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[Preprint]. 2024 Mar 26:2024.02.12.579184.

doi: 10.1101/2024.02.12.579184.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

Or-Yam Revach^{1

2

3}, Angelina M Cicerchia¹, Ofir Shorer⁴, Boryana Petrova^{2

5}, Seth Anderson^{1

3}, Joshua Park³, Lee Chen⁶, Arnav Mehta^{1

2

3}, Samuel J Wright³, Niamh McNamee^{2

7}, Aya Tal-Mason^{2

7}, Giulia Cattaneo⁸, Payal Tiwari³, Hongyan Xie^{1

2

3}, Johanna M Sweere⁹, Li-Chun Cheng⁹, Natalia Sigal⁹, Elizabeth Enrico⁹, Marisa Miljkovic⁹, Shane A Evans⁹, Ngan Nguyen⁹, Mark E Whidden⁹, Ramji Srinivasan⁹, Matthew H Spitzer^{9

10

11

12

13}, Yi Sun¹, Tatyana Sharova⁸, Aleigha R Lawless⁸, William A Michaud⁸, Martin Q Rasmussen^{1

3}, Jacy Fang^{1

3}, Claire A Palin¹, Feng Chen⁸, Xinhui Wang^{2

8}, Cristina R Ferrone^{2

8

14}, Donald P Lawrence^{1

2}, Ryan J Sullivan^{1

2}, David Liu^{2

3

15}, Uma M Sachdeva^{2

7}, Debattama R Sen^{1

2

3}, Keith T Flaherty^{1

2}, Robert T Manguso^{1

2

3}, Lloyd Bod^{1

2

3}, Manolis Kellis⁵, Genevieve M Boland^{2

3

8}, Keren Yizhak⁴, Jiekun Yang⁶, Naama Kanarek^{2

3

5}, Moshe Sade-Feldman^{1

2

3}, Nir Hacohen^{1

2

3}, Russell W Jenkins^{1

2

3}

Affiliations

¹ Mass General Cancer Center, Krantz Family Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Department of Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel.
⁵ Department of Pathology, Boston Children's Hospital, Boston, MA, USA.
⁶ Computer Science and Artificial Intelligence Lab, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁷ Division of Thoracic Surgery, Massachusetts General Hospital, Boston, MA, USA.
⁸ Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
⁹ Teiko Bio, Salt Lake City, UT, USA.
¹⁰ Department of Otolaryngology-Head and Neck Cancer, University of California, San Francisco, San Francisco, CA, USA.
¹¹ Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA.
¹² Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
¹³ Chan Zuckerberg Biohub, San Francisco, CA 94158; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
¹⁴ Department of Surgery, Cedars-Sinai Medical Center Los Angeles, CA, USA.
¹⁵ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

PMID: 38405985
PMCID: PMC10888727
DOI: 10.1101/2024.02.12.579184

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

Or-Yam Revach et al. bioRxiv. 2024.

[Preprint]. 2024 Mar 26:2024.02.12.579184.

doi: 10.1101/2024.02.12.579184.

Authors

Affiliations

¹ Mass General Cancer Center, Krantz Family Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Department of Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel.
⁵ Department of Pathology, Boston Children's Hospital, Boston, MA, USA.
⁶ Computer Science and Artificial Intelligence Lab, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁷ Division of Thoracic Surgery, Massachusetts General Hospital, Boston, MA, USA.
⁸ Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
⁹ Teiko Bio, Salt Lake City, UT, USA.
¹⁰ Department of Otolaryngology-Head and Neck Cancer, University of California, San Francisco, San Francisco, CA, USA.
¹¹ Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA.
¹² Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
¹³ Chan Zuckerberg Biohub, San Francisco, CA 94158; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
¹⁴ Department of Surgery, Cedars-Sinai Medical Center Los Angeles, CA, USA.
¹⁵ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

PMID: 38405985
PMCID: PMC10888727
DOI: 10.1101/2024.02.12.579184

Abstract

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma^1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance³. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD⁺) catabolism, is highly expressed in exhausted CD8⁺ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38^hiCD8⁺ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD⁺ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD⁺ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Keywords: 3D microfluidic culture; CD38; NAD+; PD-1; T cell exhaustion; cytokines; immunotherapy; organotypic tumour spheroids.

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Conflict of interest statement

R.W.J. is a member of the advisory board for and has a financial interest in Xsphera Biosciences Inc., a company focused on using ex vivo profiling technology to deliver functional, precision immune-oncology solutions for patients, providers, and drug development companies. R.W.J. has received honoraria from Incyte (invited speaker), G1 Therapeutics (advisory board), Bioxcel Therapeutics (invited speaker). R.W.J. has ownership interest in U.S. patents US20200399573A9 and US20210363595A1. R.W.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. A.M has served a consultant/advisory role for Third Rock Ventures, Asher Biotherapeutics, Abata Therapeutics, ManaT Bio, Flare Therapeutics, venBio Partners, BioNTech, Rheos Medicines and Checkmate Pharmaceuticals, is currently a part-time Entrepreneur in Residence at Third Rock Ventures, is an equity holder in ManaT Bio, Asher Biotherapeutics and Abata Therapeutics, and has received research funding support from Bristol-Myers Squibb. A.M.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. J.M.S., L-C.C, N.S, M.M., N.N., R.S. are current employees with Teiko.bio and own stock. E E. and S.A.E. were employed with Teiko.bio in the past 2 years and own stock. R.S. and M.H.S. are Teiko.bio’s co-founders and R.S. serves on the company board. M.H.S. serves as an advisor for Teiko.bio and owns stock. M.E.W. worked as a contractor for Teiko.bio during this project. M.H.S. has received a speaking honorarium from Standard BioTools and Kumquat Bio, has been a paid consultant for Five Prime, Ono, January, Earli, Astellas, and Indaptus, and has received research funding from Roche/Genentech, Pfizer, Valitor, and Bristol Myers Squibb.X.W. and C.R.F. report a patent on the B7-H3 CAR T cells (US10519214B2). K.T.F. serves on the Board of Directors of Clovis Oncology, Strata Oncology, Kinnate, and Scorpion Therapeutics; Scientific Advisory Boards of PIC Therapeutics, Apricity, C-Reveal, Tvardi, ALX Oncology, xCures, Monopteros, Vibliome, Karkinos, Soley Therapeutics, Alterome, Immagene, and intrECate; consultant to Nextech, Takeda, Novartis, Transcode Therapeutics, and Roche/Genentech. R.T.M. consults for Bristol Myers Squibb. G.M.B. has sponsored research agreements through her institution with: Olink Proteomics, Teiko Bio, InterVenn Biosciences, and Palleon Pharmaceuticals. She has served on advisory boards for: Iovance, Merck, Nektar Therapeutics, Novartis, and Ankyra Therapeutics. She consults for: Merck, InterVenn Biosciences, Iovance, and Ankyra Therapeutics. She holds equity in Ankyra Therapeutics. M.S.F received funding from Calico Life Sciences, Bristol-Myers Squibb, Istari Oncology and served as a consultant for Galvanize Therapeutics. N.H. holds equity in BioNTech and is an advisor for Related Sciences/Danger Bio, Repertoire Immune Medicines and CytoReason, and receives research funding from Calico Life Sciences and Bristol-Myers Squibb. D.L serves on the scientific advisory board for Oncovalent Therapeutics, and received honorariums from Genentech.

Figures

**Extended Figure 1∣. Supporting data that CD38⁺ CD8⁺ T cells are associated with ICB resistance.**
**a-d,** scRNAseq of tumour- infiltrating leukocytes (CD45⁺) from melanoma patients treated with ICB **(a)** t-SNE plot of CD45⁺ (n=16,291) with 11 distinct clusters; **(b)** feature plot demonstrating *CD8a* expression; **(c)** proportion of *CD38* expressing CD8⁺T cells from pre-ICB tumours; (ICB-R, n=9; ICB-NR, n=10) and **(d)** post ICB tumours (ICB-R, n=8; ICB-NR, n=21; Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test, **e-f,** proportion of *CD38* expressing CD8 T cells in ICB-R and ICB-NR, separated by ICB type (anti-PD-1 or anti-PD-1 plus anti-CTLA-4), (ICB-R, n=17; ICB-NR, n=31) nonparametric two sample Kolmogorov-Smirnov test. **g-i,** Receiver Operating Characteristic (ROC) curves demonstrating the predictive power of CD38⁺CD45⁺ cells for ICB resistance across different immune clusters in Extended Data Fig. 1a (clusters 6,9,11). FPR = false positive rate. TPR = true positive rate. j, summary of Receiver Operating Characteristic (ROC) curves of the predictive power of *CD38* expression across different CD45⁺ clusters for ICB resistance. k, flow cytometry immunophenotyping of CD38 surface staining in CD45⁺ and CD45⁻ cells from human melanoma tumours (n=18). Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test. **l-n,** scRNAseq analysis of CD8 T cells from melanoma validation cohort **(l)** UMAP of CD8 clusters (n=22,008) with 9 distinct clusters identified; **(m)** *CD38* expression **(n)** *TCF7* expression. ****p < 0.0001.

**Extended Figure 2∣. Supporting data that CD38⁺ CD8⁺ T cells are increased during tumour progression.**
a, proportion of *CD38* expressing CD8⁺T cells from **(a)** NSCLC (MPR-major pathological response ICB-R n=23; non-MPR, ICB-NR, n=34) b, Receiver Operating Characteristic (ROC) curve demonstrating the predictive power of CD38⁺CD8⁺ T cells for lack of ICB treatment benefit in NSCLC ; FPR = false positive rate. TPR = true positive rate. **c-d** CyTOF analysis of peripheral blood examining CD8⁺CD38⁺ T cells in melanoma patients' blood before ICB treatment **(c)** and after **(d)**. e, CyTOF analysis of peripheral blood examining CD8⁺CD38⁺IgG4⁺. f, Olink analysis of CD38 protein levels in blood plasma of ICB-R (n=64) and ICB-NR (n=53) patients before and following 6 weeks and 6 months after ICB treatment. 2-way ANOVA. g, scRNAseq of CD45⁺ cells from B16 tumours showing UMAP with 10 distinct clusters, their distribution of location (Tum), tumour draining lymph nodes (dLN), and normal lymph nodes (nLN) and the expression of *CD38* in those clusters. h, scRNAseq of T/NK tumour-infiltrating leukocytes from B16-ova tumours from Control (Vehicle/IgG, n=3) and αPD-1 (Vehicle/anti-PD-1, n=4). **i-l,** CD3⁺ TILs in Control (Vehicle/IgG, n=3) and αPD-1 (Vehicle/anti-PD-1, n=4) B16-ova treated tumours. **(i)** proportion of indicated immune populations upon PD-1 blockade (proportion from total cells; 2-way ANOVA with Sidak correction for multiple comparisons.). **j-l,** proportion of *Cd38* expressing CD3⁺TILs of indicated types; unpaired t-test. *P <0.05, ***P <0.001.

**Extended Figure 3∣. Supporting data that CD38^hiCD8⁺ T cells are dysfunctional.**
**a-c,** analysis of sorted CD38^hi and CD38^loB7-H3.CAR-T for **(a)** surface staining for PD-1⁺ **(b)** surface staining of PD-1⁺TIM-3⁺ and **(c)** gene expression of *FOXO1*. Means (bars) and individual values (open circles) are shown (n=3 (a-b); n=2 (c); 2-sided unpaired t-test). d, analysis of surface staining for PD-1⁺TIM-3⁺ in B7-H3.CAR-T cells following acute and chronic T cell stimulation. Means (bars) and individual values (open circles) are shown (n=3; 2-sided paired t-test). e, Analysis of surface staining for B7-H3 antigen in 10101 melanoma tumour cells **f-g,** cytotoxicity assay of acute and chronically stimulated B7-H3.CAR-T cells towards 10170 **(f)** and 10101 **(g)** patient derived melanoma cell lines. Two other repeats for Fig. 2k. Means +/− s.e.m. (shaded area) are shown (n=3 biological replicates; 2-way ANOVA with Sidak correction for multiple comparisons). h, surface staining for CD38⁺CD39⁺ in CD8⁺ TILs from melanoma patients, following acute and chronic T cell stimulation. Means (bars) and individual values (open circles) are shown (n=3; 2-sided paired t-test) i, cytotoxicity of 10170 CD8⁺ TILs following acute and chronic T cell stimulation, towards 10170 patient-derived cell line (n=2 biological replicates; 2-way ANOVA with Sidak correction for multiple comparisons). **j-l,** analysis of surface staining of **(j)** CD38⁺, **(k)** PD-1⁺TIM-3⁺, **(l)** CD39⁺TIM-3⁺ in chronically stimulated control sgRNA and *CD38* sgRNA B7-H3.CAR-T cells. Means (bars) and individual values (open circles) are shown (n=3; 2-sided paired t-test). *P <0.05, ***P < 0.001, ****p < 0.0001, ns, not significant.

**Extended Figure 4∣. Supporting data that CD38 blockade overcomes ICB resistance.**
a, Tumour type, tissue source (location), gender, age, clinical response, PDOTS response data, tumour mutational burden (TMB), and associated tumour mutational profile for specimens used for PDOTS profiling (n=30; samples ordered by response to dual PD-1/CD38 blockade). PDOTS response parameters defined as previously described: responder (reduction >30% compared to control), partial responder (<30% reduction and <20% growth compared to control), and non-responder (>20% growth compared to control).

**Extended Figure 5∣. Supporting data that CD38 blockade overcomes ICB resistance in human melanoma.**
**a-b**, representative images of 10213 ICB-resistant mucosal melanoma PDOTS **(a)** and 10101 ICB-resistant cutaneous melanoma PDOTS **(b)** treated with indicated treatments. Ho-Hoechst (blue), PI -propidium iodide, dead cells (red). Scale bars=100μm. c, flow cytometry analysis of indicated populations from PDOTS single cell suspension before culturing (n=25). d, immunofluorescence imaging of cutaneous melanoma PDOTS with indicated markers. Nu-nucleus, blue. White arrows denote cells with marker co-expression.

**Extended Figure 6∣. Supporting data that CD38 blockade overcomes ICB resistance.**
**a-c,** Multiple linear regression summaries for the association of immune and clinical features of melanoma patient derived tumours used for PDOTS with *ex vivo* response to PD-1/CD38 dual blockade (n=30 for clinical features and n=22 for immune features) (Fig. 3b-c). **(a)** flow cytometry measures CD8⁺ proportions; **(b)** CD4⁺ proportions; **(c)** clinical demographic features. Points are coloured by −log10(p-value). d, flow cytometry analysis of indicated populations in CD8⁺ T cells TILs from CT26-GFP tumours at day 10 and day 14. **e-f,** representative images of B16-ova murine melanoma **(e)** and CT26-GFP murine colon cancer **(f)** treated with indicated treatments. Ho-Hoechst (blue), PI- PI -propidium iodide, dead cells (red), AO- acridine orange, live cells (green). **g-i**, cell surface staining of **(g)** CD38⁺ **(h)** IL-7Ra⁺ **(i)** CD69⁺ on CT26-GFP murine tumour derived CD8⁺ TILs treated *in vitro* with CD38 blocking antibody or control IgG. Means (bars) and individual values (open circles) are shown (n=2; 2-sided unpaired t-test). *P <0.05, **P < 0.01.

**Extended Figure 7∣. Supporting data that CD38 is associated with mitochondrial dysfunction.**
**a-b,** Gene set enrichment analysis (GSEA) of **(a)** CD8⁺ TILs from human melanoma and **(b)** CD3⁺ TILs from B16-ova murine melanoma. **c-d,** mountain plots showing enrichment for OXPHOS gene set based on *CD38* expression in **(c)** CD8⁺ TILs human melanoma and **(d)** CD3⁺ TILs from B16-ova murine melanoma. e, Incucyte mitochondrial membrane potential (MMP) measurements of sorted CD38^hi and CD38_lo B7-H3.CAR-T cells. Means (bars) and individual values (open circles) are shown (n=3; 3 biological replicates; 1-way ANOVA). **f-k,** Flow cytometry analysis of mitochondrial mass (**f,h,j**) and MMP (g,i,k) in CD38^hi and CD38^lo, chronically stimulated B7-H3.CAR-T cells (**f,g**), CD8⁺ TILs (**h,i**) and JURKAT cells (**j,k**). Means (bars) and individual values (open circles) are shown (n=3; 2-sided paired t-test). **l,m,** Flow cytometry analysis of mitochondrial mass (l) and MMP (m) of chronically stimulated control and *CD38* sgRNA B7-H3.CAR-T cells (n=3; Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test). n, Extracellular acidification rate (ECAR) under basal condition in response to addition of oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A of B7-H3.CAR-T cells form indicated groups. (n=5, 2 biological replicates, 2-way ANOVA with Sidak correction for multiple comparisons). *P <0.05, **P < 0.01, ***P < 0.001.

**Extended Figure 8∣. Supporting data that CD38 inhibition restores cellular NAD⁺ levels.**
**a-b,** Analysis of relative NAD(H) in (a) control and *CD38* sgRNA B7-H3.CAR-T cells (n=3 biological repeats, 3 independent experiments; Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test) and (b) chronically stimulated B7-H3.CAR-T cells with indicated treatments (n=3 biological repeats; Means (bars) and individual values (open circles) are shown; 1-way ANOVA with Tukey correction for multiple comparisons). c, volcano plot of intracellular metabolomics of acute vs chronic B7-H3.CAR-T cells (n=3 biological repeats). d, Relative NADPH levels in B7-H3.CAR-T cells in indicated groups determined by LC-MS. Means (bars) and individual values (open circles) are shown (n=3 biological replicates, 2-sided unpaired t-test). **e-i,** Relative levels of intracellular **(e)** AMP (Adenosine mono-phosphate), **(f)** ATP (Adenosine triphosphate), **(g)** ADP(Adenosine diphosphate), **(h)** deoxyadenosine and **(i)** SAM (S-Adenosylmethionine) in chronically stimulated CAR-T cells +/− CD38i (n=6; 2 independent CAR-T donors; Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test). **j-k,** flow cytometric analysis of the indicated surface markers in chronically stimulated B7-H3.CAR-T cells (n=3 biological repeats; Means (bars) and individual values (open circles) are shown). **l-o,** Relative levels of extracellular **(l)** ADPR (Adenosine diphosphate ribose), **(m)** NAM (nicotinamide), **(n)** SAM, **(o)** Deoxyadenosine in chronically stimulated CAR-T cells +/− CD38i (n=6 biological replicates; 2 independent CAR-T donors; 2-sided unpaired t-test). p, representative images of CT26-GFP murine colon cancer treated with indicated treatments. Ho- Hoechst (blue), PI- PI -propidium iodide, dead cells (red), GFP- tumour cells. q, Viability assessment of CT26-GFP MDOTS (n=3 biological replicates, one-way ANOVA with Tukey correction for multiple comparisons), with indicated treatments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

**Figure 1∣. CD38⁺ CD8⁺ T cells are associated with ICB resistance.**
**a-g**, scRNAseq of CD45⁺ immune cells from melanoma patients with (a) t-SNE plot of CD8⁺ T cell clusters (n=6,350); (**b-d**) t-SNE plot of CD8⁺ T cell clusters showing expression of **(b)** *CD38*, **(c)** *PDCD1*, and **(d)** *TCF7*; **(e)** t-SNE plot of CD8⁺ T cell clusters by ICB response; **(f)** Proportion of *CD38* expressing CD8⁺T cells from indicated groups. Responders (ICB-R) and non-responders (ICB-NR). Means (bars) and individual values (open circles) are shown. 2-sided unpaired t-test; (g) Receiver Operating Characteristic (ROC) curve, demonstrating the predictive power of CD38⁺CD8⁺ T cells for ICB resistance. h, Validation cohort evaluating proportion of *CD38* expressing CD8⁺T cells from indicated groups; 2-sided unpaired t-test, **i-j**, CyTOF analysis of peripheral blood examining levels of **(i)** CD38⁺CD8⁺T cells and **(j)** IgG4⁺ CD38⁺CD8⁺T cells from ICB responding and non-responding patients; Means (bars) and individual values (open circles) are shown; 2-sided unpaired t-test, **k-1**, Dot plots indicating (k) *Cd38* expression in CD8⁺ TILs from B16-ova tumours (Tum), tumour draining lymph nodes (dLN), and normal lymph nodes (nLN) and (I) expression of *Cd38* and *Tcf7* (Tum, dLN, and nLN) at day 7, 10 and 16²⁰. **m-o**, scRNAseq of CD3⁺ TILs from IgG control and αPD-1 treated mice bearing B16-ova tumours showing (m) UMAP of T/NK cell clusters by condition; (n) *Cd38* gene expression in UMAP from **(m)**; (o) tracks plots of *Cd38* gene expression by T/NK cluster. 2-way ANOVA with Sidak correction for multiple comparisons. Statistics in Extended Data Fig. 2i. P < 0.05.

**Figure 2∣. CD38^hiCD8⁺ T cells are dysfunctional.**
a, Dot plot showing the expression of exhaustion and effector/memory-related genes in CD8⁺ TILs from human melanoma tumours. b, Co-expression deviation proportion plot demonstrating co-expression of exhaustion-related genes and *TCF7* from melanoma validation cohort. **c-d**, Volcano plots depicting differentially expressed genes based on CD38 expression in (c) CD8⁺ TILs from human melanoma and (d) CD3⁺ TILs from B16-ova murine melanoma. **e-f**, Surface staining (e) for PD-1⁺CD39⁺TIM-3⁺ (n=3; 2-sided paired t-test) and (f) *TCF7* expression (n=3; 2-sided unpaired t-test) in sorted CD38^hi and CD38^lo B7-H3.CAR-T cells. Means (bars) and individual values (open circles) are shown. g, Scheme depicting acute and chronic TCR stimulation. **h-k**, Acute and chronic B7-H3.CAR-T (h) proliferation assay; Means +/− s.e.m. (shaded areas) are shown (n=3; 2-way ANOVA with Sidak correction for multiple comparisons). Surface staining for CD38⁺CD39⁺ **(i)** and CD39⁺TIM-3⁺ (j); Means (bars) and individual values (open circles) are shown (n=3, 2-sided paired t-test) **(k)** cytotoxicity assay towards 10164 patient derived melanoma cell line. Representative experiment out of three is presented. Means +/− s.e.m. (shaded area) are shown (n=3 biological replicates; 3 independent experiments; 2-way ANOVA with Sidak correction for multiple comparisons). **l-n**, analysis of chronically stimulated control sgRNA and *CD38* sgRNA B7-H3.CAR-T cells; **(l)** Proliferation assay; Means +/− s.e.m. (shaded area) are shown (n=3 biological replicates; 3 independent experiments; 2-way ANOVA with Sidak correction for multiple comparisons); (m) TCF7 intracellular staining. Means (bars) and individual values (open circles) are shown (n=4; 2-sided paired t-test;); (n) Cytotoxicity assay 10164 melanoma cells. Means +/− s.e.m. (shaded area) are shown (n=3 biological replicates; 3 independent experiments; 2-way ANOVA with Sidak correction for multiple comparisons). *P < 0.05, **P < 0.01, ****P < 0.0001.

**Figure 3∣. CD38 blockade overcomes ICB resistance.**
a, Scheme of PDOTS preparation. b, Violin plot of PDOTS (n=30) viability assessment following treatment with anti-PD-1 (250 μg/mL pembrolizumab), anti-CD38 (200 μg/mL), or combined anti-PD-1+anti-CD38. Individual values (open circles) indicate mean for each PDOTS specimen; one-way ANOVA with Tukey correction for multiple comparisons. c, waterfall plots for PDOTS (n=30, indicated tumour types) with indicated treatments. Response is defined as 30% reduction from control (dashed lines). **d-e**, PDOTS viability assessment from (d) ICB-resistant cutaneous melanoma (10101) and (e) ICB-resistant mucosal melanoma (10213) with indicated treatments (n=3 biological replicates per PDOTS specimen, one-way ANOVA with Tukey correction for multiple comparisons). **f-g**, Viability assessment of (f) B16-ova MDOTS (n=6 biological replicates, 2 independent experiments), (g) CT26-GFP MDOTS (n=12 biological replicates, 4 independent experiments), (h) B16-ova MDOTS (n=3 biological replicates, 1 independent experiment) with indicated treatments. Means (bars) and individual values (open circles) are shown (one-way ANOVA with Tukey correction for multiple comparisons. *P < 0.05. **P < 0.01; ***P < 0.001; **** P < 0.0001; ns, not significant.

**Figure 4∣. CD38 inhibition restores cellular NAD⁺ and sensitivity to ICB.**
a, correlation analysis between the GSEA of CD38^+/−CD8⁺ T cells in human melanoma and CD38^+/−CD3⁺ T cells in B16-ova murine melanoma, **b-c,** Oxygen consumption rate (OCR) under basal condition in response to indicated mitochondrial inhibitors of chronically stimulated B7-H3.CAR-T cells form indicated groups. (n=5 biological replicates, 2 independent experiments; 2-way ANOVA with Sidak correction for multiple comparisons). **d-g**, Relative levels of **(d)** NAD⁺ (Nicotinamide adenine dinucleotide), **(e)** NADP⁺ (Nicotinamide adenine dinucleotide phosphate), **(f)** NAM (nicotinamide) and **(g)** ADPR (Adenosine diphosphate ribose) in B7-H3.CAR-T cells in indicated groups (n=6 biological replicates; 2 independent experiments; 2-sided unpaired t-test). h, scheme of NAD⁺ metabolism and log2 fold change (L2FC) of indicated analytes. **i-j**, surface staining of (i) TIM-3⁺PD-1⁺ and (j) CD39⁺TIM-3⁺ in B7-H3.CAR-T cells in indicated groups. Means (bars) and individual values (open circles) are shown (n≥4; 2-sided paired t-test), k, *TCF7* expression in B7-H3.CAR-T cells in indicated groups. Means (bars) and individual values (open circles) are shown (n=4; 2-sided unpaired t-test). **l-m**, Viability assessment of (l) B16-ova MDOTS (n=6 biological replicates, 2 independent experiments), (m) CT26-GFP MDOTS (n=12 biological replicates, 4 independent experiments), (n) CT26-GFP MDOTS (n=6 biological replicates, 2 independent experiments) with indicated treatments. Means (bars) and individual values (open circles) are shown (one-way ANOVA with Tukey correction for multiple comparisons). o, Viability assessment of melanoma PDOTS with indicated treatments. (n=21, 7 independent specimens. Mean values (bars) and individual values (open circles) are shown.; mixed effects one-way ANOVA with Geisser-Greenhouse correction and Holm-Sidak correction for multiple comparisons test. p, Viability assessment of melanoma PDOTS treated with indicated treatments (n=3 biological replicates). Means (bars) and individual values (open circles) are shown; one-way ANOVA with Tukey correction for multiple comparisons. *P < 0.05, **P < 0.01; ***P < 0.001, ns, not significant, q, Scheme demonstrating the effect of targeting CD38 on ICB response by increasing NAD⁺ and *TCF7* expression along with restoring mitochondrial bioenergetics.

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Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

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Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

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