NR4A3

On track to report initial clinical and translational data from lead CAR T-cell and TIL product candidates in 2024Received Orphan Drug Designation for LYL845 for the treatment of melanomaExpect to submit IND for second generation ROR1-targeted CAR T-cell product in the first half of 2024Presented nonclinical data demonstrating the ability of Lyell’s novel genetic and epigenetic reprogramming technologies to generate potent T-cell product candidates with durable cytotoxic function at multiple scientific conferencesAdvanced manufacturing innovations to reduce cost and build scale for both CAR T-cell and TIL product candidatesCash, cash equivalents and marketable securities of $562.7 million as of December 31, 2023 supports advancing pipeline into 2027 through multiple clinical milestones SOUTH SAN FRANCISCO, Calif., Feb. 28, 2024 (GLOBE NEWSWIRE) -- Lyell Immunopharma, Inc. (Nasdaq: LYEL), a clinical‑stage T-cell reprogramming company advancing a diverse pipeline of cell therapies for patients with solid tumors today reported financial results and business highlights for the fourth quarter and year ended December 31, 2023. “We are focused on generating clinical data in our two Phase 1 clinical trials and advancing new reprogramming technologies designed to generate T cells with the ability to resist exhaustion and maintain the stem-like qualities needed to drive durable cytotoxic functionality in solid tumors. This year, we plan to share clinical and translational data from our lead CAR T-cell and TIL product candidates that we expect will provide the first of several opportunities to understand the potential of our T-cell preprogramming technologies to deliver meaningful advances in cell therapy for patients with solid tumors,” said Lynn Seely, M.D., Lyell’s President and CEO. “With our strong cash position that is expected to fund operations into 2027, we can advance our lead programs through multiple clinical milestones while continuing to invest in innovative technology platforms and earlier stage product candidates.” Fourth Quarter Updates and Recent Business Highlights Lyell is advancing four wholly-owned product candidates. Two product candidates, LYL797 and LYL845 are in Phase 1 clinical development. Two additional product candidates, LYL119 and a second-generation tumor infiltrating lymphocyte (TIL) product candidate, are in preclinical development and our T-cell rejuvenation technology is in research. LYL797 – A ROR1-targeted Chimeric Antigen Receptor (CAR) T-cell product candidate genetically reprogrammed to overexpress c-Jun and epigenetically reprogrammed using Lyell’s proprietary Epi-RTM manufacturing protocol, designed for differentiated potency and durability Enrollment in the Phase 1 clinical trial of LYL797 is ongoing. The study includes patients with relapsed or refractory triple-negative breast cancer (TNBC) or non-small cell lung cancer (NSCLC).Initial clinical and translational data from at least 20 patients in the Phase 1 trial of LYL797 are expected in the first half of 2024.Initiated a CAR T-cell manufacturing proof-of-concept collaboration with Cellares as part of an overall manufacturing strategy to build scale and reduce cost. Under the collaboration, the companies have agreed on a proof-of-concept technology transfer process for the manufacture of Lyell’s LYL797 CAR T-cell therapy, using Cellares’ Cell ShuttleTM.Announced initial results from Lyell’s ROR1 screening program indicating that expression of ROR1 in TNBC and NSCLC, 53% (N=77) and 33% (N=18), respectively, is consistent with what has been reported in the literature. The screening program is designed to support Lyell’s current and future clinical trials.Presented a LYL797 Trial in Progress poster at the 38th Annual Meeting of the Society for Immunotherapy of Cancer (SITC). LYL845 – A novel epigenetically reprogrammed TIL product candidate using Lyell’s proprietary Epi-RTM manufacturing protocol, designed for differentiated potency and durability Enrollment in the Phase 1 clinical trial for LYL845 is ongoing. The study includes patients with relapsed and/or refractory metastatic or locally advanced melanoma, NSCLC and colorectal cancer.Initial clinical and translational data from the Phase 1 trial of LYL845 are expected in the second half of 2024.Received FDA Orphan Drug Designation for LYL845 for the treatment of stage IIB-IV melanoma.Presented nonclinical data at SITC highlighting the Epi-R P2 manufacturing process, which is designed to shorten TIL manufacturing time to less than three weeks without impacting cell number and phenotype. Epi-R P2 is expected to be incorporated into the Phase 1 trial of LYL845 in 2024.Presented a LYL845 Trial in Progress poster at SITC. LYL119 – A ROR1-targeted CAR T-cell product candidate incorporating Lyell’s four stackable and complementary reprogramming technologies for enhanced cytotoxicity LYL119 is a ROR1-targeted CAR T-cell product enhanced with Lyell’s four novel genetic and epigenetic reprogramming technologies: c-Jun overexpression, NR4A3 knockout, Epi-R manufacturing protocol and Stim‑RTM T-cell activation technology.An investigational new drug (IND) application for LYL119 is expected to be submitted in the first half of 2024.Presented posters highlighting preclinical development of LYL119 at the American Society for Gene and Cell Therapy (ASGCT) and at SITC. In preclinical studies, LYL119 demonstrated superior cytotoxicity and sustained cytokine production upon repeated antigen stimulation compared to various controls lacking one or more of the reprogramming technologies and showed robust in vivo antitumor efficacy and prolonged survival in a mouse xenograft tumor model at very low cell doses. Rejuvenation – Novel partial reprogramming technology designed to maintain T-cell identity while reducing cells’ epigenetic age Presented nonclinical data at the International Society for Stem Cell Research (ISSCR) 2023 Annual Meeting demonstrating that Lyell’s T-cell Rejuvenation technology generates cells with improved expansion capacity and increased expression of biomarkers associated with T-cell stemness, that also exhibit improved antitumor properties compared with non-rejuvenated T-cell controls in sequential cell-killing assays.Presented nonclinical data at SITC demonstrating that TIL generated with Lyell’s Rejuvenation technology retain a broad TCR repertoire and demonstrate improved T-cell function and antitumor properties. Corporate Updates Appointed Matt Lang, J.D., Chief Business Officer. Mr. Lang, who also serves as Lyell’s Chief Legal Officer and Corporate Secretary, is an experienced company builder who has successfully led growth in complex organizations. Fourth Quarter and Full Year 2023 Financial Results Lyell reported a net loss of $52.9 million and $234.6 million for the fourth quarter and year ended December 31, 2023, respectively, compared to a net loss of $8.4 million and $183.1 million for the same periods in 2022. Non‑GAAP net loss, which excludes non-cash stock-based compensation, non-cash expenses related to the change in the estimated fair value of success payment liabilities and certain non-cash investment gains and charges, was $43.9 million and $177.4 million for the fourth quarter and year ended December 31, 2023, respectively, compared to $0.3 million and $104.2 million for the same periods in 2022. Revenue Revenue was approximately zero and $0.1 million for the fourth quarter and year ended December 31, 2023, respectively, compared to $48.4 million and $84.7 million for the same periods in 2022. No research and development pursuant to our collaboration and license agreement with GlaxoSmithKline (GSK Agreement) was performed in 2023 due to the termination of the GSK Agreement in December 2022, which drove the decrease in revenue. GAAP and Non-GAAP Operating Expenses Research and development (R&D) expenses were $47.0 million and $182.9 million for the fourth quarter and year ended December 31, 2023, respectively, compared to $38.0 million and $159.2 million for the same periods in 2022. The increase in fourth quarter 2023 R&D expenses of $9.0 million was primarily driven by non-cash expenses related to the change in the estimated fair value of success payment liabilities. The increase in annual 2023 R&D expenses of $23.8 million was primarily driven by a $11.2 million increase in personnel-related expenses, including $4.6 million for one-time severance payments and other employee-related costs in connection with the reduction in workforce, and an increase of $8.8 million in research activities, collaborations and outside services costs primarily due to research and laboratory costs associated with clinical trials. Non‑GAAP R&D expenses, which exclude non-cash stock-based compensation and non-cash expenses related to the change in the estimated fair value of success payment liabilities for the fourth quarter and year ended December 31, 2023, were $42.9 million and $165.7 million, respectively, compared to $41.0 million and $147.6 million for the same periods in 2022. The increase in fourth quarter 2023 non-GAAP R&D expenses was driven by an increase in research and laboratory costs primarily associated with clinical trials.General and administrative (G&A) expenses were $13.2 million and $67.0 million for the fourth quarter and year ended December 31, 2023, respectively, compared to $26.3 million and $117.3 million for the same periods in 2022. The decrease in both fourth quarter 2023 and annual 2023 G&A expenses were both primarily driven by decreases in non-cash stock-based compensation. Non‑GAAP G&A expenses, which exclude non-cash stock‑based compensation, for the fourth quarter and year ended December 31, 2023 were $8.5 million and $38.1 million, respectively, compared to $12.3 million and $52.1 million for the same periods in 2022. The decrease in 2023 non-GAAP G&A expenses was driven by a decrease in legal and corporate expenses. A discussion of non-GAAP financial measures, including reconciliations of the most comparable GAAP measures to non‑GAAP financial measures, is presented below under “Non-GAAP Financial Measures.” Cash, cash equivalents and marketable securities Cash, cash equivalents and marketable securities as of December 31, 2023 were $562.7 million compared to $710.3 million as of December 31, 2022. Lyell believes that its cash, cash equivalents and marketable securities balances will be sufficient to meet working capital and capital expenditure needs into 2027. About Lyell Immunopharma, Inc. Lyell is a clinical-stage T-cell reprogramming company advancing a diverse pipeline of cell therapies for patients with solid tumors. Lyell is currently enrolling a Phase 1 clinical trial evaluating a ROR1-targeted CAR T-cell therapy in patients with relapsed refractory triple-negative breast cancer and non-small cell lung cancer (NSCLC) and a second Phase 1 clinical trial evaluating reprogrammed tumor infiltrating lymphocytes (TIL) in patients with advanced melanoma, NSCLC and colorectal cancer. The technologies powering its product candidates are designed to address barriers that limit consistent and long-lasting responses to cell therapy for solid tumors: T-cell exhaustion and lack of durable stemness, which includes the ability to persist and self-renew to drive durable tumor cytotoxicity. Lyell is applying its proprietary ex vivo genetic and epigenetic reprogramming technologies to address these barriers in order to develop new medicines with improved durable clinical outcomes. Lyell is based in South San Francisco, California with facilities in Seattle and Bothell, Washington. To learn more, please visit www.lyell.com. Forward-Looking Statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements expressed or implied in this press release include, but are not limited to, statements regarding: Lyell’s anticipated progress, business plans, business strategy and clinical trials; Lyell’s advancement of its pipeline and its research, development and clinical capabilities; the potential clinical benefits and therapeutic potential of Lyell’s product candidates; the advancement of Lyell’s technology platform; Lyell’s expectation that its financial position and cash runway will support advancement of its pipeline through multiple clinical milestones into 2027; Lyell’s plans to submit an IND for LYL119 and the timing thereof; expectations around enrollment and timing of initial clinical and translational data from Lyell’s Phase 1 trials for LYL797 and LYL845; the potential of Lyell’s technology transfer process with Cellares to build scale and reduce cost of manufacturing; and other statements that are not historical fact. These statements are based on Lyell’s current plans, objectives, estimates, expectations and intentions, are not guarantees of future performance and inherently involve significant risks and uncertainties. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, but are not limited to, risks and uncertainties related to: the effects of geopolitical instability; macroeconomic conditions, including actual or perceived changes in interest rates and economic inflation; Lyell’s ability to submit planned INDs or initiate or progress clinical trials on the anticipated timelines, if at all; Lyell’s limited experience as a company in enrolling and conducting clinical trials, and lack of experience in completing clinical trials; Lyell’s ability to manufacture and supply its product candidates for its clinical trials; the nonclinical profiles of Lyell’s product candidates not translating in clinical trials; the potential for results from clinical trials to differ from nonclinical, early clinical, preliminary or expected results; significant adverse events, toxicities or other undesirable side effects associated with Lyell’s product candidates; the significant uncertainty associated with Lyell’s product candidates ever receiving any regulatory approvals; Lyell’s ability to obtain, maintain or protect intellectual property rights related to its product candidates; implementation of Lyell’s strategic plans for its business and product candidates; the sufficiency of Lyell’s capital resources and need for additional capital to achieve its goals; anticipated benefits and financial impact of Lyell’s workforce restructuring; and other risks, including those described under the heading “Risk Factors” in Lyell’s Quarterly Report on Form 10-Q for the quarter ended September 30, 2023, filed with the Securities and Exchange Commission (SEC) on November 7, 2023, and its Annual Report on Form 10-K for the year ended December 31, 2023, being filed with the SEC later today. Forward-looking statements contained in this press release are made as of this date, and Lyell undertakes no duty to update such information except as required under applicable law. Lyell Immunopharma, Inc.Unaudited Selected Consolidated Financial Data(in thousands) Statement of Operations Data: Three Months Ended December 31, Year Ended December 31, 2023 2022 2023 2022 Revenue$13 $48,386 $130 $84,683 Operating expenses: Research and development(1) 46,995 38,032 182,945 159,188 General and administrative 13,167 26,348 66,983 117,307 Other operating income, net (641) (1,210) (2,790) (4,754)Total operating expenses 59,521 63,170 247,138 271,741 Loss from operations (59,508) (14,784) (247,008) (187,058)Interest income, net 7,084 3,453 23,453 7,053 Other income (expense), net(1) (506) 2,934 1,846 1,887 Impairment of other investments — — (12,923) (5,000)Total other income (loss), net 6,578 6,387 12,376 3,940 Net loss$(52,930) $(8,397) $(234,632) $(183,118) (1)As of December 31, 2022, the Company’s success payment liability was recognized at fair value as Fred Hutch had provided the requisite service obligation to earn the potential success payment consideration under the continued collaboration. The change in the estimated fair value of Fred Hutch success payment liabilities beginning in Q1 2023 was recognized within other income (expense), net in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. The change in the estimated fair value of Fred Hutch success payment liabilities in 2022 was recognized within research and development expenses in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. Balance Sheet Data: As of December 31, 2023 2022 Cash, cash equivalents and marketable securities$ 562,729 $ 710,269 Property and equipment, net$ 102,654 $ 123,023 Total assets$ 750,029 $ 937,561 Total stockholders’ equity$ 654,952 $ 833,252 Non-GAAP Financial Measures To supplement our financial results and guidance presented in accordance with U.S. generally accepted accounting principles (GAAP), we present non-GAAP net loss, non-GAAP R&D expenses and non-GAAP G&A expenses. Non‑GAAP net loss and non-GAAP R&D expenses exclude non-cash stock-based compensation expense and non-cash expenses related to the change in the estimated fair value of success payment liabilities from GAAP net loss and GAAP R&D expenses. Non-GAAP net loss further adjusts non‑cash investment gains and charges, as applicable. Non‑GAAP G&A expenses exclude non-cash stock-based compensation expense from GAAP G&A expenses. We believe that these non-GAAP financial measures, when considered together with our financial information prepared in accordance with GAAP, can enhance investors’ and analysts’ ability to meaningfully compare our results from period to period, and to identify operating trends in our business. We have excluded stock-based compensation expense, changes in the estimated fair value of success payment liabilities and non-cash investment gains and charges from our non‑GAAP financial measures because they are non-cash gains and charges that may vary significantly from period to period as a result of changes not directly or immediately related to the operational performance for the periods presented. We also regularly use these non-GAAP financial measures internally to understand, manage and evaluate our business and to make operating decisions. These non-GAAP financial measures are in addition to, and not a substitute for or superior to, measures of financial performance prepared in accordance with GAAP. In addition, these non-GAAP financial measures have no standardized meaning prescribed by GAAP and are not prepared under any comprehensive set of accounting rules or principles and, therefore, have limits in their usefulness to investors. We encourage investors to carefully consider our results under GAAP, as well as our supplemental non-GAAP financial information, to more fully understand our business. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP Net Loss(in thousands) Three Months Ended December 31, Year Ended December 31, 2023 2022 2023 2022 Net loss - GAAP$(52,930) $(8,397) $(234,632) $(183,118)Adjustments: Stock-based compensation expense 8,463 18,363 47,084 81,924 Change in the estimated fair value of success payment liabilities 529 (7,307) (2,780) (5,130)Impairment of other investments — — 12,923 5,000 Gain on other investments — (2,923) — (2,923)Net loss - Non-GAAP(1)$(43,938) $(264) $(177,405) $(104,247) (1)There was no income tax effect related to the adjustments made to calculate non-GAAP net loss because of the full valuation allowance on our net U.S. deferred tax assets for all periods presented. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP Research and Development Expenses(in thousands) Three Months Ended December 31, Year Ended December 31, 2023 2022 2023 2022 Research and development - GAAP$46,995 $38,032 $182,945 $159,188 Adjustments: Stock-based compensation expense (3,768) (4,320) (18,207) (16,721)Change in the estimated fair value of success payment liabilities(1) (319) 7,307 930 5,130 Research and development - Non-GAAP$42,908 $41,019 $165,668 $147,597 (1)As of December 31, 2022, the Company’s success payment liability was recognized at fair value as Fred Hutch had provided the requisite service obligation to earn the potential success payment consideration under the continued collaboration. The change in the estimated fair value of Fred Hutch success payment liabilities beginning in Q1 2023 was recognized within other income (expense), net in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. The change in the estimated fair value of Fred Hutch success payment liabilities in 2022 was recognized within research and development expenses in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP General and Administrative Expenses(in thousands) Three Months Ended December 31, Year Ended December 31, 2023 2022 2023 2022General and administrative - GAAP$13,167 $26,348 $66,983 $117,307 Adjustments: Stock-based compensation expense (4,695) (14,043) (28,877) (65,203)General and administrative - Non-GAAP$8,472 $12,305 $38,106 $52,104 Contact: Ellen Rose Senior Vice President, Communications and Investor Relations erose@lyell.com

Extended funding of operations into 2027 by prioritizing investment in core clinical candidates and research platform value drivers as well as de-prioritizing selected early-stage research programsRemain on track to release initial clinical data from two clinical-stage product candidates in 2024Entered into proof-of-concept manufacturing collaboration with Cellares for CAR T cell product candidatePresented six abstracts at SITC highlighting innovations designed to shorten TIL manufacturing, new nonclinical data on LYL119, new technologies and the design of ongoing clinical trials for LYL797 and LYL845Cash, cash equivalents and marketable securities of $598.2 million as of September 30, 2023 supports advancing multiple product candidates through key clinical milestones SOUTH SAN FRANCISCO, Calif., Nov. 07, 2023 (GLOBE NEWSWIRE) -- Lyell Immunopharma, Inc. (Nasdaq: LYEL), a clinical‑stage T‑cell reprogramming company advancing a diverse pipeline of cell therapies for patients with solid tumors, today reported financial results and business highlights for the third quarter ended September 30, 2023. “We remain on track to present initial data from each of our two lead Phase 1 programs next year and remain confident in our science, our product candidates and our ability to deliver meaningful advances in cell therapy to patients with solid tumors,” said Lynn Seely, M.D., Lyell’s President and CEO. “As we continue to generate clinical data from our two lead programs, we have also taken important steps toward reducing costs and manufacturing time for cell therapies through our proof-of-concept CAR T-cell manufacturing collaboration with Cellares and advances in our Epi-R P2 manufacturing process. We are focused on strengthening our ability to fully elucidate the potential of the currently disclosed product candidates in our pipeline through multiple clinical milestones and have extended our ability to fund operations into 2027. We have restructured our company to prioritize investment in our clinical stage programs and core research platforms and have streamlined operations. In doing so we are parting ways with talented and valued Lyellites. Tina Albertson, M.D., Ph.D., our Chief Medical Officer is also leaving the company. We thank our departing colleagues for their many contributions that have helped advance our mission.” With key infrastructure now in place and a maturing company focus, the company is prioritizing investment in its most value enhancing clinical stage product candidates and core research platforms and has scaled down investment in certain early-stage research programs and stage gated certain other expenses. An approximately 25% reduction in workforce associated with this prioritization is expected to be completed in the fourth quarter of 2023. Third Quarter Updates and Recent Business Highlights Lyell is advancing four wholly-owned product candidates with two product candidates in Phase 1 clinical development, LYL797 and LYL845. Two additional product candidates, LYL119 and a second-generation tumor infiltrating lymphocyte (TIL) product candidate, are in preclinical development. LYL797 – A ROR1-targeted Chimeric Antigen Receptor (CAR) T-cell product candidate genetically reprogrammed to overexpress c-Jun and epigenetically reprogrammed using Lyell’s proprietary Epi-R™ manufacturing protocol, designed for differentiated potency and durability Enrollment in the Phase 1 clinical trial of LYL797 is ongoing. The study includes patients with relapsed or refractory triple-negative breast cancer (TNBC) or non-small cell lung cancer (NSCLC).Initial clinical and translational data from at least 20 patients in the Phase 1 trial of LYL797 are expected in the first half of 2024.Initiated a CAR T-cell manufacturing proof-of-concept collaboration with Cellares as part of an overall manufacturing strategy to build scale and reduce cost. Under the collaboration, the companies have agreed on a proof-of-concept technology transfer process for the manufacture of Lyell’s LYL797 CAR T-cell therapy, using Cellares’ Cell Shuttle™.Announced initial results from Lyell’s ROR1 screening program indicating that expression of ROR1 in TNBC and NSCLC, 53% (N=77) and 33% (N=18), respectively, is consistent with what has been reported in the literature. The screening program is designed to support Lyell’s current and future clinical trials.LYL797 Trial in Progress poster was presented at the 38th Annual Meeting of the Society for Immunotherapy of Cancer (SITC) in San Diego on November 1-5, 2023. LYL845 – A novel epigenetically reprogrammed TIL product candidate designed for differentiated potency and durability Enrollment in the Phase 1 clinical trial for LYL845 is ongoing. The study includes patients with relapsed and/or refractory metastatic or locally advanced melanoma, NSCLC and colorectal cancer.Initial clinical data from the Phase 1 trial of LYL845 are expected in 2024.Preclinical data on the Epi-R P2 manufacturing process designed to shorten TIL manufacturing time without impacting cell number and phenotype were presented at SITC. Epi-R P2 is expected to be incorporated into the Phase 1 trial in 2024.LYL845 Trial in Progress poster was presented at SITC. LYL119 – A ROR1-targeted CAR T-cell product candidate incorporating Lyell’s four stackable reprogramming technologies for enhanced cytotoxicity LYL119 is a ROR1-targeted CAR T-cell product enhanced with Lyell’s four novel, stackable genetic and epigenetic reprogramming technologies: c-Jun overexpression, NR4A3 knockout, Epi-R manufacturing protocol and Stim-R™ T-cell activation technology.An investigational new drug (IND) application for LYL119 is expected to be submitted in the first half of 2024.An abstract highlighting preclinical development of LYL119 was presented at SITC. Rejuvenation – Novel partial reprogramming technology designed to maintain T-cell identity while reducing cells’ epigenetic age An abstract highlighting rejuvenation of TIL through partial reprogramming was presented at SITC. Third Quarter Financial Results Lyell reported a net loss of $50.9 million for the third quarter ended September 30, 2023, compared to a net loss of $70.3 million for the same period in 2022. Non‑GAAP net loss, which excludes non-cash stock-based compensation, changes in the estimated fair value of success payment liabilities and certain non-cash investment charges, was $43.0 million for the third quarter ended September 30, 2023, compared to $43.7 million for the same period in 2022. Revenue Revenue was approximately zero for both the third quarter ended September 30, 2023 and 2022. No research and development pursuant to our collaboration and license agreement with GlaxoSmithKline (GSK Agreement) was performed in the third quarter of 2023 due to the termination of the GSK Agreement in December 2022. GAAP and Non-GAAP Operating Expenses Research and development (R&D) expenses were $43.8 million for the third quarter ended September 30, 2023, compared to $41.6 million for the same period in 2022. The increase in third quarter 2023 R&D expenses was primarily driven by personnel-related expenses, mainly due to an increase in headcount to expand our research, development and manufacturing capabilities to support increases in clinical trial enrollment, and an increase in research and laboratory costs primarily associated with clinical trials. Non‑GAAP R&D expenses, which exclude non-cash stock-based compensation and changes in the estimated fair value of success payment liabilities, for the third quarter ended September 30, 2023, were $40.5 million, compared to $34.7 million for the same period in 2022. The increase in third quarter 2023 non-GAAP R&D expenses was driven by increased personnel-related expenses, primarily related to an increase in headcount to expand our clinical development and manufacturing capabilities in support of our ongoing clinical trials.General and administrative (G&A) expenses were $15.5 million for the third quarter ended September 30, 2023, compared to $26.1 million for the same period in 2022. The decrease in third quarter 2023 G&A expenses was primarily driven by changes in non-cash stock-based compensation. Non‑GAAP G&A expenses, which exclude non-cash stock-based compensation, for the third quarter ended September 30, 2023, were $9.5 million, compared to $11.4 million for the same period in 2022. The decrease in third quarter 2023 non-GAAP G&A expenses was driven by a decrease in legal, consulting and other administrative expenses. A discussion of non-GAAP financial measures, including reconciliations of the most comparable GAAP measures to non‑GAAP financial measures, is presented below under “Non-GAAP Financial Measures.” Cash, cash equivalents and marketable securities Cash, cash equivalents and marketable securities as of September 30, 2023 were $598.2 million, compared to $710.3 million as of December 31, 2022. Lyell believes that its cash, cash equivalents and marketable securities balances will now be sufficient to meet working capital and capital expenditure needs into 2027 while continuing to advance its clinical programs. About Lyell Immunopharma, Inc. Lyell is a clinical-stage T-cell reprogramming company advancing a diverse pipeline of cell therapies for patients with solid tumors. Lyell is currently enrolling a Phase 1 clinical trial evaluating a ROR1-targeted CAR T-cell therapy in patients with relapsed refractory triple-negative breast cancer and non-small cell lung cancer (NSCLC) and a second Phase 1 clinical trial evaluating reprogrammed tumor infiltrating lymphocytes (TIL) in patients with advanced melanoma, NSCLC and colorectal cancer. The technologies powering its product candidates are designed to address barriers that limit consistent and long-lasting responses to cell therapy for solid tumors: T-cell exhaustion and lack of durable stemness, which includes the ability to persist and self-renew to drive durable tumor cytotoxicity. Lyell is applying its proprietary ex vivo genetic and epigenetic reprogramming technologies to address these barriers in order to develop new medicines with improved durable clinical outcomes. Lyell is based in South San Francisco, California with facilities in Seattle and Bothell, Washington. To learn more, please visit www.lyell.com. Forward-Looking Statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements expressed or implied in this press release include, but are not limited to, statements regarding: Lyell’s anticipated progress, business plans, business strategy and clinical trials; Lyell’s advancement of its pipeline and its research, development and clinical capabilities; the potential clinical benefits and therapeutic potential of Lyell’s product candidates; the advancement of Lyell’s technology platform; Lyell’s expectation that its financial position and cash runway will support advancement of its pipeline through multiple clinical milestones into 2027; Lyell’s expectations related to its planned workforce restructuring in the fourth quarter of 2023; Lyell’s plans to submit an IND for LYL119 and the timing thereof; expectations around enrollment and timing of initial clinical data from Lyell’s Phase 1 trials for LYL797 and LYL845; the potential of Lyell’s partnership with Cellares to reduce cost and manufacturing time for CAR T-cell and the effect of the steps Lyell has taken to reduce cost and manufacturing time for Epi-R P2; and other statements that are not historical fact. These statements are based on Lyell’s current plans, objectives, estimates, expectations and intentions, are not guarantees of future performance and inherently involve significant risks and uncertainties. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, but are not limited to, risks and uncertainties related to: the effects of geopolitical instability; macroeconomic conditions and the lingering effects of the COVID-19 pandemic; Lyell’s ability to submit planned INDs or initiate or progress clinical trials on the anticipated timelines, if at all; Lyell’s limited experience as a company in enrolling and conducting clinical trials, and lack of experience in completing clinical trials; Lyell’s ability to manufacture and supply its product candidates for its clinical trials; the nonclinical profiles of Lyell’s product candidates not translating in clinical trials; the potential for results from clinical trials to differ from nonclinical, early clinical, preliminary or expected results; significant adverse events, toxicities or other undesirable side effects associated with Lyell’s product candidates; the significant uncertainty associated with Lyell’s product candidates ever receiving any regulatory approvals; Lyell’s ability to obtain, maintain or protect intellectual property rights related to its product candidates; implementation of Lyell’s strategic plans for its business and product candidates; the sufficiency of Lyell’s capital resources and need for additional capital to achieve its goals; Lyell’s ability to successfully implement the workforce restructuring; and other risks, including those described under the heading “Risk Factors” in its Annual Report on Form 10-K for the year ended December 31, 2022, filed with the Securities and Exchange Commission (SEC) on February 28, 2023, and the Quarterly Report on Form 10-Q for the quarter ended September 30, 2023, being filed with the SEC today. Forward-looking statements contained in this press release are made as of this date, and Lyell undertakes no duty to update such information except as required under applicable law. Lyell Immunopharma, Inc.Unaudited Selected Consolidated Financial Data(in thousands) Statement of Operations Data: Three Months Ended September 30, Nine Months Ended September 30, 2023 2022 2023 2022 Revenue$25 $3 $117 $36,297 Operating expenses: Research and development(1) 43,849 41,607 135,950 121,156 General and administrative 15,507 26,084 53,816 90,959 Other operating income, net (292) (1,251) (2,149) (3,544)Total operating expenses 59,064 66,440 187,617 208,571 Loss from operations (59,039) (66,437) (187,500) (172,274)Interest income, net 6,608 2,251 16,369 3,600 Other income (expense), net(1) 1,578 (1,068) 2,352 (1,047)Impairment of other investments — (5,000) (12,923) (5,000)Total other income (loss), net 8,186 (3,817) 5,798 (2,447)Net loss$(50,853) $(70,254) $(181,702) $(174,721) (1) As of December 31, 2022, the Company’s success payment liability was recognized at fair value as Fred Hutchinson Cancer Center (Fred Hutch) had provided the requisite service obligation to earn the potential success payment consideration under the continued collaboration. The change in the estimated fair value of Fred Hutch success payment liabilities beginning in Q1 2023 was recognized within other income (expense), net in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. The change in the estimated fair value of Fred Hutch success payment liabilities in 2022 was recognized within research and development expenses in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. Balance Sheet Data: As of September 30, 2023 As of December 31, 2022 Cash, cash equivalents and marketable securities$598,160 $710,269Property and equipment, net$108,096 $123,023Total assets$794,989 $937,561Total stockholders’ equity$697,573 $833,252 Non-GAAP Financial Measures To supplement our financial results and guidance presented in accordance with U.S. generally accepted accounting principles (GAAP), we present non-GAAP net loss, non-GAAP R&D expenses and non-GAAP G&A expenses. Non‑GAAP net loss and non-GAAP R&D expenses exclude non-cash stock-based compensation expense and non-cash expenses related to the change in the estimated fair value of success payment liabilities from GAAP net loss and GAAP R&D expenses. Non-GAAP net loss further adjusts non‑cash investment gains and charges, as applicable. Non‑GAAP G&A expenses exclude non-cash stock-based compensation expense from GAAP G&A expenses. We believe that these non-GAAP financial measures, when considered together with our financial information prepared in accordance with GAAP, can enhance investors’ and analysts’ ability to meaningfully compare our results from period to period, and to identify operating trends in our business. We have excluded stock-based compensation expense, changes in the estimated fair value of success payment liabilities and non-cash investment gains and charges from our non‑GAAP financial measures because they are non-cash gains and charges that may vary significantly from period to period as a result of changes not directly or immediately related to the operational performance for the periods presented. We also regularly use these non-GAAP financial measures internally to understand, manage and evaluate our business and to make operating decisions. These non-GAAP financial measures are in addition to, and not a substitute for or superior to, measures of financial performance prepared in accordance with GAAP. In addition, these non-GAAP financial measures have no standardized meaning prescribed by GAAP and are not prepared under any comprehensive set of accounting rules or principles and, therefore, have limits in their usefulness to investors. We encourage investors to carefully consider our results under GAAP, as well as our supplemental non-GAAP financial information, to more fully understand our business. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP Net Loss(in thousands) Three Months Ended September 30, Nine Months Ended September 30, 2023 2022 2023 2022 Net loss - GAAP$ (50,853) $ (70,254) $ (181,702) $ (174,721)Adjustments: Stock-based compensation expense 10,516 19,123 38,621 63,561 Change in the estimated fair value of success payment liabilities (2,706) 2,441 (3,309) 2,177 Impairment of other investments — 5,000 12,923 5,000 Net loss - Non-GAAP(1)$ (43,043) $ (43,690) $ (133,467) $ (103,983) (1) There was no income tax effect related to the adjustments made to calculate non-GAAP net loss because of the full valuation allowance on our net U.S. deferred tax assets for all periods presented. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP Research and Development Expenses(in thousands) Three Months Ended September 30, Nine Months Ended September 30, 2023 2022 2023 2022 Research and development expense - GAAP$43,849 $41,607 $135,950 $121,156 Adjustments: Stock-based compensation expense (4,548) (4,442) (14,439) (12,401)Change in the estimated fair value of success payment liabilities(1) 1,246 (2,441) 1,249 (2,177)Research and development expense - Non‑GAAP$40,547 $34,724 $122,760 $106,578 (1) As of December 31, 2022, the Company’s success payment liability was recognized at fair value as Fred Hutch had provided the requisite service obligation to earn the potential success payment consideration under the continued collaboration. The change in the estimated fair value of Fred Hutch success payment liabilities beginning in Q1 2023 was recognized within other income (expense), net in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. The change in the estimated fair value of Fred Hutch success payment liabilities in 2022 was recognized within research and development expenses in the unaudited Condensed Consolidated Statements of Operations and Comprehensive Loss. Lyell Immunopharma, Inc.Unaudited Reconciliation of GAAP to Non-GAAP General and Administrative Expenses(in thousands) Three Months Ended September 30, Nine Months Ended September 30, 2023 2022 2023 2022 General and administrative expense - GAAP$15,507 $26,084 $53,816 $90,959 Adjustments: Stock-based compensation expense (5,968) (14,681) (24,182) (51,160)General and administrative expense - Non‑GAAP$9,539 $11,403 $29,634 $39,799 Contact:Ellen RoseSenior Vice President, Communications and Investor Relationserose@lyell.com

不知道大家发现没有，一些研究方向跟着大牛肯定是没有任何问题的，比如之前推广过的张泽民老师的中性粒细胞，组织驻留记忆T细胞。这次终于卷到NK细胞了，敲黑板，5+IF文章，爱要不要。NK细胞在肿瘤的预后，治疗中发挥作用巨大，这一次又是大佬叠加cell推荐，必然会成为文章发表的新热点，大家一定要引起关注。什么？有人担心结果做不出来？大佬都总结好了！秘密就在图片中！图中的排序就是肿瘤相关NK细胞占比！肝癌、结肠癌、甲状腺癌、胰腺癌和头颈部鳞状细胞癌荣获前五！这五个方向科研人冲就对了！大家好，最近张泽民教授又出新成果了，团队成员联合中国科学技术大学彭慧教授课题组，在《Cell》杂志上发表了题目为“A pan-cancer single-cell panorama of human natural killer cells”的论文，该研究收集整理了大量公开的单细胞转录组数据，涉及24种癌症类型，包括来自716名患者的1223个样本的NK细胞单细胞表达谱数据，首次在泛癌水平系统地鉴定到了5类CD56 bright CD16 lo 和9类CD56 dim CD16 hi NK细胞亚群，并详细地刻画了各类群的表型和功能多样性。数据来源1. 大量公开的单细胞转录组数据公开发表的70个数据集的scRNA-seq数据，来自669名患者的1146个样本，包括肿瘤、邻近非肿瘤组织、外周血和其他组织(如淋巴结)。2.新生成的scRNA-seq数据作者从其团队之前的研究中收集和分析了来自47名癌症患者的77个样本，涉及8种癌症类型。分析流程将公开数据库的数据和研究团队新生成的scRNA-seq数据，进行整合，得到来自共计716名患者的1223个样本的NK细胞单细胞表达谱数据，涉及24种癌症类型，本研究最终获得了160,011个高质量NK细胞，其中包括11,963个新生成的NK细胞。分析流程图结果解读1.在单细胞水平上构建人类泛癌NK细胞景观为了更好地定义NK细胞的泛癌群体结构，作者整合了数据集之间批次效应最小的scRNA-seq数据，并进行了两轮无监督聚类。第一轮分析是基于高表达的典型细胞标记NCAM1和FCGR3A，分别对应于先前报道的“NK_1”和“NK_2”群体，区分两种特征明确的主要细胞类型为CD56brightCD16lo和CD56dimCD16hi。在第二轮聚类中，CD56brightCD16lo区可以进一步细分为5个细胞群， CD56dimCD16hi区进一步被确定为9个细胞群，其中包括同时表达高水平的NCAM1和FCGR3A的CD56brightCD16hi NK细胞，其表现出介于CD56brightCD16lo和CD56dimCD16hi NK细胞之间的中间特征，表明不同的细胞亚群具有不同的转录表型。接下来，作者分析了所有亚群的组织分布，观察到不同的组织富集模式，表明本研究的综合分析可以保留不同组织的异质性。为了进一步证实聚类的稳定性，作者还将血液、肿瘤和邻近非肿瘤组织中的NK细胞分别重新聚类，聚类结果高度一致。此外，作者还发现不同发育状态的NK细胞亚群在肿瘤中同时存在，表明NK细胞向肿瘤的迁移可能与NK细胞成熟解耦。        作者进一步检查了基因表达特征，以破译不同细胞亚群之间的功能差异。研究发现，CD56dimCD16hi NK细胞高表达细胞毒性效应基因，包括穿孔素(PRF1)和除GZMK外的大多数颗粒酶(GZMB、GZMA和GZMH)，而GZMK只在CD56brightCD16lo NK细胞中表达，CD56brightCD16lo NK细胞还表达多种细胞因子基因，如IL-8等。此外，作者还描述了NK细胞亚群中激活或抑制受体的表达情况，结果表明不同细胞亚群的转录和功能表型存在显著差异。   总之，作者在单细胞水平上构建了人类泛癌NK细胞的详细转录组谱，深刻剖析了不同NK细胞亚群的分子特异性，并揭示了其未被重视的异质性。图1 NK细胞泛癌单细胞图谱及其特征2. 不同肿瘤类型NK细胞的组织异质性接下来，作者评估了不同癌症类型中肿瘤浸润NK细胞群的偏好，观察到不同癌症类型中，NK细胞亚群的分布存在明显差异。如，未成熟的CD56brightCD16lo NK细胞在鼻咽癌和基底细胞癌中占主导地位，而成熟的CD56dimCD16hi NK细胞在肾癌和肺癌中占主导地位，这与先前的报道一致。其他如结直肠癌和肝癌中没有明显的差异(图2A)。 为了进一步检验上述现象是否可以用器官特异性来解释，作者分析了邻近非肿瘤组织中主要NK细胞类型的内在组成及其在肿瘤中的相应变化。结果发现，在包括肺癌、肾癌在内的多种癌症类型中，CD56dimCD16hi NK细胞在肿瘤组织中的比例明显降低(图2B)，乳腺癌和食管癌等癌症类型中的优势NK细胞群发生了逆转(图2C)。这些观察结果表明，器官特异性和肿瘤因素共同影响NK细胞群的分布。作者进一步从细胞亚群的角度探索NK细胞的癌症类型特异性，根据NK细胞亚群比例对所分析的癌症类型进行分层。结果发现，NK细胞亚群如c2-CX3CR1在胰腺癌、乳腺癌和黑色素瘤中表现出强烈的偏好。在某些癌症类型中观察到高变异性，如c4-NFKBIA和c7-NR4A3从头颈部鳞状细胞癌和甲状腺癌到食管癌和鼻咽癌的中位数频率显著降低(图2D)。 此外，作者还发现，罕见CD56brightCD16hi NK细胞亚群在黑色素瘤和白血病中大量存在，特别是在急性髓性白血病(AML)中(图2D)，与AML患者的总生存率和无复发生存率的降低有关。与其他NK细胞群相比，这些低成熟阶段的CD56brightCD16hi细胞在激活和抑制受体方面表现出独特的表型和功能变化(图2E)。综上所述，作者认为，目前基于NK细胞的治疗侧重于增强NK细胞的活化和延长其寿命，但通常忽略了癌症类型之间的异质性和TME对NK细胞细胞毒性功能的抑制作用，这些都应该在未来的治疗策略中加以考虑。图2 肿瘤浸润NK细胞在癌症类型中的异质性3. RGS1是组织浸润NK细胞的标志如上所述，NK细胞成分从血液到组织显著改变。同样，在组织浸润NK细胞与其血液对应物之间检测到广泛的转录变化(图3A)。先前的研究已经确定了组织驻留NK细胞的几种标记物，如CD69、CD103、CXCR6和CD49a。然而，作者发现ITGA1 (CD49a)、ITGAE (CD103)和CXCR6在单细胞转录组水平的检测很少，CD69在NK细胞(包括血液细胞)中广泛表达(图3E)。作者选择了血液和组织之间的差异表达基因，并进一步评估了它们的敏感性和特异性，以区分NK细胞的组织来源。RGS1 (G蛋白信号1的调节因子)被明确识别，其只在肿瘤和邻近非肿瘤组织的NK细胞中表达，但在血液中几乎检测不到(图3C和3D)。此外，RGS1的表达与KLF2、SELL等迁移信号相反(图3E)。与上述常规组织驻留标志物相比，RGS1具有更高的敏感性和特异性。接下来，作者直接比较了RGS1与ITGAE、CD69等在血液中的表达模式，发现RGS1单独在血液中的检出率最低，而RGS1/CD69组合可以进一步提高组织中的阳性检出率(图3F)， 还可以更好地区分血液和非血液NK细胞。值得注意的是，RGS1在癌症患者中广泛表达(图3G-H)。综上所述，这些特征暗示了RGS1单独或与CD69结合的潜在作用，在转录组水平上是组织浸润NK细胞的优秀标记物。作者推测RGS1的表达可能减弱G蛋白的信号活性，导致NK细胞趋化迁移能力减弱，促进NK细胞驻留。RGS1在NK细胞中的作用机制有待进一步研究。 图3 RGS1作为NK细胞关键组织浸润标志物的鉴定4. 肿瘤相关NK细胞程序及其特征接下来，作者试图阐明NK细胞在肿瘤中的具体特征。利用RNA 速率分析，作者分析了NK细胞的转录动力学，在CD56brightCD16lo和CD56dimCD16hi NK细胞中观察到从血液富集亚群到肿瘤浸润亚群的明确定向流动，作者发现CD56dimCD16hi c6-DNAJB1 NK细胞位于速率分析末端，因此推断为终末状态(图4A)。值得注意的是，来自邻近非肿瘤组织的CD56dimCD16hi NK细胞主要出现在富含c7-NR4A3细胞的均匀歧形近似和投影(UMAP)区域；相比之下，肿瘤来源的CD56dimCD16hi NK细胞在富含c6-DNA JB1细胞的UMAP区域占主导地位(图4B)。与此一致的是，肿瘤富集的c6-DNAJB1 NK细胞的标记物，如DNAJB1和HSPA1A在肿瘤浸润的CD56dimCD16hi NK细胞群中高度表达(图4C)。作者进一步关注肿瘤微环境，发现肿瘤组织中高度富集一群DNAJB1+ CD56dimCD16hi NK细胞。数据分析发现，该群细胞具有功能失调的表型，包括杀伤性下降、抑制受体上升、高表达应激反应相关蛋白等，因此将这一群细胞命名为肿瘤相关NK细胞（Tumor-associated NK cells, TaNK cells）(图4D)。作者还利用流式细胞术验证了TaNK细胞(CD56dimCD16hi HSP40+)在体内肿瘤的富集情况。结果显示，在1例肝内胆管癌和6例HCC样本中发现了TaNK细胞，其在肿瘤浸润性CD56dimCD16hi NK细胞中的比例高于匹配的邻近肝组织，与作者的scRNA-seq数据一致(图4E)。为了检验TaNK细胞的新特征，作者通过拟时序分析来研究CD56dimCD16hi NK细胞的动态，其结果和RNA速率分析结果一致。有趣的是，CD56dimCD16hi NK细胞在过渡过程中表现出细胞毒性降低，抑制受体和应激基因表达升高(图4F)。值得注意的是，在所有肿瘤浸润的CD56dimCD16hi NK细胞亚群中，终端TaNK细胞具有最低的细胞毒性和最高的应激评分。相比之下，相应的c7-NR4A3在邻近非肿瘤组织中富集，具有高细胞毒性(图4G)。通过对几种癌症类型进行多重免疫荧光染色，作者观察到TaNK细胞表现出较低水平的GZMB(图4H)。肝癌患者的流式细胞术分析进一步证实，与CD56dimCD16hi HSP40−NK细胞相比，TaNK细胞在肿瘤部位的细胞毒性颗粒(颗粒酶B和穿孔素)表达较低，抑制受体CD158a (KIR2DL1)和CD158e (KIR3DL1)表达较高(图4I和4J)。肝癌患者的流式细胞术分析进一步证实，与CD56dimCD16hi HSP40−NK细胞相比，TaNK细胞在肿瘤部位的细胞毒性颗粒(颗粒酶B和穿孔素)表达较低，抑制受体CD158a (KIR2DL1)和CD158e (KIR3DL1)表达较高(图4I和4J)。这些结果表明，TaNK细胞可能与功能失调状态有关。此外，作者还观察到了NR4A核受体家族随着拟时序的增加而呈现的差异动态趋势(图4F)。c7-NR4A3 NK细胞高表达NR4A2和NR4A3，而TaNK细胞高表达NR4A1。有趣的是，NR4A1已被确定为T细胞功能障碍的关键介质，并被认为有助于限制实体瘤中CAR - T细胞的功能。总之，该研究数据表明，肿瘤中的TaNK细胞可能发生终末功能障碍，并可能在TME中发挥关键作用。图4肿瘤相关NK细胞的特征5. TaNK细胞与不良预后和免疫治疗耐药的关系由于NK细胞和CD8+ T细胞表现出广泛的表型和功能相似性，作者接下来研究了靶向CD8+ T细胞的免疫检查点阻断(ICB)疗法是否也会影响NK细胞。在肿瘤浸润性NK细胞和CD8+ T细胞中，TaNK细胞和耗竭T细胞(Tex)表现出明显的应激状态(图5A)，提示它们参与肿瘤免疫应答。两者都高度表达一系列抑制性受体分子，然而它们在各种免疫调节基因上具有不同的表达谱。传统的免疫检查点基因，如PDCD1和CTLA4，在TaNK细胞上几乎不表达(图5A)，这意味着它们不是抗PD-1/CTLA-4治疗的直接靶点。在TME和当前的ICB治疗中，TaNK细胞可能与Tex细胞发挥不同的作用。作者观察到不同癌症类型的TaNK细胞丰度存在显著差异(图5B)，肿瘤分期对TaNK细胞比例的影响很小(图5C)。值得注意的是，在TCGA数据集中，肿瘤中的高TaNK细胞信号与大多数癌症类型的低生存率相关(图5D和5E)。作者进一步应用基于深度学习的模型进行反卷积和细胞组成分析，发现高TaNK细胞频率表明癌症患者预后不良。作者还通过分析先前乳腺癌和黑色素瘤的ICB治疗研究中预处理肿瘤的scRNA-seq数据 (图5F)，研究了TaNK细胞是否与ICB治疗应答相关。值得注意的是，在两种癌症类型中，无应答患者中观察到的TaNK细胞比例高于应答患者。进一步利用来自各种癌症(包括黑色素瘤、肺癌、转移性尿路上皮癌)的已发表的大量数据，作者证实了无应答患者比应答患者表现出更强的TaNK细胞信号(图5G)。作者推测，长期浸润可能赋予肿瘤中TaNK细胞的功能状态，导致其对恶性细胞的无效杀伤。TaNK细胞的富集与对肿瘤的免疫反应受损以及对当前ICB治疗的低敏感性有关。该研究发现揭示了TaNK细胞在肿瘤中的潜在作用，并为NK细胞免疫疗法的合理设计提供了参考。图5  TaNK细胞与临床结局的关系6. TME塑造肿瘤浸润NK细胞功能的潜在介质为了深入了解NK细胞在TME中的调控程序，我们利用CellPhoneDB来探测NK细胞和其他CD45+免疫细胞(包括T细胞和髓细胞)之间潜在的细胞-细胞相互作用。与T细胞相比，除肥大细胞外，大多数髓细胞类型与CD56dimCD16hi NK亚群表现出强烈的潜在相互作用(图6A)。特别有趣的是，TaNK细胞被预测通过ANXA1调节多髓细胞类型，ANXA1是一种在炎症反应中与免疫抑制和诱导巨噬细胞重编程相关的蛋白(图6B)，这表明功能失调的NK细胞可能具有抑制TME中促炎巨噬细胞的潜力。为了进一步阐明NK细胞来源的ANXA1在巨噬细胞中的作用，作者对肺癌和肝癌的肿瘤样本进行了多重免疫荧光染色，发现了一个ANXA1+ NK细胞群。与远离ANXA1+ NK细胞的巨噬细胞相比，靠近ANXA1+ NK细胞的巨噬细胞表现出较低的活化标志物CD86(图6C、6D)和较高的抗炎标志物转化生长因子β (TGF-β)的表达水平(图6E、6F)。值得注意的是，在树突状细胞(DC)亚群中，LAMP3+ DC，最近表征的成熟cDCs(也称为mregDC)显示出与CD56dimCD16hi NK细胞最强的相互作用潜力(图6A)。多重免疫荧光分析显示，LAMP3+ DCs与NK细胞共定位(图6J)。此外，预测它们之间的相互作用通过IL-15-IL-15受体和NECTIN2-TIGIT相互作用轴介导(图6B)。      重要的是，LAMP3+ DCs在转录组水平上表达免疫群体中最高水平的IL15、PVRL2 (NECTIN2)和PVR(图6G和S6F)。流式细胞术也证实了IL-15在LAMP3+ dc中的高表达(图6H)。IL-15已被确定为维持NK细胞寿命的稳态相关细胞因子，并用于NK细胞输注和体外繁殖。相反，TIGIT作为一种抑制性受体有助于抑制NK细胞介导的免疫反应。此外，在TCGA数据集中，LAMP3+ DCs的丰度与CD56dimCD16hi NK细胞相关(图6I)。        接下来，作者研究了LAMP3+ DCs在肿瘤中的具体调控过程，发现肿瘤浸润的LAMP3+ DCs与邻近非肿瘤组织相比，IL15的表达较低，表明LAMP3+ DCs可能对TME中CD56dimCD16hi NK细胞的激活作用受损。事实上，物理上接近LAMP3+ DCs的NK细胞表达颗粒酶B的水平较低(图6J、6K)。   总之，以上数据分析表明，在TME中，LAMP3+ DCs对CD56dimCD16hi NK细胞的异常调节。图6  在不同肿瘤类型中，LAMP3+ DCs与CD56dimCD16hi NK细胞的关系 7.外周血NK细胞亚群的不同转录组模式通过分析来自35名健康供者的血液来源NK细胞的9个scRNA数据集，能够探测肿瘤患者外周血中NK细胞的特异性改变。作者首先比较了来自健康供体和肿瘤患者的循环NK细胞的转录组特征。循环NK细胞在来自不同数据集的健康供体中表现出高度的相似性(图S7B)。相比之下，对于来自肿瘤患者的循环CD56brightCD16lo细胞，观察到与健康供者的转录组存在显著差异；在所分析的所有癌症类型中，循环CD56dimCD16hi细胞的差异甚至更为显著(图S7A)。值得注意的是，肿瘤患者在NK细胞亚群中表现出显著的组成变化，这种模式似乎是癌症类型特异性的(图S7C)。例如，在结直肠癌、头颈部鳞状细胞癌、肾癌和HCC中，循环CD56brightCD16lo c3-CCL3 NK细胞的比例增加，但在其他被分析的癌症类型中没有增加。接下来，作者将重点放在CD56dimCD16hi c8-KLRC2适应性NK细胞上，该细胞在某些癌症类型如结直肠癌和胃癌中富集(图S7D)。适应性NK细胞被认为是CAR NK细胞的一个有吸引力的来源，因为它们具有增强细胞因子反应的效应特性和对免疫抑制效应的内在抗性特别有趣的是，与其他循环NK细胞相比，这些细胞特异性表达MHC II类基因(图S7E)。作者还检查了这些NK细胞在肿瘤患者中的功能变化，发现与健康供体相比，肿瘤来源的适应性NK细胞的功能基因和MHC II类基因的表达明显更高(图S7F和S7H)，这意味着它们在肿瘤患者中处于高度激活状态。作者通过流式细胞术进一步证实，与健康供者相比，HCC患者循环NK细胞中MHC II类分子的高表达(图S7G)。因此，在患者源性适应性NK细胞中上调的基因参与了免疫效应过程的正调控等途径(图S7I)。综上所述，研究分析表明，循环NK细胞参与了肿瘤进展过程中外周免疫环境的系统性变化。图S7患者循环NK细胞的改变（与图1相关）     讨论本研究中，作者收集了大量的单细胞转录组数据，首次在泛癌水平系统地鉴定到了5类CD56brightCD16lo和9类CD56dimCD16hi NK细胞亚群，并详细地刻画了各类群的表型和功能多样性。这些NK细胞可能参与了广泛的抗肿瘤反应，如直接杀死癌细胞、分泌促炎细胞因子和募集其他免疫成分(图7A和7B)。促进NK细胞在实体恶性肿瘤中的浸润一直是开发治疗性NK细胞产品的关键焦点。我们在转录组水平上发现RGS1是组织浸润NK细胞的关键标志物(图7C)。促进NK细胞在实体恶性肿瘤中的浸润一直是开发治疗性NK细胞产品的关键焦点。作者在在转录组水平上发现RGS1是组织浸润NK细胞的关键标志物(图7C)。此外，作者发现了一个潜在功能失调状态的肿瘤富集NK细胞亚群，称为TaNK细胞(图7D)。TaNK细胞的高丰度与多种癌症类型的不良预后和免疫治疗耐药性有关，暗示了其在临床环境中的作用。作者推测，TaNK细胞富集可能反映或影响TME中的肿瘤免疫反应，尽管这些细胞可能不是ICB治疗的直接靶点。本研究的分析和实验证据支持骨髓细胞是NK细胞的核心介质。具体来说，LAMP3+ DCs可以作为关键的调节剂，并可能抑制CD56dimCD16hi NK细胞在TME中的功能，但需要进一步的功能验证 (图7E)。总之，作者通过寻找细胞内在和TME相关的因子，为维持NK细胞在体内的抗肿瘤活性提供了线索。图7研究的主要发现总结本研究聚焦TME中与T细胞功能相似、但具有更直接的识别和杀伤癌细胞能力的NK细胞。作者创新性地整合利用大规模单细胞数据，克服数据整合的多项难点，包括高精度分离NK细胞、去除复杂的批次效应等，首次在泛癌水平系统地鉴定到5类CD56brightCD16lo和9类CD56dimCD16hiNK细胞亚群，并详细地刻画了各类群的表型和功能多样性，揭示了NK细胞中的基因表达模式转变，捕捉了肿瘤免疫微环境的NK细胞亚群组分变化，为未来通过更全面的整合分析手段探索新的生物标志物和治疗靶点提供了助力，也为药物研发提供更准确、更全面的数据支持。参考文献：Tang F, Li J, Qi L, Liu D, Bo Y, Qin S, Miao Y, Yu K, Hou W, Li J, Peng J, Tian Z, Zhu L, Peng H, Wang D, Zhang Z. A pan-cancer single-cell panorama of human natural killer cells. Cell. 2023 Aug 15:S0092-8674(23)00849-8. doi: 10.1016/j.cell.2023.07.034. Epub ahead of print. PMID: 37607536.识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。