Summary of Study ST003779

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002357. The data can be accessed directly via it's Project DOI: 10.21228/M8ZN7S This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003779
Study Title	Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants - Intracellular metabolomics of B7.H3 human CAR-T cells
Study Type	Intracellular metabolomics
Study Summary	CD38 is an ectoenzyme on the surface of T cells and controlling both intracellular and extracellular NAD+ levels, which are important for T cell function. In this study we profiled the intracellular metabolomics of B7.H3 human CAR-T cells. Results can be found in Fig. 4 and Fig. S7 in the paper Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants (Revach et al.)
Institute	Massachusetts General Hospital
Department	KF-CCR
Laboratory	Jenkins lab
Last Name	Revach
First Name	Or-Yam
Address	55 Fruit street, Boston, MA, 02114, USA
Email	oryamush@gmail.com
Phone	(617) 726-5130
Submit Date	2025-01-14
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-05-14
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002357
Project DOI:	doi: 10.21228/M8ZN7S
Project Title:	Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants
Project Summary:	T cell exhaustion is a major driver of immune checkpoint blockade (ICB) resistance and clinically effective strategies to prevent or reverse itT cell exhaustion to restore ICB sensitivity are lacking. CD38, an ecto-enzyme involved in NAD+ catabolism, is highly expressed in exhausted CD8+ T cells in human melanoma, yet its role in T cell exhaustion remains to be elucidated. Here we show that CD38+CD8+ T cells are enriched during tumor progression and are strongly associated with ICB resistance in melanoma. Chronic TCR activation and type I interferon stimulation upregulate CD38 in human T cells, leading to mitochondrial dysfunction and reduced anti-tumor activity. Disrupting CD38 restored cellular NAD+ pools and T cell bioenergetics, leading to increased TCF7 expression, improved T cell proliferation, and enhanced effector function. Importantly, targeting CD38 in a cohort of patient-derived organotypic tumor spheroid (PDOTS), human living tumor explants, demonstrates that CD38-directed therapies can overcome ICB resistance in clinically resistant human melanoma, an effect that is furthered induced by supplementation with NAD+. These results emphasize the need for further preclinical and clinical evaluation of CD38 directed therapies in melanoma and underscore the importance of NAD+ as a vital metabolite to enhance those therapies.
Institute:	Massachusetts General Hospital
Department:	KF-CCR
Laboratory:	Jenkins lab
Last Name:	Revach
First Name:	Or-Yam
Address:	55 Fruit street, Boston, MA, 02114, USA
Email:	oryamush@gmail.com
Phone:	(617) 726-5130

Subject:

Subject ID:	SU003913
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Stimulation
SA410029	OYR88	acute
SA410030	OYR89	acute
SA410031	OYR90	acute
SA410032	OYR91	chronic
SA410033	OYR92	chronic
SA410034	OYR93	chronic
SA410035	OYR97	chronic
SA410036	OYR98	chronic
SA410037	OYR99	chronic
SA410038	OYR94	chronic+78c
SA410039	OYR95	chronic+78c
SA410040	OYR96	chronic+78c
SA410041	OYR100	chronic+78c
SA410042	OYR101	chronic+78c
SA410043	OYR102	chronic+78c

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO003906
Collection Summary:	Four million cultured CAR-T cells (donor 1-d1) or two million cultured CAR-T cells (donor 2-d2), in three technical replicates were collected, centrifuged, washed with 0.9% NaCl, and resuspended in 300 μL extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labeled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]).
Sample Type:	T-cells

Treatment:

Treatment ID:	TR003922
Treatment Summary:	Acutely or chronically stimulated CAR-T cells or chronically stimulated +3 days treatment of CD38 inhibitor 1uM 78c (6391, Toris) of two different donors (d1 and d2) were analyzed. In vitro T cell chronic stimulation. For chronic stimulation assays B7-H3.CAR-T cells were activated with washed αCD3/αCD28 Dynabeads ® (ThermoFisher Scientific, 11161D), according to the manufacturer protocols, in a 1:1 ratio of beads to cells, or 3 μg anti-human CD3 and CD28 antibodies (Biolegend, 300438, 377604), 20 ng/mL of IL-2 (PeproTech, 200-02), 0.6 μg/mL aFAS neutralizing antibody (EMD Millipore, 05-338) and RPMI (Corning, 10-040-CV), supplemented with 10% FBS and 1% penicillin/streptomycin. After 3 days, the beads were removed using a magnet/antibodies were washed and cells were divided into acute and chronic conditions. Acute and chronic were cultured in the media above, and only the chronic got restimulated with 1:1 Dynabeads ® to T cells ratio or anti-human CD3 and CD28 antibodies. Media and beads were replaced every 2-4 days for a total of 10-14 days. Donor 1 conditions: acute stimulation, chronic stimulation, chronic+CD38i(78c) Donor 2 conditions: chronic stimulation, chronic+CD38i(78c)

Treatment ID:

TR003922

Treatment Summary:

Acutely or chronically stimulated CAR-T cells or chronically stimulated +3 days treatment of CD38 inhibitor 1uM 78c (6391, Toris) of two different donors (d1 and d2) were analyzed. In vitro T cell chronic stimulation. For chronic stimulation assays B7-H3.CAR-T cells were activated with washed αCD3/αCD28 Dynabeads ® (ThermoFisher Scientific, 11161D), according to the manufacturer protocols, in a 1:1 ratio of beads to cells, or 3 μg anti-human CD3 and CD28 antibodies (Biolegend, 300438, 377604), 20 ng/mL of IL-2 (PeproTech, 200-02), 0.6 μg/mL aFAS neutralizing antibody (EMD Millipore, 05-338) and RPMI (Corning, 10-040-CV), supplemented with 10% FBS and 1% penicillin/streptomycin. After 3 days, the beads were removed using a magnet/antibodies were washed and cells were divided into acute and chronic conditions. Acute and chronic were cultured in the media above, and only the chronic got restimulated with 1:1 Dynabeads ® to T cells ratio or anti-human CD3 and CD28 antibodies. Media and beads were replaced every 2-4 days for a total of 10-14 days. Donor 1 conditions: acute stimulation, chronic stimulation, chronic+CD38i(78c) Donor 2 conditions: chronic stimulation, chronic+CD38i(78c)

Sample Preparation:

Sampleprep ID:	SP003919
Sampleprep Summary:	At the day of the metabolomic analysis cells were cultured in fresh full RPMI media in equal concentrations for 2h. Four million cultured CAR-T cells (donor 1) or two million cultured CAR-T cells (donor 2), in three technical replicates were collected, centrifuged, washed with 0.9% NaCl, and resuspended in 300 μL extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labeled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was dried on ice using a liquid nitrogen dryer. Dried samples were resuspended in 25 μL water and 2 μL was injected into a ZIC-pHILIC 150 x 2.1 mm (5 um particle size) column (EMD Millipore). Operated on Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA).

Sampleprep ID:

SP003919

Sampleprep Summary:

At the day of the metabolomic analysis cells were cultured in fresh full RPMI media in equal concentrations for 2h. Four million cultured CAR-T cells (donor 1) or two million cultured CAR-T cells (donor 2), in three technical replicates were collected, centrifuged, washed with 0.9% NaCl, and resuspended in 300 μL extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labeled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was dried on ice using a liquid nitrogen dryer. Dried samples were resuspended in 25 μL water and 2 μL was injected into a ZIC-pHILIC 150 x 2.1 mm (5 um particle size) column (EMD Millipore). Operated on Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA).

Chromatography:

Chromatography ID:	CH004708
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	25 °C
Flow Gradient:	0–20 min: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B
Flow Rate:	150 uL/min
Solvent A:	100% acetonitrile
Solvent B:	100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN006207
Analysis Type:	MS
Chromatography ID:	CH004708
Num Factors:	3
Num Metabolites:	116
Rt Units:	Minutes
Units:	a.u.