1–[41]5). A preexisting, tumor-reactive T cell infiltrate
   is prognostic for ICB response in most cancers ([42]6–[43]8).
   Nonsquamous NSCLC is a heterogeneous disease whose treatment is
   determined by driver mutations ([44]9, [45]10). Whereas EGFR-, ALK-, or
   ROS1-mutated tumors receive targeted small-molecule therapies that
   inhibit the activity of the corresponding oncogenic proteins, the
   remaining patients with NSCLC, dominated by KRAS mutations, receive ICB
   ([46]9–[47]11). Mutations that co-occur with KRAS play a role in
   shaping the T cell infiltrate ([48]11, [49]12). Tumors with mutations
   in KRAS and LKB1/STK11 frequently lack T cell infiltration and are
   resistant to ICB ([50]11, [51]12). Tumors with mutations in both KRAS
   and TP53 are frequently infiltrated with T cells ([52]11, [53]12). Yet,
   in T cell–infiltrated KRAS/TP53-mutant tumors, the ICB response rate is
   only ~35% ([54]11, [55]12). This discrepancy suggests that in KRAS/TP53
   mutant NSCLC, not all tumor-infiltrating T cells respond to ICB, and a
   better understanding of the functional states of tumor-reactive T cells
   is needed.

   That the functional state of antitumor T cells determines ICB response
   is supported by clinical reports correlating programmed cell death
   ligand 1 (PD-L1) expression to ICB outcome in NSCLC ([56]3). PD-L1
   expression within the tumor microenvironment (TME) is indicative of T
   cell function because PD-L1 is induced by the effector cytokine
   interferon-γ (IFN-γ) ([57]13). Patients with PD-L1–positive, T
   cell–infiltrated tumors comprise about 40% of the ICB-treated patients
   with NSCLC and are the group most likely to respond to ICB ([58]3,
   [59]9, [60]14). In contrast, patients with NSCLC with PD-L1–negative, T
   cell–infiltrated tumors respond poorly to ICB. Clinically, these T cell
   responses were described as “nonfunctional,” referring to their
   inability to produce IFN-γ ([61]3). However, our understanding of the
   resistance mechanisms in T cell–infiltrated, ICB-refractory patients is
   limited.

   To better understand ICB resistance in T cell–infiltrated NSCLC, we
   used an orthotopic model for NSCLC derived from an autochthonous
   genetically engineered mouse model driven by Kras^G12D expression and
   deletion of Tp53 (KP cell line) ([62]15). We found that antitumor T
   cell responses in this model entered into a T cell dysfunctional state
   that was distinct from T cell exhaustion (T[ex]). This dysfunctional
   state shared aspects of T cell responses found in ICB-refractory, T
   cell–infiltrated patients with NSCLC, including a lack of up-regulation
   of effector molecules after ICB ([63]3). We found that the functional
   state of the tumor-infiltrating T cell response was determined during
   priming in the tumor-draining lymph node (TdLN) in our model system.
   ICB-refractory T cell responses were characterized by a lack of
   effector and exhaustion molecule expression despite robust T cell
   activation and migration into the KP tumors. Understanding how
   ICB-refractory T cell responses differ from ICB-sensitive,
   conventionally exhausted T cell responses is of high clinical interest.
   An improved understanding of the impact of T cell states on antitumor
   immunity will enable the development of targeted therapies to engage
   all T cell subsets in NSCLC.

RESULTS

ICB resistance in KP lung tumors despite a brisk T cell infiltrate

   We inoculated KP tumor cells intravenously to establish lung tumors or
   subcutaneously to establish flank tumors and subsequently administered
   anti–cytotoxic T lymphocyte–associated protein 4 (anti–CTLA-4) and
   anti–PD-L1 combination ICB beginning on day 7 after tumor inoculation
   ([64]Fig. 1A). KP lung tumors were unaffected by ICB ([65]Fig. 1, [66]B
   and [67]C), but KP flank tumors had significantly delayed tumor
   outgrowth ([68]Fig. 1D). Calculation of the fold change in tumor size,
   as a ratio of the treated tumor areas to the average of the control
   tumor areas, confirmed that flank tumors responded significantly better
   to ICB than lung tumors ([69]Fig. 1E). Quantification of
   tumor-infiltrating lymphocyte (TIL) density in tumor areas using
   immunofluorescence (IF) staining, identified by keratin staining,
   confirmed that KP lung tumors had TIL (280.6 ± 29.66 T cells/ mm^2,
   means ± SEM; [70]Fig. 1F and [71]fig. S1). The density of TIL in lung
   tumors was 29.6-fold higher compared with ICB-sensitive flank tumors
   ([72]Fig. 1, [73]F and [74]G), indicating that ICB resistance was not
   driven by a lack of T cell infiltration. However, TIL in lung tumors
   expanded only 1.4-fold, whereas the density of TIL in flank tumors
   increased 11-fold after ICB ([75]Fig. 1, [76]F to [77]H). This model
   system allowed us to gain insights into ICB resistance by contrasting
   ICB-resistant and ICB-sensitive T cell responses in T cell–infiltrated
   NSCLC driven by the same oncogenic mutations.

Fig. 1. Orthotopic KP tumors in the lung are resistant to ICB.

   Fig. 1.
   [78]Open in a new tab

   (A) Scheme of tumor inoculation and ICB. (B) Representative example and
   (C) quantification of lung tumor burden on day 21 in control and
   ICB-treated mice assessed by H&E stain (control n = 9, ICB n = 9,
   combined data from three experiments). (D) Outgrowth of flank KP tumors
   treated with ICB or control (control n = 9, ICB n = 9, combined data
   from three experiments). (E) Comparison between ICB efficacy in lung
   and flank tumors shown as percent change of tumor size over control
   treatment (lung n = 9, flank n = 9, combined data from three
   experiments). Dotted line represents no change from the untreated
   controls. (F) Representative IF images of lung and flank tumors on day
   21 from control mice or mice treated with ICB. For uncropped images,
   see [79]fig. S1. PI, propidium iodide. Scale bar, 100 mm. (G) Density
   of CD8^+ T cells in control and ICB-treated lung and flank KP tumors on
   day 21 determined by IF. (H) Fold change of tumor-infiltrating CD8^+ T
   cells between ICB-treated and control mice. Dashed line is at 1, which
   is no change from the control. Regions analyzed in (G) and (H): flank,
   n = 12 from three mice; flank + ICB, n = 14 from four mice; lung, n =
   58 from three mice; and lung + ICB, n = 73 from four mice. Data are
   shown as means ± SEM, and statistical analysis was conducted using a
   two-way ANOVA (D), MWU test (C, E, and H), or one-way ANOVA (G) with
   **P < 0.01, ***P < 0.001, and ****P < 0.0001.

TIL from KP lung tumors do not exhibit conventional T[ex]

   We performed single-cell RNA sequencing (scRNA-seq) on TIL ([80]16).
   All analyses excluded circulating CD45^+ cells using intravenous
   injection of a phycoerythrin (PE)–labeled anti-CD45 antibody before
   euthanasia, followed by magnetic depletion of PE-labeled cells. We
   sequenced 122,554 cells from eight flank and eight lung tumor–bearing
   mice on day 14 with and without ICB, including 10,774 T cells ([81]Fig.
   2A) and identified nine distinct T cell phenotypes ([82]Fig. 2, [83]B
   and [84]C). Cluster proportions were not significantly different
   between control and ICB ([85]fig. S2A) but were present at different
   proportions between sites of tumor growth ([86]Fig. 2D). We focused our
   subsequent analysis on the two non-naïve CD8^+ T cell clusters (CD8^+
   c1 and CD8^+ c2) ([87]6, [88]17, [89]18). CD8^+ c1 were dominant in
   flank TIL, whereas CD8^+ c2 were dominant in lung TIL ([90]Fig. 2,
   [91]A, [92]B, and [93]D). CD8^+ c1 had increased Pdcd1, Lag3, Tnfrsf9,
   Tnfrsf4, Havcr2, Cd160, and Nrgn gene expression, all associated with
   conventional T cell activation and exhaustion ([94]Fig. 2E and
   [95]table S1) ([96]19–[97]22). CD8^+ c2 had higher expression of
   transcripts associated with CD8^+ T cell activation, including Klrg1
   and Ccl5, and T cell survival and tissue homing, including Klf2, Klf3,
   S1pr1, S1pr4, Itga4, and Itgb1 ([98]Fig. 2E) ([99]23–[100]31). Pathway
   analysis suggested interleukin-2 (IL-2)/signal transducer and activator
   of transcription 5 signaling to be enriched in CD8^+ c1 ([101]Fig. 2F,
   [102]fig. S2B, and [103]table S2). These data suggested that two
   distinct CD8^+ T cell responses were induced against the KP tumor cell
   line depending on the location of the tumor growth; CD8^+ T cell
   responses against flank tumors were characterized by effector function
   and conventional T[ex], whereas CD8^+ TIL in lung tumors lacked
   exhaustion molecules but expressed a distinct set of effector
   molecules.

Fig. 2. Lung tumor–infiltrating CD8^+ T cells do not acquire effector or
exhausted phenotypes observed in flank TIL.

   Fig. 2.
   [104]Open in a new tab

   (A) UMAP plots of scRNA-seq indicating treatment status and tumor
   location and (B) curated clusters. T[H], T helper. MAIT, mucosal
   associated invariant T cell. (C) Bubble plot of gene expression
   analysis of curated clusters. (D) Stack charts of contributions to each
   curated cluster based on treatment condition and tumor location. (E)
   Volcano plot of DEGs between CD8^+ c1 and CD8^+ c2 CD8^+ T cells.
   Selected DEGs in CD8^+ c2 (green) or CD8^+ c1 (orange) TIL are
   highlighted. (F) Pathway analysis of DEGs showing enriched MSigDb
   hallmarks pathways for CD8^+ c2 (green) and CD8^+ c1 (orange) TIL. (G)
   Validation of differentially expressed key effector/exhaustion markers
   on lung and flank CD8^+ TIL (lung, n = 9 and flank, n = 9, combined
   data from three experiments). Data are shown as means ± SEM, and
   statistical analysis was conducted using an MWU test (G) with **P <
   0.01, ***P < 0.001, and ****P < 0.0001, or as indicated in Materials
   and Methods (A to F). MFI, mean fluorescence intensity.

   Flow cytometry on TIL from lung or flank tumors was performed to
   validate differentially expressed genes (DEGs). Lung and flank TIL
   expressed programmed cell death 1 (PD-1), indicative of antigen
   recognition occurring at both tumor sites ([105]Fig. 2G) ([106]32).
   Flank TIL had higher expression of molecules associated with T[ex]:
   LAG-3 (Lag3) and 4–1BB (Tnfrsf9), effector T cell differentiation, CD25
   (Il2ra), and granzyme B (Gzmb) ([107]Fig. 2G), whereas CD49d (Itga4)
   was higher on lung TIL ([108]Fig. 2H). A second model of lung cancer
   (LL/2) provided similar results ([109]fig. S2C). See [110]fig. S2D for
   example flow cytometry plots. Thus, in our model, lung tumor TIL
   exhibited signs of T cell activation yet lacked hallmarks of effector T
   cell differentiation and T[ex]. We therefore concluded that lung
   tumor–reactive TIL in our models were in a dysfunctional state distinct
   from T[ex]. For ease, we refer to this T cell state as lung
   cancer–specific T cell dysfunction, abbreviated T[Ldys], throughout the
   manuscript.

TIL with T[Ldys] are tumor antigen specific

   Differences in TIL between lung and flank KP tumors could be driven by
   differences in antigen specificities or T cell receptor (TCR) usage. We
   recovered Tcra and Tcrb sequences from single-cell libraries ([111]33).
   CD8^+ c1 and CD8^+ c2 both showed clonal expansion in lung and flank KP
   tumors, suggesting that both sites of tumor growth induced a
   tumor-reactive T cell response ([112]fig. S2E). Using grouping of
   lymphocyte interactions by paratope hotspots 2 (GLIPH2) ([113]34) to
   analyze similarities in the TCR repertoire, we identified seven
   specificity groups that contained CDR3 sequences from three or more
   mice and that were shared across flank and lung tumors and both CD8^+
   phenotypes ([114]fig. S2F). Identification of public clonotypes
   suggests a shared antigen present in both lung and flank KP tumors
   ([115]table S3) capable of inducing clonal expansion.

   To directly determine whether CD8^+ T cell responses against the same
   antigen could enter the CD8^+ c1 and CD8^+ c2 states, we engineered KP
   cells to express the model antigen SIYRYYGL (SIY, KP.SIY cell line),
   which is presented on H-2K^b. Using SIY-loaded pentamers, we sorted
   SIY-reactive CD8^+ TIL from day 14 lung and flank KP.SIY tumors. To
   elucidate whether SIY-reactive CD8^+ T cells were transcriptionally
   similar to CD8^+ T cell populations from KP parental lung and flank
   tumors, we conducted an integrated analysis of both datasets. We
   determined a high degree of overlap in the clustering of lung and flank
   SIY-reactive CD8^+ TIL with their respective counterparts from KP
   parental tumors ([116]Fig. 3A). Unsupervised analysis of the integrated
   data yielded two clusters consistent with the CD8^+ c1 and CD8^+ c2 in
   the KP parental cell line ([117]Fig. 3B). The proportion of CD8^+ c1
   and CD8^+ c2 within TIL from KP.SIY and KP parental tumors was similar
   ([118]Fig. 3C), with CD8^+ c1 being 47 and 59% of KP and KP.SIY flank
   TIL, respectively. Lung TIL consisted of 90 and 89% CD8^+ c2 from KP
   and KP.SIY tumors, respectively, demonstrating that the T[Ldys]
   phenotype is dominant in lung TIL ([119]Fig. 3C). Next, we determined
   the DEGs between CD8^+ c1 and CD8^+ c2 from the KP.SIY TIL ([120]Fig.
   3D and [121]table S4). Consistently, CD8^+ c1 from KP.SIY tumors had
   higher expression of exhaustion markers, including Tnfrsf9, Tnfrsf4,
   Lag3, Cd160, and Havcr2, whereas CD8^+ c2 had higher expression of
   lung-homing factors, including Ccl5 and Itga4. A comparison of DEGs
   between CD8^+ c1 and CD8^+ c2 from both the KP parental and KP.SIY
   CD8^+ TIL datasets showed a highly significant degree of similarity (R
   = 0.55, P < 0.001; [122]Fig. 3E), indicating that the transcriptional
   differences between CD8^+ c1 and CD8^+ c2 were conserved in TIL
   isolated from KP parental and KP.SIY tumors. This suggested that
   T[Ldys](CD8^+ c2) was not a result of weak or absent reactivity to
   tumor-associated antigens because T cell responses against SIY induced
   differentiation to this T cell state.

Fig. 3. T[Ldys] T cells in lung tumors and T[ex] T cells in flank tumor
respond to the same antigen.

   Fig. 3.
   [123]Open in a new tab

   (A) Combined UMAP plot of scRNA-seq of polyclonal CD8^+ T cells (lung
   and flank KP tumors) and SIY-pentamer^+ CD8^+ T cells (lung and flank
   KP.SIY tumors) indicating tumor cell line and location. (B) UMAP plot
   showing clusters CD8^+ c1 and CD8^+ c2 of combined datasets from (A).
   (C) Proportions of cluster CD8^+ c1 or CD8^+ c2 within flank and lung
   TIL. (D) Volcano plot of DEGs between CD8^+ c1 and CD8^+ c2
   SIY-reactive CD8^+ T cells. Genes of interest are highlighted. (E) The
   log fold change values of DEGs between CD8^+ c1 and CD8^+ c2 were
   compared between SIY-reactive CD8^+ T cells from KP.SIY tumors and
   polyclonal CD8^+ T cells from KP tumors. (F) Flow cytometry of
   SIY-reactive TIL from KP.SIY lung and flank tumors (lung intravenous, n
   = 12; lung intratracheal, n = 6; and flank n = 11; data pooled from
   four independent experiments, two with intratracheal tumor
   administration). Data are shown as means ± SEM, and statistical
   analysis was conducted using an MWU test (F) with *P < 0.05, **P <
   0.01, and ***P < 0.001, or as indicated in Materials and Methods (A to
   E).

   Flow cytometric analysis of SIY-reactive TIL ([124]Fig. 3F) confirmed
   that the phenotypes of lung and flank TIL were similar to those
   observed in KP parental tumors ([125]Figs. 2G and [126]3F). We also
   performed intratracheal administration of KP.SIY cells as a second
   method of tumor inoculation into the lung. We detected a difference in
   only CD49d between intravenously and intratracheally induced lung
   tumors, suggesting that intravenous tumor inoculation was not driving
   T[Ldys] ([127]Fig. 3F). In summary, differences between lung and flank
   TIL were preserved in the presence of a strong neoantigen and were
   consistent between intravenous and intratracheal lung tumor induction.

T[Ldys] is not driven by the lung TME

   Because separation of tumors from normal lung tissue before analysis
   was not feasible, we used IF staining to determine whether T[Ldys] T
   cells were in the TME or within the adjacent lung. Using keratin to
   identify tumor areas, we found that CD8^+ T cells were significantly
   enriched in lung tumors compared with the adjacent normal tissue
   ([128]fig. S3, [129]A and [130]B). This was true for lung tumors
   inoculated intravenously or intratracheally ([131]fig. S3, [132]A and
   [133]B), indicating that most CD8^+ T cells from lung tumor–bearing
   mice resided in the tumor (see also [134]fig. S1). We next quantified
   PD-1 expression on CD8^+ T cells infiltrating lung tumors or the
   adjacent normal tissue because PD-1 is expressed upon antigen encounter
   ([135]32). PD-1^+CD8^+ T cells were significantly enriched in lung
   tumors compared with adjacent tissue ([136]fig. S3, [137]A and [138]C).
   PD-1 was expressed on 74.5, 78.3, and 73.7% of CD8^+ T cells from
   intravenous lung, intratracheal lung, and flank tumors, respectively
   ([139]fig. S3, [140]A and [141]D). These data indicate that most PD-1^+
   CD8^+ T cells are within tumors and suggest that the differences
   between T[ex] and T[Ldys] TIL are not due to failed infiltration of TIL
   with T[Ldys] into the tumor.

   To further determine whether T[Ldys] were independent of the lung TME,
   we adoptively transferred 20,000 Thy1.2^+ fluorescence-activated cell
   sorting (FACS)–sorted TIL from flank or lung tumors into Rag2^−/− mice.
   Eight to 16 weeks later, we inoculated the reconstituted Rag2^−/− mice
   with KP flank tumors. Adoptively transferred TIL from KP flank tumors
   led to slower tumor growth compared with those from KP lung tumors
   ([142]fig. S3E), suggesting that TIL from KP lung and flank tumors are
   functionally different and that T[Ldys] is a persistent T cell state
   that cannot be reversed by a flank tumor or homeostatic proliferation.

   To probe for earlier differences in T cell responses against lung and
   flank tumors, we performed a concomitant immunity assay. Mice bearing
   flank or lung KP tumors were challenged with a second KP tumor
   implanted on the contralateral flank 7 days after the first tumor
   inoculation. Mice bearing an initial flank tumor experienced
   significantly improved tumor control of the second tumor compared with
   naïve mice ([143]fig. S3F), but mice with an initial lung tumor showed
   no protection against a second flank tumor ([144]fig. S3F). Similar
   results were obtained with the KP.SIY cell line ([145]fig. S3G).
   Antibody-mediated depletion confirmed that this protection was due to
   CD8^+ T cells ([146]fig. S3H). To determine whether the lung TME was
   sufficient to induce T[Ldys] after initial T cell activation, we
   assessed the effect of an initial flank tumor on the growth of
   subsequent lung tumors and observed protection against a second lung
   tumor challenge ([147]fig. S3I), suggesting that the lung TME was not
   directly imposing T[Ldys]. These data suggest that the T[Ldys] induced
   by KP and LL/2 lung tumors is a persistent state of T cell dysfunction
   established early during the anti–lung tumor immune response.

T[Ldys] is established during T cell priming in the mediastinal LN

   We next postulated that the differences in CD8^+ T cell responses were
   imposed during T cell priming in the lung tumor–draining mediastinal LN
   (mLN) or flank tumor–draining inguinal LN (iLN). To determine whether
   differences in antitumor T cell responses could be detected in the
   periphery, we performed an IFN-γ enzyme-linked immune absorbent spot
   (ELISpot) assay. KP lung tumors had significantly fewer
   IFN-γ–producing, SIY-reactive splenocytes compared with KP flank tumors
   ([148]fig. S4A). To determine whether this was due to a lack of T cell
   proliferation during priming, we used an adoptive T cell transfer
   approach using TCR-transgenic 2C T cells (2C T cells) specific for SIY
   ([149]35). Naïve, carboxyfluorescein diacetate succinimidyl ester
   (CFSE)–labeled 2C T cells were transferred into mice with KP.SIY lung
   or flank tumors on day 7, and the degree of T cell activation was
   determined in TdLN on day 10. The 2C T cells proliferated robustly in
   both the mLN and the iLN ([150]fig. S4, [151]B and [152]C), suggesting
   that differences in antitumor CD8^+ T cell responses were not due to a
   lack of T cell expansion during priming.

   Next, we adoptively transferred 2C T cells to mice with KP.SIY lung or
   flank tumors on day 7. On day 10, 2C T cells from the mLN of lung
   tumor–bearing mice and the iLN of flank tumor–bearing mice were
   isolated using FACS, and scRNA-seq was performed using Smart-Seq to
   determine transcriptional differences between 2C T cells primed in the
   mLN or iLN ([153]Fig. 4A). A total of 1744 genes were differentially
   expressed between 2C T cells priming in iLN or mLN ([154]table S5).
   Consistent with the scRNA-seq data of endogenous flank TIL, 2C T cells
   primed in iLN showed high levels of genes associated with effector
   function (Gzma, Gzmb, Il2ra, Il12rb1, Il12rb2, Prdm1, Bhlhe40, and Id2)
   ([155]36–[156]39) and exhaustion (Pdcd1, Havcr2, Tnfrsf4, and Cd160)
   ([157]19–[158]22) ([159]Fig. 4B). In contrast, 2C T cells primed in the
   mLN showed high levels of genes associated with inhibition of effector
   T cell differentiation (Sell, Pecam1, Bcl6, Id3, Lef1, Ctla4, Bach2,
   Tox, and Tox2) ([160]36–[161]44) and genes associated with lung T cell
   responses (Ccl5, Itga4, and Itgb1) ([162]23–[163]25) ([164]Fig. 4B).
   Direct comparison of 2C T cells and endogenous CD8^+ TIL revealed that
   42 genes were consistently regulated between these early and late CD8^+
   T cell responses ([165]fig. S4D and [166]table S6), suggesting that
   differences in T cell responses between lung and flank KP tumors
   originated from differences during T cell priming. Flow cytometry
   affirmed that CD25 and GzmB were significantly reduced on T cells
   activated in mLN ([167]Fig. 4, [168]C and [169]D), whereas mLN-primed
   2C T cells showed higher levels of CD49d ([170]Fig. 4E).

Fig. 4. CD8^+ T cells primed in the mLN fail to acquire an effector
phenotype.

   Fig. 4.
   [171]Open in a new tab

   (A) Schematic of experimental setup. Mice were inoculated with KP.SIY
   lung or flank tumors, and on day 7 of tumor growth, 1 × 10^6 naïve,
   congenically marked 2C T cells were transferred to tumor-bearing mice.
   On day 10 of tumor growth, the 2C T cells were isolated from the mLN of
   lung tumor–bearing mice and iLN of flank tumor–bearing mice using FACS.
   (B) Volcano plot of DEGs between 2C T cells primed in the mLN and iLN
   of tumor-bearing mice. mLN, n = 3 and iLN, n = 4 biological replicates.
   Genes of interest are highlighted. (C to E) Representative example and
   quantification (means ± SEM) of CD25 (C), GzmB (D), and CD49d (E)
   expression levels on CFSE-labeled 2C T cells primed in the mLN and iLN
   of tumor-bearing mice at 72 hours after adoptive transfer, day 10 of
   tumor growth (mLN, n = 6 and iLN, n = 6; data pooled from two
   experiments). CFSE dilution is shown on the x axis. (F to I)
   Representative example and quantification (means ± SEM) of the percent
   and absolute number of SIY+-reactive T cells (F) as well as CD25 (G),
   GzmB (H), and Tox (I) expression levels on endogenous SIY-reactive T
   cells in the mLN and iLN of tumor-bearing mice 7 days after tumor
   inoculation. NS, not significant; SSC, side scatter. (J) Mice were
   inoculated with KP.SIY cells either intravenously, intratracheally, or
   subcutaneously. On day 7 of tumor growth, mLNs and iLNs were isolated,
   and endogenous SIY-reactive CD8^+ T cells were analyzed with flow
   cytometry for the expression of CD25, GzmB, and CD49d (n = 9 for CD25
   and GzmB and n = 6 for CD49d comparisons, data pooled from three
   experiments). Data are shown as means ± SEM, and statistical analysis
   was conducted using an MWU test (C to J) with *P < 0.05, **P < 0.01,
   ***P < 0.001, and ****P < 0.0001 or as indicated in Materials and
   Methods (B).

   To determine whether differences in antigen-presenting cells (APCs) in
   the TdLN were responsible for the differences in T cell priming, we
   examined tumor antigen–containing APC using KP tumor cells engineered
   to express ZsGreen, allowing extended detection of APC populations
   carrying tumor cell debris ([172]45, [173]46). Flow cytometry of
   ZsGreen^+ cells in the mLN of lung tumor–bearing mice and the iLN of
   flank tumor–bearing mice on day 7 after tumor inoculation revealed that
   conventional dendritic cells (DCs) were the dominant APC that acquired
   tumor antigen and migrated to the TdLN ([174]fig. S4E). Furthermore, we
   detected equal numbers of ZsGreen^+ DCs in both mLN and iLN ([175]fig.
   S4F). To assess functional abilities of DCs, KP cells expressing
   ZsGreen and the SIIN peptide were inoculated into mice, and 7 days
   later ZsGreen^+ DCs were sorted from TdLNs. ZsGreen^+ DCs were
   cocultured with naïve TCR-transgenic CD8^+ T cells specific for SIIN
   peptide (OT-I T cells). We detected identical T cell activation using
   ZsGreen^+ DCs from mLN or iLN ([176]fig. S4G), indicating that both DC
   populations have similar stimulatory capacity ex vivo, highlighting
   that differences between CD8^+ T cell responses against lung and flank
   tumors were not due to differences in DC-mediated antigen presentation.

   We next evaluated the priming of endogenous SIY-reactive T cells in
   response to KP.SIY lung and flank tumors. We found no significant
   differences in either the percentage or number of SIY-reactive CD8^+ T
   cells on day 7 after tumor inoculation ([177]Fig. 4F). However,
   SIY-reactive CD8^+ T cells in the iLN of flank tumor–bearing mice
   expressed higher CD25 and GzmB ([178]Fig. 4, [179]G and [180]H).
   SIY-reactive CD8^+ T cells in the mLN of lung tumor–bearing mice
   expressed higher Tox ([181]Fig. 4I), consistent with higher Tox
   transcript levels in 2C T cells primed in mLN ([182]Fig. 4B). We next
   assessed the T cell phenotype of SIY-reactive CD8^+ T cells in the mLN
   after intravenous or intratracheal KP tumor administration.
   SIY-reactive CD8^+ T cells in the mLN had reduced CD25 and GzmB and
   increased CD49d compared with SIY-reactive CD8^+ T cells in the iLN,
   indicating that these differences in priming occurred with both
   intravenous and intratracheal inoculation ([183]Fig. 4J). To determine
   whether these differences in T cell activation were due to the
   orthotopic nature of KP lung tumor, we inoculated mice with the
   melanoma cell line B16.SIY. Responses in the mLN were similar to
   responses in the iLN ([184]fig. S5, [185]A and [186]B), indicating that
   CD8^+ T cell priming in the mLN in response to orthotopic tumors in the
   lung led to T[Ldys].

T cells with T[Ldys] retain T cell factor 1 but are functionally distinct
from precursor-exhausted T cells

   Studies in chronic lymphocytic choriomeningitis virus (LCMV) have
   described two subsets of exhausted CD8^+ T cells: terminally exhausted
   T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3^+)
   CD8^+ T cells, which gradually lose their functional properties and
   eventually undergo apoptosis, and precursor-exhausted T cell factor
   1–positive (TCF-1^+) CD8^+ T cells that lack cytotoxicity but retain a
   stem-like capacity to differentiate into TIM-3^+ T cells ([187]36,
   [188]47). Because Havcr2, which encodes TIM-3, was differentially
   expressed on tumor-reactive CD8^+ T cells in mLN and iLN and the
   corresponding TIL populations, we determined the expression of TIM-3
   and TCF-1 during CD8^+ T cell responses to lung and flank KP.SIY
   tumors. TIM-3 was up-regulated on CD8^+ T cells in the iLN, both on
   transferred 2C T cells ([189]Fig. 5A) and on endogenous SIY-specific
   CD8^+ T cells ([190]Fig. 5B). In contrast, CD8^+ T cells activated in
   the mLN did not up-regulate TIM-3 and remained TCF1^+ ([191]Fig. 5,
   [192]A and [193]B). These differences were specific for orthotopic lung
   KP tumors because no differences were detected using B16.SIY tumors
   ([194]fig. S5C).

Fig. 5. T[Ldys] T cell state is functionally distinct from conventional T[ex]
T cell state.

   Fig. 5.
   [195]Open in a new tab

   (A to D) Flow cytometry analysis of TCF-1 and TIM-3 expression on
   adoptively transferred 2C T cells in the mLN and iLN of KP.SIY
   tumor–bearing mice 72 hours after adoptive transfer (10 days after
   tumor inoculation; n = 14 for mLN and n = 10 for iLN, data pooled from
   two experiments). (B) Endogenous, SIY-reactive CD8^+ T cells in KP.SIY
   tumor–bearing mice 7 days after intravenous, intratracheal, or
   subcutaneous inoculation (n = 9 for IV mLN, n = 10 for IT mLN, and n =
   9 for iLN, data pooled from three experiments). (C) Adoptively
   transferred 2C T cells in lung or flank KP.SIY tumors 14 days after
   tumor inoculation and 7 days after adoptive transfer (n = 6 for lung
   and n = 5 for flank, data pooled from two experiments). (D) Endogenous,
   SIY-reactive CD8^+ TIL in KP.SIY tumor–bearing mice 14 days after tumor
   inoculation (n = 6 for all conditions, data pooled from two
   experiments). (E to H) Analysis of SIY-reactive CD8^+ T cells in KP.SIY
   tumor untreated or treated with ICB given on days 7 and 10 after tumor
   inoculation. TCF-1 and TIM-3 expression in mLN and iLN (E) and lung and
   flank tumors (F) 14 days after tumor inoculation (n = 6, data pooled
   from two experiments). GzmB expression on SIY-reactive CD8^+ T cells in
   (G) mLN and iLN and lung and flank tumors (H) 14 days after tumor
   inoculation (n = 6, data pooled from two experiments). (I and J)
   Comparison of CD8^+ c2 (green) or CD8^+ c1 (orange) signatures from KP
   parental (I) and KP. SIY (J) datasets with previously published CD8^+ T
   cell signatures from acute and chronic LCMV infection. (K and L) Flow
   cytometric analysis of TCF1–1, TIM-3 (K), and GzmB (L) expression in
   serially transferred 2C T cells on day 5 after transfer to RAG2^−/− (n
   = 6 for mLN and n = 7 for iLN, data pooled from two experiments) Data
   are shown as means ± SEM, and statistical analysis was conducted using
   an MWU test with **P < 0.01, ***P < 0.001, and ****P < 0.0001 (A to H
   and K to L).

   We next analyzed TIL on day 14 after tumor inoculation. Both adoptively
   transferred 2C T cells and endogenous T cells acquired a terminally
   exhausted TIM-3^+ TCF1^− phenotype in flank tumors ([196]Fig. 5, [197]C
   and [198]D). However, neither 2C T cells nor SIY-reactive TIL in lung
   tumors acquired significant TIM-3^+ expression and remained either
   TCF-1^+ or became TIM-3^− TCF-1^− ([199]Fig. 5, [200]C and [201]D)
   despite up-regulation of PD-1 ([202]Figs. 2G and [203]3F and [204]fig.
   S3, [205]A, [206]C, and [207]D), suggesting that T cells were
   responding to their cognate antigen without acquiring T cell effector
   function. TCF-1 expression by SIY-reactive CD8^+ T cells in mLN and
   lung tumors raised the possibility that KP lung tumors had an increased
   proportion of precursor-exhausted CD8^+ T cells. Because ICB targets
   TCF-1^+ precursor-exhausted CD8^+ T cells and induces their
   differentiation into TIM-3^+ TCF-1^− effector T cells ([208]37,
   [209]40, [210]48–[211]51), we determined the potential of TCF-1^+ T
   cells in mLN to differentiate to TIM3^+ T cells. We treated mice on
   days 7 and 10 of KP.SIY tumor growth with ICB and evaluated the
   phenotypes of SIY-reactive CD8^+ T cells. The proportion of TIM3^+
   TCF1^− SIY-reactive CD8^+ T cells in the iLN of flank tumor–bearing
   mice increased after ICB, with no differences in T cell phenotype
   detected in the mLN of lung tumor–bearing mice ([212]Fig. 5E).
   Consistent with this observation, we detected no accumulation of TIM3^+
   TCF1^− TIL in KP lung tumors; instead, ICB resulted in increased
   TIM-3^− TCF-1^− CD8^+ TIL ([213]Fig. 5F). SIY-specific CD8^+ T cells in
   mLN and KP lung tumors also failed to up-regulate GzmB after ICB,
   whereas ICB enhanced GzmB expression in SIY-reactive CD8^+ T cells in
   the iLN ([214]Fig. 5, [215]G and [216]H). Lack of TIM-3 and GzmB
   up-regulation in SIY-reactive CD8^+ T cells from mLN and lung tumors
   after ICB was consistent with the lack of ICB efficacy in KP lung
   tumors ([217]Fig. 1, [218]A to [219]E) and suggested that the TCF-1^+
   CD8^+ T cells in mLN and lung tumors are functionally distinct from
   precursor-exhausted CD8^+ T cells.

   We compared the transcriptional profiles of TIL with a published
   dataset of T cell exhaustion ([220]36). CD8^+ c2 from both the KP and
   KP. SIY scRNA-seq datasets, which constitutes T[Ldys], was most
   enriched for naïve and acute effector gene signatures despite low
   expression of effector molecules such as GzmB and CD25 ([221]Fig. 5,
   [222]I and [223]J). CD8^+ c1 from both the KP and KP.SIY scRNA-seq
   datasets were most similar to signatures from exhausted effectors and
   TCF-1^+ precursor-exhausted T cells ([224]Fig. 5, [225]I and [226]J).
   These data suggest that despite TCF-1 expression, lung tumor–reactive
   T[Ldys] CD8^+ T cells are transcriptionally and functionally distinct
   from precursor-exhausted T cells. Thus, T cells with T[Ldys] are
   activated and gain tissue-homing capabilities but minimally
   differentiate into TIM-3^+ CD8^+ T cells and fail to acquire
   appreciable effector T cell functions even when exposed to ICB.

T[Ldys] is associated with decreased T cell functionality

   To measure the functionality of the T[Ldys] state, we assessed cytokine
   production from SIY-reactive TIL from KP.SIY tumors. Although neither
   lung nor flank TIL produced high levels of IL-2, we detected
   significantly lower production of tumor necrosis factor–α and IFN-γ in
   lung TIL compared with flank TIL ([227]fig. S6A). To evaluate
   cytotoxicity, we performed an in vivo cytotoxicity assay ([228]52).
   Flank tumor–bearing mice showed increased eradication of SIY-pulsed
   cells, whereas no significant cytotoxicity was detected in lung
   tumor–bearing animals ([229]fig. S6B). These results demonstrated that
   T cells with T[Ldys] fail to differentiate into cytotoxic effector T
   cells despite robust T cell activation in the mLN ([230]Fig. 4, [231]C
   to [232]E) and successful infiltration into lung tumors ([233]fig. S3,
   [234]A to [235]C).

   To determine the persistence of the T[Ldys] state after T cell priming,
   naïve 2C T cells were transferred to lung or flank KP.SIY–bearing mice
   on day 7, isolated from mLN or iLN on day 10, and transferred to
   RAG2^−/− mice bearing flank KP.SIY tumors. The 2C T cells primed in the
   mLN retained a TCF-1^+ TIM-3^− phenotype with decreased GzmB after
   infiltration into flank tumors, whereas the 2C T cells primed in iLN
   retained increased TIM-3 and GzmB expression ([236]Fig. 5, [237]K and
   [238]L). This further affirmed that T[Ldys] is a persistent state
   induced during T cell priming in the mLN rather than a consequence of
   differential stimulation in the TME.

T[Ldys] is detectable in patients with NSCLC

   To determine whether a T[Ldys] T cell gene signature identifies subsets
   of human NSCLC TIL, we generated a uniform manifold approximation and
   projection (UMAP) plot from a scRNA-seq study of human NSCLC ([239]Fig.
   6A) ([240]53) and mapped signatures corresponding to T[ex] and T[Ldys]
   phenotypes onto this UMAP ([241]Fig. 6B and [242]table S7). The T[ex]
   signature enriched among the terminally differentiated CD8-LAYN
   cluster, whereas the T[Ldys] signature was enriched among the less
   differentiated CD8-KLF2 (Kruppel like factor 2), CD8-GZMK, and CD8-XCL1
   clusters ([243]Fig. 6B). Pseudotemporal ordering revealed a trajectory
   from the CD8-KLF2, CD8-GZMK, and CD8-XCL1 populations to the terminally
   differentiated CD8-LAYN phenotype ([244]Fig. 6C). Along this
   trajectory, the T[Ldys] signature was down-regulated as a function of
   pseudotime, whereas the T[ex] signature was up-regulated ([245]Fig.
   6D). Thus, less differentiated CD8^+ T cell populations in human NSCLC
   demonstrated transcriptomic similarity to the T[Ldys] T cell state
   found in KP lung tumors, whereas terminally exhausted CD8^+ T cell were
   closely related to the T[ex] T cells that dominate KP flank tumors.
   These observations suggest that both T[ex] and T[Ldys] T cells are
   abundant, transcriptionally distinct, and mutually exclusive in the TIL
   of human NSCLC samples. Gueguen et al. ([246]53) found minimal TCR
   sequence overlap between the KLF2 and GZMK clusters with the LAYN
   cluster, suggesting that, similar to our observations made in the mouse
   model, human CD8^+ T cells enriched for the T[Ldys] signature
   inefficiently differentiated to terminally exhausted TIL.

Fig. 6. T[Ldys] T cell state is detectable in human NSCLC TIL populations.

   Fig. 6.
   [247]Open in a new tab

   (A) UMAP plot of CD8^+ T cell clusters from Gueguen et al. ([248]53).
   (B) CD8^+ c2 or CD8^+ c1 signatures mapped onto the CD8^+ T cell
   clusters from Gueguen et al. in (A). (C) Pseudotemporal analysis of
   CD8^+ T cell clusters from Gueguen et al. in (A). (D) Pseudo-temporal
   analysis of CD8^+ c2 or CD8^+ c1 signatures and CD8^+ T cell clusters
   from Gueguen et al. in (A). (E) UMAP feature plots from three human
   datasets analyzed for expression of CD8^+ c2 (top) and CD8^+ c1
   (bottom) signatures. (F) Proportions of CD8^+ T cells from individual
   patients that have CD8^+ c1 (black) or CD8^+ c2 (gray) signatures.

   To validate that the T[Ldys] gene signature could also be detected in
   other NSCLC datasets, we mapped gene signatures of T[ex] or T[Ldys] T
   cell states onto three additional NSCLC datasets ([249]54–[250]56). We
   found that across NSCLC datasets, most of the CD8^+ TIL appeared
   enriched for the T[Ldys] signature, whereas only a fraction of TIL were
   enriched for the T[ex] signature ([251]Fig. 6E). For individual
   patients, the contribution of the T[Ldys] and T[ex] phenotype varied
   drastically, supporting the notion that a fraction of patients with
   NSCLC completely lack a population of T cells in the T[ex] state found
   in KP flank tumors and are dominated by the T[Ldys] phenotype found in
   KP lung tumors ([252]Fig. 6F). In summary, we were able to validate
   that the T[Ldys] T cell state is detectable in human NSCLC TIL, and
   similar to our observation in the mouse model, they have limited
   differentiation to the T[ex] state despite signs of T cell activation.

IL-2 and IL-12 therapy prevents T[Ldys]

   Our data suggested that the T[Ldys] state was induced during T cell
   priming in the mLN, where transcriptional profiling indicated
   significantly lower expression of Il2ra, Il12rb, and Il12rb2 ([253]Fig.
   4B). In addition, pathway analysis had found that IL-2 signaling was
   significantly enriched in flank TIL compared with lung TIL ([254]Fig.
   2F). Given these observations, we determined whether cytokine therapy
   with IL-2 or IL-12 could prevent T[Ldys] induction in our model. To
   deliver IL-2 or IL-12 to tumor-bearing mice, we used fusion proteins of
   IL-2 or IL-12 to murine serum albumin (MSA) (MSA-IL2 and MSA-IL12,
   respectively), which lengthens the half-life of IL-2 or IL-12 in vivo
   and has potent antitumor effects in mouse flank tumor models ([255]57,
   [256]58). We transferred 2C T cells to mice bearing KP.SIY lung tumors
   and, on the same day, administered MSA-IL2 or MSA-IL12. MSA-IL2
   treatment slightly increased CD25 and GzmB expression, whereas MSA-IL12
   had no effect ([257]Fig. 7B). Combined administration of MSA-IL2 and
   MSA-IL12 led to a significant increase in both CD25 and GzmB ([258]Fig.
   7B). To determine whether differentiation of endogenous T cells to the
   T[Ldys] state could be overcome, we treated mice bearing KP.SIY lung
   tumors with MSA on day 7 after tumor inoculation and analyzed the
   endogenous SIY-reactive CD8^+ T cells in mLN and tumors on day 10 after
   tumor inoculation. SIY-reactive CD8^+ T cells in the mLN down-regulated
   TCF-1 and up-regulated TIM-3 ([259]Fig. 7C), CD25, GzmB, and 4–1BB,
   whereas CD49d was unchanged ([260]Fig. 7D). We also found a notable
   up-regulation of TIM-3, CD25, GzmB, and 4–1BB ([261]Fig. 7, [262]E and
   [263]F) and a modest down-regulation of CD49d expression ([264]Fig. 7F)
   in SIY-reactive CD8^+ TIL, indicating that IL-2 and IL-12 were
   sufficient to overcome T[Ldys] in our model.

Fig. 7. T[Ldys] can be overcome by combined IL-2 and IL-12 therapy.

   Fig. 7.
   [265]Open in a new tab

   (A) Schematic of experimental design. Mice were inoculated with KP.SIY
   lung tumors. On day 7 of tumor growth, 2C T cells were adoptively
   transferred to tumor-bearing mice. Immediately after adoptive transfer,
   mice were given either IL-2 or IL-12 fusions to MSA or the combination.
   Seventy-two hours after adoptive 2C T cell transfer, the mLNs were
   analyzed with flow cytometry. (B) Percentage of CD25 (left) and Gzmb
   (right) on 2C T cells in mLNs for control or cytokine treatments
   (control, IL-2, and IL-12, n = 6 and IL-2 + IL-12, n = 7, data pooled
   from two experiments). (C and D) Endogenous SIY-reactive CD8^+ T cells
   in the mLNs were analyzed for (C) TCF-1 and TIM-3 expression or (D) for
   PD-1, 4–1BB, CD25, GzmB, and CD49d expression. (E and F) Analysis of
   endogenous SIY-reactive CD8^+ TIL from the same mice as (C) and (D).
   (E) TCF-1 and TIM-3 expression. (F) PD-1, 4–1BB, CD25, GzmB, and CD49D
   expression. [(C to F) n = 7 each condition, data pooled from two
   experiments] (G) Schematic of experiment combining ICB with MSA-IL2 +
   MSA-IL12 therapy. Mice were inoculated with KP.SIY lung tumors. Mice
   received either control treatments, ICB on days 7, 10, 13, and 16,
   MSA-IL2 + MSA-IL12 on days 7 and 10, or the combination of ICB and
   MSA-IL2 and MSA-12. (H and I) Lung tumor burden assessed on day 21 with
   (H) showing a representative H&E example and (I) showing the
   quantification of tumor area per lung lobe (control and ICB, n = 5 and
   MSA-IL2 + MSA-IL12 and ICB + MSA-IL2 + MSA-IL12, n = 6, data pooled
   from two experiments). (J) Mice were inoculated with lung KP tumors,
   treated as in (G), and monitored for survival (control, n = 9; ICB, n =
   6; MSA-IL2 + MSA-IL12, n = 6; and ICB + MSA-IL2 + MSA-IL12, n = 3; data
   pooled from three experiments). Data are shown as means ± SEM, and
   statistical analysis was conducted using an MWU test with *P < 0.05,
   **P < 0.01, and ***P < 0.001 (B to I), or with a Kaplan-Meier analysis
   (J).

   On the basis of the significant change in T cell differentiation
   induced by MSA-IL2 and MSA-IL12, we next assessed whether this therapy
   could induce control of KP lung tumors. Mice were inoculated with KP
   parental lung tumors and treated with ICB, MSA-IL2, and MSA-IL12 or the
   combination on day 7 ([266]Fig. 7G). ICB had no effect on tumor growth
   ([267]Fig. 7, [268]H and [269]I), whereas MSA-IL2 and MSA-IL12 and the
   combination of ICB + MSA-IL2 and MSA-IL12 induced marked reductions in
   lung KP tumors on day 21 ([270]Fig. 7, [271]H and [272]I). In addition,
   a longitudinal study using micro–computed tomography found that MSA-IL2
   and MSA-IL12 treatment even reduced tumor size over time ([273]fig. S7,
   [274]A and [275]B, and [276]movies S1 to [277]S4). Consistently,
   MSA-IL2– and MSA-IL12–treated mice had significantly longer survival
   ([278]Fig. 7J). Consistent with previous reports ([279]57), KP flank
   tumors also benefitted from MSA-IL2 and MSA-IL12 ([280]fig. S8),
   suggesting that this therapy also enhances T cell responses in flank
   TIL. In summary, our data suggest that CD8^+ T cells with T[Ldys] fail
   to respond to ICB but respond to combined IL-2 and IL-12 treatment in
   the KP lung tumor model.

DISCUSSION

   We identified a state of T cell dysfunction induced during T cell
   priming in the mLN of KP lung tumor–bearing mice, which we termed
   T[Ldys]. T[Ldys] was characterized by reduced expression of effector
   and exhaustion molecules on CD8^+ T cells and ICB resistance ([281]Fig.
   8). T[Ldys] resembles T cell responses in an ICB-refractory sub-set of
   human NSCLCs that have been referred to as nonfunctional. In this
   subset of patients, tumors are infiltrated by T cells, but these T
   cells fail to up-regulate effector molecules and PD-L1 in response to
   ICB ([282]3). Therefore, our observations on the induction and
   characteristics of the T[Ldys] T cell state may allow for the rational
   development of new treatment strategies for T cell–infiltrated but
   ICB-refractory NSCLC.

Fig. 8. Schematic summary.

   Fig. 8.
   [283]Open in a new tab

   (Left) In response to flank tumors, tumor-reactive CD8^+ T cells primed
   in the iLN up-regulate CD25 and IL-12R, express effector molecules,
   show signs of conventional exhaustion, and respond to CBT. (Right) In
   response to lung tumors, however, tumor-reactive CD8^+ T cells primed
   in the mLN activate a lung-specific dysfunctional program (T[Ldys]), do
   not up-regulate CD25 and IL-12R, fail to gain effector molecule
   expression, and do not acquire a conventional T[ex] phenotype. CD8^+
   TLdys cells do not respond to CBT. This suggests that differentiation
   of CD8^+ T cells into dysfunctional states other than conventional
   T[ex]drives CBT resistance in a subset of T cell–infiltrated NSCLCs.
   T[eff], effector T cell. Created with [284]Biorender.com.

   T cell infiltration into tumors is predictive for ICB response, but the
   quality of T cells is critical ([285]40). We provide evidence that not
   all tumor-reactive T cells are functionally interchangeable and that
   not all tumor-specific CD8^+ T cells will react to ICB. T cells with
   T[Ldys], induced in response to orthotopic lung cancer, failed to
   produce IFN-γ, a feature of antitumor immune responses that mediate
   PD-L1 up-regulation, a biomarker in NCSLC. ICB failed to expand TIL
   T[Ldys] and did not affect the phenotype or function of lung
   tumor–reactive T cells. Furthermore, T cells with T[Ldys] had low GzmB
   and poor cytotoxicity in vivo. Thus, T[Ldys] closely resembles the
   nonfunctional T cell responses described in an ICB-refractory NSCLC
   patient population ([286]3, [287]59–[288]62). In contrast, flank TIL
   gained effector function and responded to ICB with T cell expansion and
   tumor control, suggesting that T[Ldys] was specific to orthotopic lung
   tumors.

   We determined that T cell fate decisions leading to T[ex] or T[Ldys]
   occurred during priming in the TdLN. Transcriptional differences
   occurred within 72 hours after T cell activation. T cells primed in the
   mLN never gained robust CD25 expression, whereas priming in the iLN
   resulted in CD25 up-regulation. CD8^+ T cells that express high levels
   of CD25 differentiate into effector T cells ([289]38, [290]39),
   suggesting that CD25 expression is required for differentiation into
   effector and exhausted T cell states. Consistently, T cells primed in
   the mLN had low GzmB, a phenotype that was preserved in lung TIL. These
   data suggested that T cell fate decisions are made early during T cell
   priming, depend on the local cytokine milieu, and can predetermine the
   quality of the resulting TIL.

   T cell activation is affected by antigen drainage to the TdLN and the
   functional capacity of APC. We found comparable antigen load and
   stimulatory capacity of DC between lung and flank tumors. T[Ldys] was
   also observed with intratracheal administration of tumor cells, a
   condition used to control for potential systemic dissemination of tumor
   cells or different tumor loads induced between intravenous and
   subcutaneous settings. However, it remains a possibility that greater
   immunogenic cell death is induced in flank tumors, leading to a more
   inflammatory environment and thus effective CD8^+ T cell activation.
   Alternatively, the lung could be a tolerogenic environment ([291]63),
   requiring a greater degree of immunogenic cell death for innate immune
   activation. It should be noted, however, that only inoculation of lung
   cancers KP and LL/2 into the lung induced T[Ldys], whereas inoculation
   of B16 melanoma cells resulted in T[ex], suggesting that only
   orthotopic lung tumors may induce T[Ldys]. Additional tumor cell lines
   and modes of cancer induction will be needed to fully elucidate the
   details resulting in T[ex] or T[Ldys] induction and whether induction
   of T[Ldys] is restricted to the mLN. Our analyses indicate the presence
   of both phenotypes in flank TIL, suggesting that T cell differentiation
   may be complex.

   Adoptive transfer studies suggested that T[Ldys] persisted after T cell
   activation. Neither homeostatic proliferation in RAG2^−/− mice nor ICB
   treatment induced effector function, supporting that epigenetic
   imprinting might mediate T[Ldys] ([292]64, [293]65). We found increased
   Tox levels in T cells primed in the mLN. Tox is an epigenetic modifier,
   previously found to be a critical factor for not only epigenetic
   remodeling of the chromatin during induction and maintenance of T[ex]
   but also long-lived memory T cell responses ([294]42, [295]66). In
   addition, Tox expression was found in stem-like memory CD8^+ T cells
   induced after intravenous nanoparticle vaccine administration
   ([296]67). Therefore, we speculate that Tox up-regulation during
   induction of the T[Ldys] suggests a context-specific effect of Tox.
   Further assessment of the epigenetic state of T[Ldys] and T[ex] will be
   needed to fully elucidate whether Tox or additional epigenetic
   modifiers are important for the induction of T[Ldys] and how the
   epigenetic state of T[Ldys] compares with other T cell states.
   Consistent with decreased expression of CD25 and GzmB after priming in
   the mLN, we found decreased differentiation of CD8^+ T cells into
   TIM-3^+ effector cells. TCF-1 and TIM-3 define precursor- and
   terminally exhausted CD8^+ T cells, respectively, in chronic LCMV and
   melanoma models ([297]36, [298]37, [299]40, [300]47, [301]51).
   Expression of TCF1^+ in SIY-reactive T cells in the mLN together with
   elevated levels of Tox, Bach2, and Bcl6 suggested a relation to
   precursor-exhausted CD8^+ T cells ([302]47). However, adoptive transfer
   studies of 2C T cells primed in mLN clearly demonstrated that T cells
   with T[Ldys] failed to differentiate into effector or exhausted T
   cells. Direct comparison of CD8^+ TIL from KP lung tumors to
   transcriptional signatures of precursor-exhausted CD8^+ T cells
   suggested that T cells with T[Ldys] do not transcriptionally resemble
   precursor-exhausted CD8^+ T cells. These data suggest that T[Ldys] is
   induced early and does not give rise to conventionally exhausted T
   cells.

   Integration of our findings with clinical datasets suggested that a
   fraction of T cell–infiltrated patients lacked exhausted T cells and
   were dominated by T[Ldys] T cells. A recent publication described five
   CD8^+ TIL populations in NSCLC: blood-derived CD8-KLF2 and CD8-GZMK
   precursors, tumor-infiltrating CD8-XCL1 precursors, a transitory
   CD8-GZMH population, and terminally exhausted CD8-LAYN T cells
   ([303]53). Mapping the T[Ldys] signature onto this dataset suggested a
   close association of T cells with T[Ldys] with KLF2 and GZMK precursor
   clusters, whereas the exhaustion signature derived from CD8^+ c1 T
   cells mapped to the exhausted LAYN cluster. Gueguen et al. ([304]53)
   found limited differentiation of KLF2 and GZMK precursor populations to
   exhausted LAYN T cells, consistent with our data that T cells with
   T[Ldys] failed to acquire effector and exhaustion molecules. Multiple
   studies found that exhausted CD8^+ T cells correlate with good
   responses to ICB in patients with NSCLC ([305]60–[306]62), and the
   presence of a T cell–derived IFN-γ signature and PD-L1 up-regulation is
   predictive for ICB response in NSCLC. This is supported by our studies
   in which T[Ldys] T cells failed to respond to ICB or make IFN-γ;
   therefore we propose that the patient populations dominated by T[Ldys]
   T cells may represent patients that would be ICB refractory. However,
   this requires clinical validation. We also showed that IL-2/IL-12
   cytokine treatment could mediate differentiation of T[Ldys] T cells
   into highly potent effector T cells. This observation demonstrates that
   whereas T[Ldys] T cells fail to spontaneously differentiate into
   effector T cells, cytokine therapy could facilitate this
   differentiation. Further studies will be required to determine the
   phenotype of T cells responding to IL-2/IL-12 therapy in flank tumors
   because about 50% of flank TIL were of T[Ldys] T cell state. How
   natural cytokine signals might affect the spontaneous differentiation
   into T[ex] and T[Ldys] state will also require additional
   interrogations. However, immunosuppressive populations such as
   regulatory T cells or tumor-associated macrophages might affect T cell
   differentiation by cytokine production or consumption ([307]68,
   [308]69).

   Whereas Gueguen et al. ([309]53) found limited evidence for transition
   of T cell clonotypes from the KLF2 or GZMK clusters to the LAYN
   cluster, our TCR clonotype analyses identified shared clonotypes
   between T[ex] and T[Ldys]. Furthermore, we were able to demonstrate the
   induction of T[Ldys] and exhausted T cell phenotypes among endogenous
   SIY-reactive T cells in KP.SIY tumors and in TCR-transgenic T cells. In
   addition, only subtle differences between the CD8^+ TIL responses
   against KP and KP.SIY tumors were found. Together, these observations
   provide strong support that decisions between T[ex] and T[Ldys] are
   independent of TCR usage or affinity. Whether TCR signal strength
   affects T cell states in the TME requires further investigation. In
   summary, analyses of both clinical samples and our mechanistic studies
   agree that T cell priming in LN is involved in inducing different T
   cell states and highlights the importance of further understanding the
   determinants of early T cell fate decisions.

   To determine whether the T[Ldys] phenotype could be therapeutically
   manipulated, we assessed whether cytokine therapy might be effective
   when administered during priming. Combined treatment with IL-2 and
   IL-12 induced differentiation of T[Ldys] to an effector T cell state.
   This raises questions about IL-2 and IL-12 signals during priming. IL-2
   availability can be significantly affected by immunosuppressive
   populations such as regulatory T cells ([310]70, [311]71). Regulatory T
   cells, in turn, are frequently localized to tissue-draining LN based on
   tissue-specific antigen expression patterns ([312]72, [313]73).
   Lung-specific regulatory T cells may therefore affect effector T cell
   activation in the mLN. IL-12 is produced by myeloid cells ([314]74),
   but the exact cellular source of IL-12 is still unclear. It is
   conceivable that IL-12 is induced once CD8^+ T cells gain the ability
   to produce IFN-γ ([315]75). IL-12 has also been shown to induce
   down-regulation of Klf2 and S1pr1 expression in T cells ([316]76).
   Down-regulation of KLF2 and S1PR1 is a common feature of CD8^+ T cells
   that express high levels of PD-1, LAG-3, TIM-3, and 4–1BB both in human
   TIL ([317]59–[318]62) and TIL isolated from KP flank tumors. This
   together with the requirement of IL-2 might suggest that IL-2 and IL-12
   signaling sensitizes T cells during priming for the optimal acquisition
   of effector function. Thus, IL-2– and IL-12–based immunotherapies may
   help differentiate TIL toward an ICB-responsive state. In a recent
   study, new derivatives of IL-12 were generated to reduce natural killer
   cell–mediated toxicity but retain the ability of IL-12 to induce T cell
   effector function, primarily IFN-γ and GzmB ([319]77). This preclinical
   work, together with our observations, points to a potential use for
   cytokine therapy in patients with nonfunctional T cell responses who
   fail to respond to current treatments.

   One shortcoming of our studies was that we could not separate lung
   tumors and normal lung tissue, and therefore, we cannot fully exclude
   that T cells from healthy lung affect our analysis; however, our IF
   experiments suggest that TIL were strongly enriched in the TME. Another
   shortcoming was that our study is based on a limited number of
   transplantable cell lines and tumor inoculation methods. In addition,
   there were insufficiently available human NSCLC data to determine
   whether the gene signature associated with CD8^+ c2 is predictive of
   ICB resistance. Future longitudinal human studies comparing ICB outcome
   with pretreatment CD8^+ TIL scRNA-seq will be required to address this
   question.

   Together, our data suggest that responsiveness to ICB in NSCLC can be
   predetermined during T cell priming in the mLN. If T cells enter
   T[Ldys], a T cell response lacking cytotoxic effector functions becomes
   established in tumors. It is conceivable that this early decision point
   precludes T cells with T[Ldys] from reinvigoration by ICB. This study
   provides mechanistic insight into the induction of ICB-refractory T
   cell responses and provides rationale for potential strategies to
   increase effector CD8^+ T cell differentiation in NSCLC, potentially
   benefiting ICB-refractory patients with nonfunctional immune responses,
   improving outcomes for patients with NSCLC.

MATERIALS AND METHODS

   A full description of the methods can be found in [320]Supplementary
   Methods.

Study design

   The purpose of this study was to determine how differences in T cell
   activation between lung and flank KP tumors led to ICB resistance in KP
   lung tumors. We characterized T cell differentiation in these two tumor
   settings and defined how this contributed to ICB resistance. We used
   transplantable syngeneic tumor mouse models and publicly available
   human datasets. Mice were randomized into study groups after tumor
   inoculation and before treatments. All experiments were performed at
   least twice independently with at least three mice per group. All data
   were included unless there were technical issues with experimental
   setup or data collection. Outliers were not removed. All tumor
   outgrowth studies were planned to end on day 21 after tumor injection.
   Early termination occurred if tumors grew past the allowed size or if
   mice were flagged by veterinarians for tumor ulceration, changes in
   body condition, or other health reasons that required euthanasia before
   day 21. Other tumor analysis time points were days 7 and 14 after tumor
   injection or 3 days after adoptive transfer of transgenic T cells.

Mice

   All mice were housed and bred under specific pathogen–free conditions
   at the Koch Institute animal facility. All experimental animal
   procedures were approved by the Committee on Animal Care at the
   Massachusetts Institute of Technology (MIT).

Generation of expression vectors

   The pLV-EF1a-IRES-puro vector (Addgene no. 85132) was used in
   conjunction with the cerulean-SIY insert generated using the
   Cerulean-N1 template (Addgene no. 54742) or the ZsGreen insert
   zsGreen_minOVA (a gift from M. Krummel).

Tumor cell lines and tumor outgrowth studies

   KP, LL/2, KP.SIY, and B16.SIY were cultured under standard conditions.
   Tumor cells (2.5 × 10^5) were injected. Subcutaneous tumor area
   measurements were collected two to three times a week, whereas lung
   tumor areas of hematoxylin and eosin (H&E) sections were measured using
   QuPath software.

Immunohistochemistry

   Flank tumors and tumor-bearing lungs were fixed in 10% formalin. Fixed
   tissues were processed, paraffin-embedded, sectioned, and stained by
   the Hope Babette Tang Histology Facility at the Koch Institute at MIT.
   For anti-CD8 staining, a 1:200 dilution of anti-CD8 (clone 4SM16,
   eBioscience 14–0195-82) was used.

Immunofluorescence

   Tumors were processed as previously described ([321]78). Tissues were
   stained with antibodies (see the [322]Supplementary Materials) and
   washed with phosphate-buffered saline (PBS), and nuclei were
   counterstained with propidium iodide. Tissue sections were imaged using
   the TissueFAXS whole-slide scanning system (TissueGnostics, USA) using
   a Zeiss 20x Plan-Neofluar air, 0.5 numerical aperture and analyzed
   using the TissueQuest image analysis software (TissueGnostics, USA).
   Images were processed with the image processing package FIJI ([323]79).

Tumor dissociation

   Spleens and LNs were dissected from mice and physically dissociated.
   Tumors were dissociated using the gentleMACS Octo Dissociator
   (Miltenyi).

Flow cytometry and cell sorting

   All analyses excluded circulating CD45^+ cells using intravenous
   injection of a PE-CF594–labeled anti-CD45 antibody before euthanasia,
   which allowed circulating cells to be excluded from analyses. Cells
   were resuspended in antibody-containing staining buffer plus
   eBioscience Fixable Viability Dye eFluor 780 or eFluor 506 to
   distinguish live and dead cells and with anti-CD16/CD32 to prevent
   nonspecific antibody binding. Cell surface proteins were stained for 20
   min on ice with fluorophore-conjugated antibodies at a 1:200 dilution.
   Cells were washed and resuspended in fixation/permeabilization buffer
   followed by staining with antibodies against intracellular targets. To
   obtain absolute counts of cells, Precision Count Beads (BioLegend) were
   added. Flow cytometry sample acquisition was performed on an
   LSRFortessa cytometer (BD Biosciences), and the collected data were
   analyzed using FlowJo v10.5.3 software (TreeStar). For cell sorting,
   the surface staining was performed as described above under sterile
   conditions, and cells were acquired and sorted into TRIzol or buffer
   using a FACSAria III sorter (BD Biosciences). For TIL analysis, cells
   were pregated on live, CD45^+, CD45-IV^−, TCRb^+, single cells, CD4^−,
   and CD8^+. See the [324]Supplementary Materials for all antibodies. See
   [325]fig. S9 for example gating strategy of CD8^+ and SIY-reactive
   CD8^+ cells.

Seq-Well sequencing and analysis

   Single-cell suspensions were prepared as described above. Mice received
   anti–CD45-PE antibody retro-orbitally 3 min before euthanasia, followed
   by bead-mediated depletion of antibody-labeled circulating cells. SIY
   pentamer–reactive CD8^+ T cells were isolated using FACS. Cells were
   then processed using the Seq-Well platform with second strand
   chemistry, as previously described ([326]16, [327]33). Libraries were
   barcoded and amplified using the Nextera XT kit (Illumina) and were
   sequenced on a NovaSeq 6000 or a NextSeq 550 (Illumina).

Single-cell data processing and visualization

   Raw read processing of scRNA-seq reads was performed as previously
   described ([328]80). Principal components analysis was performed. The
   number of principal components used for visualization was determined by
   examination of the elbow plot, and two-dimensional embeddings were
   generated using UMAP. Clusters were determined using Louvain
   clustering, as implemented in the FindClusters function in Seurat, and
   clusters that contained T cells were selected for further analysis.
   These cells were reprocessed with the same processing and clustering
   steps described above. Differential gene expression was performed for
   each cluster, and clusters corresponding to similar phenotypes were
   merged.

Pathway enrichment analysis

   Gene sets representing relevant pathways were obtained from MSigDB
   ([329]81, [330]82). A score for the expression of genes in each pathway
   was calculated for each cell using the AddModuleScore function in
   Seurat ([331]83). Pathways in the hallmarks gene collection were used
   to analyze CD8^+ T cells and KP.zsGreen tumor cells.

Paired single-cell TCR sequencing

   Paired TCR sequencing and read alignment was performed as previously
   described ([332]84). Processing of reads was performed using the
   Immcantation software suite ([333]85, [334]86). GLIPH2 analysis was
   performed as described previously ([335]34) using the provided data
   from mouse CD8^+ T cells as a reference. For the analysis of global
   motifs, sub-stitutions between any pair of amino acids were permitted.
   GLIPH specificity groups that were subsets of other specificity groups
   were not analyzed further.

Integrated analysis of KP parental and KP.SIY single-cell data

   Single-cell sequencing data from KP parental and KP.SIY TIL were
   integrated using Seurat v3 ([336]87). Genes that were detected in less
   than 3% of cells in either the KP TIL or KP.SIY TIL were excluded.
   Default parameter values and the top 30 canonical correlation analysis
   (CCA) components were used to find transfer anchors between datasets
   and generate an integrated matrix.

Adoptive TIL transfer

   Flank and lung tumors were dissociated and prepared for FACS as
   described above. TIL were identified as Live, CD45^+, noncirculating
   Thy1^+ cells. Recipient RAG2^−/− mice received equal numbers (20,000)
   of either lung or flank TIL intravenously. TIL were allowed more than
   12 weeks to reconstitute RAG2^−/− mice, after which the reconstituted
   mice were inoculated with flank tumors.

Concomitant immunity assay

   Mice were injected with KP tumors subcutaneously or intravenously as
   outlined above. Seven days after tumor injection, mice were injected
   with a second KP tumor on the contralateral flank or intravenously.

CD8 antibody depletion

   Mice were injected with 200 µg of anti-CD8 antibody (clone 53–6.7 Bio X
   Cell) weekly for the duration of the experiment, beginning 48 hours
   before tumor inoculation.

IFN-γ–ELISpot

   Splenocytes (1 × 10^6) were plated on coated and washed ELISpot plates
   and stimulated with 160 nM SIY peptide or a mixture of phorbol
   12-myristate 13-acetate (100 ng/ml; Sigma-Aldrich) and ionomycin (1
   µg/ml; Sigma-Aldrich) as a positive control or medium only as negative
   control. Plates were developed the next day using the BD Biosciences
   mouse IFN-γ–ELISpot kit, following the manufacturer’s protocol.

2C T cell adoptive transfer

   T cells were isolated from 2C RAG2^−/−CD45.1^+ mice (spleen and LN) and
   labeled with CFSE or CellTrace Violet (Life Technologies). T cells
   (10^6) were transferred to mice bearing KP.SIY tumor 7 days after tumor
   inoculation and isolated 72 hours later. For CFSE analysis, the percent
   proliferated was calculated as the percentage of cells that had
   undergone one or more rounds of division based on CFSE staining
   intensity using an unstimulated, CFSE-labeled sample as an undivided
   reference control. For transfers into secondary recipients, 2C T cells
   were transferred into primary recipients as described above, isolated
   from mLN or iLN, and enriched using the Miltenyi CD8^+ T cell isolation
   kit before CD45.1^+ 2C T cells were sorted using FACS. Sorted cells
   were transferred intravenously into secondary recipient RAG2^−/− mice
   bearing day 7 flank KP.SIY tumors. Secondary recipients were analyzed 5
   days after adoptive transfer.

DC analysis and ex vivo coculture assay

   LN processing procedure was adapted from Ruhland et al. ([337]45), and
   CD19^+ and CD3^+ cells were depleted using magnetic beads before flow
   analysis or sort of DC. For ex vivo cocultures, OT-I T cells were
   isolated and CFSE-labeled as described above. CD8^+ OT-I cells (5 ×
   10^4) and FACS-sorted ZsGreen^+ DCs (5 × 10^3) were cocultured and
   analyzed by flow cytometry at 72 hours.

Smart-seq bulk RNA-seq and analysis

   2C T cells were adoptively transferred to tumor-bearing mice as
   described above. Seventy-two hours after transfer, TdLN cells were
   isolated and prepared for FACS. 2C T cells were FACS-sorted directly
   into TRIzol reagent and RNA-extracted. RNA library preparation and
   sequencing were performed at the KI Genomics Core / MIT BioMicro
   Center. Sequencing was performed using the HiSeq 2000 sequencing system
   (Illumina). Sequence alignment and .bam file generation were performed
   by the KI Genomics Core / MIT BioMicro Center. The resulting .bam files
   were sorted using Samtools. Differential gene expression analysis was
   performed on the sorted .bam files with Cufflinks.

Analysis of data from Chen et al.

   Data from the scRNA-seq of P14 cells published by Chen et al. ([338]36)
   were downloaded from the Gene Expression Omnibus (GEO) database under
   accession number [339]GSE131535. The data were processed using the
   processing pipeline described above, and clusters were determined using
   the FindClusters function in Seurat. Gene signature scores were
   calculated using the AddModuleScore function in Seurat.

Cytokine and detection

   Single-cell suspensions were isolated from tumors as described above.
   Round-bottom 96-well plates were coated overnight in PBS with anti-CD3
   (0.2 mg/ml; clone 145–2C11, BD Biosciences), and anti-CD28 (0.5 µg/ml;
   clone 37.51, BD Biosciences). Cells were plated in coated round-bottom
   96-well plates for 4 hours in the presence of BrefeldinA (BioLegend).
   After 4 hours, the cells were stained for extra-cellular and
   intracellular antigens.

In vivo cytotoxicity assay

   The protocol was adapted from Kim et al. ([340]52). Wild-type
   splenocytes were ammonium-chloride-potassium–lysed and pulsed for 1
   hour at 37°C with 0.2 µM SIY peptide. SIY-pulsed splenocytes were then
   labeled with 0.5 µM CFSE, whereas control, unpulsed splenocytes were
   labeled with 5 µM CFSE for 10 min at 37°C. After CFSE labeling,
   SIY-pulsed and control splenocytes were mixed at a 1:1 ratio, and 10 ×
   10^6 of each were intravenously injected into mice bearing KP.SIY lung
   or flank tumors on day 7 of tumor growth or into naïve control mice.
   Four hours after injection of CFSE-labeled splenocytes, spleens from
   tumor-bearing mice were isolated, single-cell splenocyte suspensions
   were generated by physical dissociation, and splenocytes were stained
   with a live/ dead discriminator dye and analyzed for CFSE^+ populations
   by flow cytometry.

Analysis of human NSCLC datasets

   Human NSCLC single-cell datasets were downloaded from the GEO database,
   under accession numbers [341]GSE99254 ([342]54), [343]GSE127465
   ([344]56), [345]GSE123902 ([346]55), and [347]GSE162500 ([348]53). Data
   from Gueguen et al. were processed using integration in Seurat 3, as
   described by Stuart et al. ([349]87). Gene signature scores were
   computed using the AddModuleScore function in Seurat. Pseudotemporal
   ordering using diffusion pseudotime was performed using sc.tl.dpt
   function in SCANPY ([350]88, [351]89). Data from Guo et al. ([352]54),
   Zillonis et al. ([353]56), and Laughney et al. ([354]55) were processed
   using the processing pipeline described above to identify clusters
   corresponding to CD8^+ T cells. Gene signature scores were computed
   using the AddModuleScore function in Seurat. Clusters were identified
   using the FindClusters function in Seurat and were classified as CD8^+
   c1 or CD8^+ c2 according to their expression of the corresponding gene
   signatures.

ICB treatment

   Mice were injected intraperitoneally with anti–CTLA-4 (clone
   UC10–4F10–11) and anti–PD-L1 (clone 10F.9G2) antibodies on days 7, 10,
   13, and 16 after tumor inoculation. Each mouse received 100 µg of each
   antibody per treatment.

MSA-IL2 and MSA-IL12 generation

   MSA-cytokine fusions were generated as previously described ([355]57,
   [356]58). Human embryonic kidney (HEK) 293 cells were transfected with
   plasmid DNA using polyethylenimine in OptiPro serum-free medium (Thermo
   Fisher Scientific). His-tagged proteins were isolated from HEK293
   supernatant using TALON Metal Affinity Resin (Takara Bio Inc.).
   Cytokine fusion proteins were then further purified by size exclusion
   chromatography using a HiLoad 16/600 Superdex 200 pg column on an ÄKTA
   fast protein liquid chromatography system (GE Healthcare). All proteins
   were stored at 4°C, but before therapeutic injection, cytokine fusion
   proteins were warmed to room temperature.

MSA treatment

   Each mouse was injected retro-orbitally with MSA-IL2 (5.94 × 10^−10 mol
   per mouse) and/or MSA-IL12 (1.42 × 10^−11 mol per mouse) per treatment.
   For experiments phenotyping 2C T cells or endogenous SIY-reactive CD8^+
   T cells, mice received one dose of MSA-cytokine fusions on day 7 after
   tumor inoculation and were analyzed on day 10 after tumor inoculation.
   For therapeutic experiments, mice were dosed with MSA-IL2 and/or
   MSA-IL12 on days 7 and 14 after tumor inoculation and analyzed on day
   21 after tumor inoculation and monitored for survival after treatment.

Micro–computed tomography

   A Bruker Skyscan 1276 was used to acquire a series of images with a
   rotation step of 0.65° over a 360° rotation. Images were acquired with
   x-ray tube settings of 100 kV, 200 µA, and an exposure time of 90 ms
   with a 0.5-mm aluminum beam filter. A 4 × 4 detector binning was used
   for an isotropic resolution of 40.16 µm. Anesthesia was induced at 3%
   isoflurane and maintained at 2.0 to 2.5% during imaging, which lasted
   76 s. Image reconstruction was performed using the Bruker NRecon
   software.

Statistical analysis

   Statistical analyses were performed using GraphPad Prism (GraphPad) and
   R. All data are shown as means ± SEM. For flow cytometry,
   immunohistochemistry, and tumor outgrowth studies, statistical analyses
   were performed with Mann-Whitney U (MWU) test for comparisons of two
   groups or two-way analysis of variance (ANOVA) for multiple comparisons
   over time, with *P < 0.05; **P < 0.01; ***P < 0.001; and ****P <
   0.0001. For differential gene expression between T cell clusters, P
   values were calculated using a two-sided Wilcoxon rank-sum test and
   were corrected with Bonferroni correction. For comparison of gene
   modules with previously published T cell states, false discovery rate q
   values were calculated using a one-tailed hypergeometric test and were
   corrected with Bonferroni correction. For pathway analysis, P values
   were calculated using a two-tailed Wilcoxon rank-sum test and were
   corrected with Bonferroni correction.

Supplementary Material

   Suppl material
   [357]NIHMS1776231-supplement-Suppl_material.pdf^ (2.2MB, pdf)
   table S3
   [358]NIHMS1776231-supplement-table_S3.docx^ (16KB, docx)
   table S2
   [359]NIHMS1776231-supplement-table_S2.xlsx^ (17.3KB, xlsx)
   table S1
   [360]NIHMS1776231-supplement-table_S1.xlsx^ (18.6KB, xlsx)
   table S6
   [361]NIHMS1776231-supplement-table_S6.xlsx^ (27.7KB, xlsx)
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   [362]NIHMS1776231-supplement-table_S5.xlsx^ (148.6KB, xlsx)
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   table S8
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   table S7
   [365]NIHMS1776231-supplement-table_S7.xlsx^ (66.1KB, xlsx)
   Movie S1
   [366]Download video file^ (20.1MB, avi)
   movie S2
   [367]Download video file^ (19.6MB, avi)
   Movie S3
   [368]Download video file^ (19.1MB, avi)
   Movie S4
   [369]Download video file^ (21MB, avi)

Acknowledgments: