Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion

May combine two claims.

The study performed pooled Perturb-CITE-Seq screens targeting 248 ICR signature genes with 744 guides and profiled single-cell transcriptomes and 20 surface proteins in more than 218,000 cells.

“We performed pooled Perturb-CITE-Seq screens of the ICR program genes in a patient-derived tumor-TIL co-culture model, targeting 248 genes of the ICR signature (744 targeting guides) and profiling single-cell transcriptomes and 20 surface proteins in >218,000 cells.”

May combine two claims.

The CD58 finding was validated in additional patient models and in clinical scRNA-seq data from patients with ICR.

“and we validate this finding in additional patient models and in clinical scRNA-seq data of patients with ICR.”

May combine two claims.

The study developed an integrated analytical framework and recovered known clinically relevant immune resistance mutations along with novel ones.

“We developed an integrated analytical framework and recover the known landscape of clinically relevant immune resistance mutations along with novel ones.”

May combine two claims.

Perturbations of B2M, HLA-A, JAK1, JAK2, STAT1, IFNGR1, and IFNGR2 conferred immune evasion, consistent with clinical resistance mechanisms.

“perturbations of B2M, HLA-A, JAK1, JAK2, STAT1, IFNGR1 and IFNGR2 (Fig. 2d,e, Extended Data Fig. 3e,f, Supplementary Table 2) conferred immune evasion, consistent with clinical mechanisms of resistance to immunotherapy7–9.”

• cites [7] Zaretsky, J. M. et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N. Engl. J. Med. 375, 819–829 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [8] Sade-Feldman, M. et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat. Commun. 8, 1136 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [9] Jerby-Arnon, L. et al. A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade. Cell 175, 984–997 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Tumor cell lysis was highly reproducible and specific to TCR/MHC Class I interactions.

“Tumor cell lysis was highly reproducible and specific to T cell receptor (TCR)/MHC Class I interactions”

May combine two claims.

At the protein level, MHC proteins and PD-L1 increased in IFNγ-treated versus control cells, and MHC proteins globally increased in co-culture versus control cells.

“At the protein level, we observe increases in MHC proteins and PD-L1 (CD274) in IFNγ-treated vs. control cells; a global increase in MHC proteins in co-culture vs. control cells”

May combine two claims.

CD58 KO conferred a different molecular phenotype than IFNγ-JAK/STAT pathway perturbations, belonged to a distinct module, and did not impact expression of antigen presentation genes.

“the CD58 KO conferred a different molecular phenotype than IFNγ-JAK/STAT pathway perturbations, belonged to a distinct module, and did not impact the expression of antigen presentation genes”

May combine two claims.

MaMel-134 TILs produce IFNγ, TNFα, and Granzyme-B.

“MaMel-134 TILs produce IFNγ, TNFa, and Granzyme-B.”

May combine two claims.

Integrating RNA and protein measurements highlighted gene programs common across conditions or unique to different conditions, with co-cultured cells uniquely enriched for induction of immune escape pathway genes and the ICR signature.

“integrating RNA and protein measurements for joint analysis (Methods) highlighted gene programs that are either common across conditions, or unique to different conditions (Fig. 3i), with the co-cultured cells uniquely enriched for induction of immune escape pathway genes and the ICR signature.”

May combine two claims.

CD58 emerged as a compelling immune evasion mechanism because KO conferred resistance to T cell killing in the viability screen and CD58 RNA/protein were downregulated in cells surviving TIL co-culture.

“CD58 emerged as a compelling mechanism of immune evasion: KO of CD58 conferred resistance to T cell killing in the viability screen and CD58 RNA/protein were down regulated in cells surviving TIL co-culture.”

May combine two claims.

Neither B2M nor HLA-A KO impacted CD58 protein levels in the Perturb-CITE-Seq screen.

“Conversely, neither B2M nor HLA-A KO impacted CD58 protein levels in our Perturb-CITE-Seq screen”

May combine two claims.

Flow cytometry showed CD58 KO did not alter MHC protein expression at baseline or their induction by IFNγ.

“Using flow-cytometry, we show that compared to parental cells, CD58 KO did not alter expression of MHC proteins at baseline or their induction in response to IFNγ”

May combine two claims.

The Perturb-CITE-Seq model correctly reconstructed impacts of genes known to affect resistance to immunotherapy, especially the effect of perturbing the IFNγ response machinery, along with novel pathways.

“The Perturb-CITE-Seq model correctly reconstructed impacts of genes known to affect resistance to immunotherapy, especially the effect of perturbing the IFNγ response machinery (Fig. 4b), along with novel pathways.”

May combine two claims.

CD58, CD47, and IFNGR1 protein levels were reduced in co-culture, concordant with enrichment of their genetic KO in the viability screen.

“In particular, CD58, CD47, and IFNGR1 (CD119) protein levels were reduced in co-culture, concordant with enrichment of their genetic KO in the viability screen”

Probably not a substantial claim: may not pass S3 (load-bearing).

In patient 2686 Cas9-expressing melanoma cells carrying the ICR library, pre-selection mKATE2+ fraction and post-puromycin survival correlate with Pearson R2=0.90.

“Proportion of mKATE2+ cells prior to selection (x axis) and survival after puromycin selection (y axis) in 2686 melanoma Cas9 transgenic cells transduced with the ICR library. Line: Linear regression, Pearson R2=0.90.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Empirically, a second EM iteration had only a very modest effect on sgRNA/target assignments.

“Empirically, adding a second iteration had only a very modest effect on sgRNA/target assignments.”

May combine two claims.

The downregulated IFNγ program included key antigen presentation components, chemokines, inflammatory cytokines, interferon response elements, surface checkpoints, and differentiation genes.

“The downregulated program included key components of antigen presentation and associated machinery (e.g., PSMB4, PSMB8, PSMB9, PSMA4, HLA-A,B,C gene and protein, HLA-E, HLA-F, HLAD-DPB1), chemokines associated with anti-tumor immunity (CXCL1, CXCL2, CXCL8, CXCL10, CXCL11), inflammatory cytokines (STAT3, IL1B, IL6), interferon response elements (STAT1, IRF1, IRF3, IFITM3, IFIT6), surface checkp”

May combine two claims.

In all three patient-derived melanoma cell line models, CD58 KO and B2M KO conferred resistance to T cell mediated killing, while PD-L1 KO sensitized cells.

“In all models, CD58 KO and B2M KO conferred resistance (Fig. 5b-d) while PD-L1 KO sensitized cells to T cell mediated killing”

May combine two claims.

Within each condition, cell cycle and cell complexity affected RNA profiles.

“Within each condition, both the cell cycle (G1/0, S, G2/M) (Fig. 3f) and complexity (Extended Data Fig. 4c) impacted RNA profiles”

May combine two claims.

Several programs, including cell cycle regulation, DNA repair, and antigen presentation, were shared across conditions, whereas immune escape programs were uniquely recovered in co-culture and interferon response genes in IFNγ stimulation.

“Several programs, including cell cycle regulation, DNA repair, and antigen presentation, were shared across conditions, whereas immune escape programs were uniquely recovered in co-culture data, and interferon response genes in IFNγ stimulation”

May combine two claims.

TILs from patient 2686 still induce IFNγ and TNFα, and reactivation with OKT-3 boosts Granzyme-B relative to IL2-only culture.

“TILs from 2686 retain ability to induce IFNγ and TNFa, and OKT-3 reactivation leads to an increase in Granzyme-B production compared to TILs grown in IL2 alone.”

May combine two claims.

The viability screen recovered known immune evasion mechanisms in a dose-dependent manner and identified several novel candidates.

“Overall, the viability screen recovered known immune evasion mechanisms in a dose-dependent manner, and identified several novel candidates.”

May combine two claims.

In the co-culture condition, one EM iteration moved 1,741 target assignments to near zero (confidence <25%), while a second iteration changed only 97 target-cell assignments.

“For example, in the co-culture condition, a single iteration of EM adjusts the assignment of 1,741 targets to near zero (confidence that the cell contains an effective sgRNA < 25%), while an additional iteration only adjusted 97 target-cell assignments.”

May combine two claims.

MaMel-80 TILs produce IFNγ, TNFα, and Granzyme-B.

“MaMel-80 TILs produce IFNγ, TNFa, and Granzyme-B.”

May combine two claims.

CITE profiles of 20 cell surface proteins do not reflect cell cycle phases or UMI count.

“CITE profiles of 20 cell surface proteins do not reflect cell cycle phases (d) or UMI count (e).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Control and knockout melanoma cells show comparable induction of apoptosis in response to Staurosporin and resistance to DTIC.

“Comparable induction of apoptosis in response to Staurosporin and resistance to DTIC in control and KO melanoma cells.”

May combine two claims.

In co-culture, perturbing CD58 leaves B2M and HLA expression unchanged at both RNA and protein levels but increases CD274.

“CD58 perturbation in co-culture does not affect B2M and HLA expression at the RNA and protein level but induces CD274.”

• AssumptionRests on an assumption: The patient-derived melanoma-TIL co-culture with IFNγ pretreatment models T cell-mediated immune selection in human melanoma.

Probably not a substantial claim: may not pass S3 (load-bearing).

A G2M cell cycle control program was learned from the control dataset.

“G2M program learned from control dataset.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CD58, also known as LFA3, is an understudied adhesion molecule typically expressed on antigen-presenting cells that binds CD2 on T and NK cells.

“CD58 (also known as LFA3) is an understudied adhesion molecule, typically expressed on antigen-presenting cells, that binds CD2 on T cell and Natural Killer (NK) cells22.”

• cites [22] Arulanandam, A. R. et al. The CD58 (LFA-3) binding site is a localized and highly charged surface area on the AGFCC’C” face of the human CD2 adhesion domain. Proc. Natl Acad. Sci. USA 90, 11613–11617 (1993). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Activated T cells also express SIRPa, and interaction with CD47 increases T cell activation and cytokine production, which may affect the clinical benefit of CD47 blockade.

“Recent studies revealed that activated T cells also express SIRPa and that interaction with CD47 results in increased T cell activation and cytokine production20, which may impact the potential clinical benefit of CD47-blockade21.”

• cites [20] Myers, L. M. et al. A functional subset of CD8+ T cells during chronic exhaustion is defined by SIRPα expression. Nat. Commun. 10, 794 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [21] Zhang, W. et al. Advances in anti-tumor treatments targeting the CD47/SIRPα Axis. Front. Immunol. 11, 18 (2020).Not checked yet

  Not checked in the free run.

May combine two claims.

A proof-of-concept ECCITE-seq study detected protein, RNA, and guide RNAs in individual cells but was small-scale and lacked the analytical framework needed for large screens.

“A proof-of-concept study using ECCITE-seq5 demonstrated simultaneous detection of protein, RNA, and guide RNAs in individual cells and was applied at a small scale without the analytical framework required for large scale screens.”

• cites [5] Mimitou, E. P. et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat. Methods 16, 409–412 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Clinically relevant ICR mechanisms include B2M mutations causing MHC Class I loss, antigen presentation downregulation, and IFNγ-JAK/STAT mutations that impair response to T cell anti-tumor activity.

“Clinically relevant mechanisms of include mutations in beta-2-microglobulin (B2M) resulting in loss of MHC Class I presentation, downregulation of the antigen presentation machinery, and mutations in the IFNγ-JAK/STAT pathway that impair response to T cell mediated anti-tumor activity7–9.”

• cites [7] Zaretsky, J. M. et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N. Engl. J. Med. 375, 819–829 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [8] Sade-Feldman, M. et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat. Commun. 8, 1136 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [9] Jerby-Arnon, L. et al. A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade. Cell 175, 984–997 (2018). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

CRISPR/Cas9 KO screens in murine models or engineered human cell lines identified putative novel immune evasion mechanisms involving chromatin regulators, TNFα signaling, SOX4, PTPN2, and APLNR, but their clinical relevance is unclear.

“CRISPR/Cas9 KO screens in murine models or engineered human cell lines testing tumor-T cell interactions identified putative novel mechanisms of immune evasion involving chromatin regulators10, TNFα signaling11, SOX412, PTPN213, and APLNR10, yet their clinical relevance is unclear.”

• cites [10] Patel, S. J. et al. Identification of essential genes for cancer immunotherapy. Nature 548, 537–542 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [11] Kearney, C. J. et al. Tumor immune evasion arises through loss of TNF sensitivity. Sci. Immunol. 3, eaar3451 (2018).Not checked yet

  Not checked in the free run.

• cites [12] Pan, D. et al. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science 359, 770–775 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Many relevant phenotypes are best understood at the protein level and differ between RNA and protein, so prior Perturb-Seq schemes needed expansion.

“However, many relevant phenotypes are functionally best understood at protein level, and are reflected differently at the RNA and protein level, thus requiring expansion of prior Perturb-Seq schemes.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CD47 is a don't-eat-me signal on tumor cells that interacts with SIRPa on phagocytic cells, and blockade of the CD47/SIRPa axis improves tumor control in vivo.

“CD47 is a “don’t eat me” signal on tumor cells that interacts with SIRPa on phagocytic cells; and blockade of the CD47/SIRPa axis improves tumor control in vivo19.”

• cites [19] Veillette, A. & Chen, J. SIRPα–CD47 immune checkpoint blockade in anticancer therapy. Trends Immunol. 39, 173–184 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

ICIs produce durable responses in some patients, but most patients either do not benefit or develop resistance over time.

“While ICIs produce durable responses in some patients most patients either do not benefit or develop resistance over time.”

• cites [6] Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Perturb-CITE-Seq analysis rediscovered key immune evasion mechanisms, organized them into modules, related them to impacted genes and programs, and recovered new modules that may confer immune evasion beyond IFNγ-JAK/STAT and antigen presentation defects.

“Taken together, our Perturb-CITE-Seq analysis rediscovered key mechanisms of immune evasion, organized them into modules and related them to the genes and programs they impact, as well as recovered new modules that may confer immune evasion beyond defects in the IFNγ-JAK/STAT pathway and antigen presentation.”

May combine two claims.

Multiple transcripts and proteins implicated in modifying immunotherapy response are directly regulated by the IFNγ-JAK/STAT axis, suggesting some mechanisms are a consequence of defective IFNγ-JAK/STAT signaling rather than independent modes of resistance.

“Thus, multiple transcripts and proteins implicated in modifying the response to immunotherapies are directly regulated by the IFNγ-JAK/STAT axis, suggesting that some of these mechanisms are a consequence of defective IFNγ-JAK/STAT signaling, rather than independent modes of resistance.”

May combine two claims.

PD-L1 is upregulated in CD58 KO, suggesting CD58 loss could confer immune evasion directly through reduced adhesion or indirectly through inhibitory PD-L1.

“Furthermore, PD-L1 is upregulated in CD58 KO, suggesting that CD58 loss could confer immune evasion directly (reduced adhesion) or indirectly (inhibitory PD-L1)”

May combine two claims.

The study presents a large-scale CRISPR/Cas9 screen with multi-modal single-cell readouts, providing a general approach and addressing a critical clinical challenge in a patient-derived cell culture model system.

“Our study presents a large-scale CRISPR/Cas9 screen with multi-modal single-cell readouts, providing a general approach, as well as addressing a critical clinical challenge in a unique patient-derived cell culture model system.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Melanoma cells tested repeatedly negative for contamination with mycoplasma and other contaminants.

“Melanoma cells tested repeatedly negative for contamination with mycoplasma and other contaminants using PlasmoTest (InvivoGen, San Diego, CA).”

May combine two claims.

CD58 KO also impacted NK-mediated killing, which may have implications for NK cell-based therapies in tumors with loss of antigen presentation.

“CD58 KO also impacted NK-mediated killing (Fig. 5g), which may have implications for NK cell-based therapies in tumors with loss of antigen presentation.”

May combine two claims.

The permutation test partially corrects for bias from sample size and effect size in regulatory coefficients and allows direct comparison of empirical p values across targets.

“This procedure partially corrects for the bias of sample size and effect size in regulatory coefficients, and enables direct comparison of the empirical p values across targets.”

May combine two claims.

Loss of CD58 confers immune evasion to a similar extent as loss of MHC Class I expression through B2M KO, but through an independent path and without impacting antigen presentation genes and proteins.

“Our experiments and analysis show that loss of CD58 confers immune evasion to a similar extent as loss of MHC Class I expression itself (through B2M KO), but through an independent path, and without impacting the expression of antigen presentation genes and proteins”

The quote only partly states this claim.

Including cell cycle and cell complexity as covariates improved the model fit quality.

“Including these covariates improved the model fit quality (residuals approach mean zero, independent and identically distributed; Extended Data Fig. 6b,c).”

May combine two claims.

Some genes down-regulated by IFNγ pathway perturbations, such as CXCL10 and CXCL11, were previously associated with immune evasion, suggesting these do not represent salient mechanisms of immune evasion.

“Some of the genes down-regulated by IFNγ pathway perturbations, such as CXCL10 and CXCL11, are genes whose downregulation was previously associated with immune evasion, suggesting that these do not represent a salient mechanisms of immune evasion.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A full expectation-maximization approach is not guaranteed to converge to a global minimum in high-dimensional linear regression with noisy and missing data.

“A full EM approach is not guaranteed to converge to a global minimum in a high-dimensional linear regression problem with noisy and missing data36.”

• cites [36] Loh, P.-L. & Wainwright, M. J. High-dimensional regression with noisy and missing data: provable guarantees with nonconvexity. Ann. Stat. 40, 1637–1664 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Upregulation of MYC and CDK4/6 and their transcriptional targets was associated with increased resistance to immunotherapy, and CDK4/6 inhibitors reduced resistance in human and pre-clinical melanoma models.

“consistent with our previously reported patient and pre-clinical data, where we demonstrate that upregulation of MYC and CDK4/6 (and their transcriptional targets) was associated with increased resistance to immunotherapy, and CDK4/6 inhibitors reduced resistance in human and pre-clinical melanoma models9.”

• cites [9] Jerby-Arnon, L. et al. A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade. Cell 175, 984–997 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

All experiments were performed in triplicates in each of at least two independent experiments.

“All experiments were performed in triplicates in each of at least two independent experiments.”

• Also stated in: § Extended Data · plain

  “All experiments were performed in triplicates in each of at least two independent experiments.”

The quote only partly states this claim.

The CRISPR/Cas9 viability screen is reproducible across triplicates.

“Screen reproducibility across triplicates.”

May combine two claims.

The CD58/CD2 axis is a potent co-stimulatory pathway in CD8+ T cells that lack CD28, which are common in the tumor microenvironment and peripheral blood of solid tumor patients and were predominant in the Perturb-CITE-Seq screen.

“Notably, the CD58/CD2 axis is a potent co-stimulatory pathway in CD8+ T cells that lack expression of CD28 (CD8+CD28- T cells)29, which are common in the tumor-microenvironment and peripheral blood of solid tumor patients30, and were the predominant T cells in our Perturb-CITE-Seq screen”

• cites [29] Leitner, J., Herndler-Brandstetter, D., Zlabinger, G. J., Grubeck-Loebenstein, B. & Steinberger, P. CD58/CD2 is the primary costimulatory pathway in human CD28–CD8+ T cells. J. Immunol. 195, 477–487 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [30] Strioga, M., Pasukoniene, V. & Characiejus, D. CD8+CD28– and CD8+CD57+ T cells and their role in health and disease. Immunology 134, 17–32 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The study recovers the landscape of resistance mechanisms to immunotherapies and validates a novel mechanism of immune resistance.

“We recover the landscape of resistance mechanisms to immunotherapies and, guided by our high-dimensional data, validate a novel mechanism of immune resistance.”

May combine two claims.

Other genes whose perturbation enhanced immune evasion in the screen, such as CD58 and CD59, appear to reflect distinct mechanisms because their expression was not impaired by IFNγ signaling defects and their molecular phenotype following perturbation is distinct.

“In contrast, other genes whose perturbation enhanced immune evasion in our screen (e.g., CD58, CD59) appear to reflect distinct mechanism, both because their expression was not impaired by IFNγ signaling defects, and because the molecular phenotype following their perturbation is distinct.”

May combine two claims.

Reduced CD58 does not directly impair antigen presentation or JAK/STAT signaling, but lowers T cell co-stimulation and raises PD-L1-mediated co-inhibition, leading to resistance to T cell killing.

“Loss or downregulation of CD58 does not interfere with antigen presentation or JAK/STAT-signaling per se, but reduces T cell co-stimulation while simultaneously promoting increased co-inhibitory signaling via PD-L1, and therefore overall confers resistance to T cell mediated killing.”

• AssumptionRests on an assumption: The patient-derived melanoma-TIL co-culture with IFNγ pretreatment models T cell-mediated immune selection in human melanoma.