Asparagine is critical for early activation and proliferation of CD4+ T cells.

(A) Schematic of experimental design. Naive CD4+ T cells were stimulated for either 24 hours or 72 hours with plate-bound anti-CD3 and anti-CD28 mAbs in DMEM media with glutamine or DMEM media with glutamine supplemented with 0.38 mM asparagine (Asn), 0.38 mM alanine (Ala), 0.15 mM aspartate (Asp), 0.13 mM glutamate (Glu), or 0.17 mM proline (Pro). (B) Representative flow cytometry histogram depicting CTV dye dilution in naive CD4+ T cells following 3 days of stimulation with plate-bound anti-CD3/CD28 mAbs in DMEM media with glutamine or DMEM media with glutamine supplemented with the indicated amino acids. (C) Quantification of division index in B. (D-I) Quantification of the proportions of CD4+ T cells expressing the cell surface activation markers PD-1 (D), CD44 (F) CD25 (H) as well as expression levels of each respective marker on a per cell basis (E,G,I) following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs in DMEM media with glutamine or DMEM media with glutamine supplemented with 0.38 mM Asn, 0.38 mM Ala, 0.15 mM Asp, 0.13 mM Glu, or 0.17 mM Pro. (J-K) Expression levels of activation markers CD44 (J), CD25 (K) following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs in RPMI lacking the indicated individual amino acids shown in red. Non-essential amino acids (NEAA) include asparagine (Asn), aspartate (Asp), glutamate (Glu), proline (Pro), arginine (Arg), glutamine (Gln), glycine (Gly), serine (Ser), and tyrosine (Tyr). (L) Quantification of the proportions of viable CD4+ T cells following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs in complete RPMI (RPMI), Asn-deficient RPMI or RPMI with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at the start of culture. (M-N) Expression level of CD25 (M) and CD44 (N) on a per cell basis following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs under the same conditions as in L. (O) Representative flow cytometry histograms showing CTV dye dilution in naive CD4+ T cells following 3 days of stimulation with plate-bound anti-CD3/CD28 mAbs in RPMI, Asn-deficient RPMI or RPMI with 10 IUs/L PEG-AsnASE added at the start of culture. Grey histogram represents unstimulated control. (P) Quantification of division index in O. Each dot represents cells obtained from an individual animal (L-N, P). Results are shown as mean ± SD (C-N, P) and are representatives of 2 independent experiments (B-K, O) or pooled from 2 independent experiments (L-N, P). Not significant (n.s), *p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test (C-K) or Tukey’s multiple comparison test (L-N, P). Schematics were created using BioRender.com.

Extracellular asparagine is needed for sustained CD4+ T cell activation and proliferation at later stages following initial activation.

(A) Schematic of Asn metabolism in mammalian cells. (B-C) Bulk RNA-seq analysis showing expression of Asns (B), and Asrgl1 (C) in naive CD4+CD62L+CD44− T cells at baseline (gray dot) or following stimulation with anti-CD3/CD28 mAbs under T helper 1 (TH1), pathogenic T helper 17 (pTH17) and non-pathogenic T helper 17 (npTH17) conditions for 1, 6, 12, 20 and 48 hours. Results are shown as average (n=3 for each condition, per time point). (D-E) qPCR analysis showing the expression kinetics of Asns (D) and Asrgl1 (E) over time in naive CD4+ T cells at baseline or stimulated with anti-CD3/CD28 mAbs for 24 and 48 hours. (F) Western blot analysis of ASNS protein expression in CD4+ T cells activated with anti-CD3/CD28 mAbs in either RPMI or Asn-deficient RPMI at 24 or 48 hours after activation. Naive CD4+ T cells are shown as controls. (G) Densitometry analysis of F showing the relative Asns to actin ratio. (H) Schematic of experimental design. Purified naive CD4+ T cells were activated in vitro with plate-bound anti-CD3/CD28 mAbs in complete RPMI media (RPMI), Asn-deficient RPMI, or RPMI with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at 0, 6, 12, 24, 36, 48 and 60 hours. (I) Representative flow cytometry histogram showing cell trace violet (CTV) dye dilution in naive CD4+ T cells following 3 days of stimulation. (J) Quantification of division index in I. (K-L) Quantification of the gMFI of CD4+ T cells expressing CD25(K) and CD44(L) following 2 days of stimulation with plate-bound anti-CD3/CD28 mAbs in complete RPMI, Asn-deficient RPMI or RPMI with 10 IUs/L PEG-AsnASE added at 0, 6, 12, 24, 36 hours. Each dot represents cells from an individual animal (J-L). Results are shown as average (n=3 per condition, per time point) (B-C) or mean ± SD (D-E, G, J-L) and are representative of 2 independent experiments (D-G, I-L). non-significant (n.s), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test (J-L) or Tukey’s multiple comparison test (G). Schematics were created using BioRender.com.

Asparagine is taken up by CD4+ T cells and incorporated into their proteome.

(A) Schematic of experimental design. Purified naive CD4+ T cells were activated in vitro with plate-bound anti-CD3/CD28 mAbs in complete RPMI media (RPMI) or Asn-deficient RPMI for 24 hours. At 24 hours Asn was added to cells stimulated in Asn-deficient media at a final concentration of 0.38 mM for 4 hours. At 28 hours protein synthesis was measured using the O-propargyl-puromycin (OPP) probe. As a positive control T cells cultured in RPMI were treated with 50 μg/mL of the protein synthesis inhibitor cycloheximide (CHX). (B) Representative flow cytometry histogram showing mean fluorescent intensity (MFI) of the nascent protein synthesis reporter Click-iT OPP. Naive CD4+ T cells are shown as controls. (C) Quantification of Click-iT OPP gMFI. (D) Experimental design for measuring the incorporation of heavy labeled 15N2-Asn into proteins following naive CD4+ T cell activation. Naive CD4+ T cells were stimulated with plate-bound anti-CD3/CD28 mAbs in Asn-deficient media reconstituted with 0.38 mM 15N2-Asn for 24 or 48 hours. (E) Quantification of the 15N2-Asn labeled fraction in the T cell proteome. Results are shown as mean ± SD (C and E) and are representative of at least two independent experiments (B-C, E). ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test (C). Schematics were created using BioRender.com.

Extracellular asparagine depletion impairs CD4+ T cell bioenergetics upon activation.

(A) Schematic of experimental design. Naive CD4+ T cells were stimulated for 48 hours with plate-bound anti-CD3/CD28 mAbs in either complete RPMI media (RPMI), Asn-deficient RPMI or RPMI treated with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at 0, 6, 12, 24, or 36 hours and stained with Mitotracker green (MTG) and Tetramethylrhodamine methyl ester (TMRM). (B) Representative flow cytometry contour plots depicting TMRM and MTG in CD4+ T cells. (C) Quantification of proportion of TMRM/MTG low cells. (D) Schematic of experimental design. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in naive CD4+ T cells stimulated for 48 hours with plate-bound anti-CD3/CD28 mAbs in either complete RPMI media (RPMI), Asn-deficient RPMI or RPMI with 10 IUs/L PEG-AsnASE added at 0, 6, 12, 24, or 36 hours. (E) OCR under mitochondrial stress test conditions. (F) Quantification of basal respiration and (G) ATP production. (H) ECAR under glycolysis stress test conditions. (I) Quantification of glycolysis. (J) Schematic of experimental design. OCR was measured in naive CD4+ T cells stimulated for 24 hours with plate-bound anti-CD3/CD28 mAbs in DMEM with glutamine or DMEM with glutamine supplemented with 0.38 mM of either asparagine (Asn), alanine (Ala), aspartate (Asp), glutamate (Glu), or proline (Pro). (K) OCR under mitochondrial stress test conditions. (L) Quantification of basal respiration and (M) maximal respiration. Each dot in panels C, F-G, and I represent cells obtained from an individual animal. Results are shown as mean ± SD and are representative of at least 2 independent experiments. non-significant (n.s), *p < 0.05, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test. Schematics were created using BioRender.com.

Asparagine deficiency subdues lineage-specific cytokine production in CD4+ T helper subsets.

(A) Schematic of experimental design. Purified naive CD4+ T cells were activated in vitro with plate-bound anti-CD3/CD28 mAbs under T helper 1 (TH1), pathogenic T helper 17 (pTH17), and non-pathogenic T helper 17 (npTH17) conditions in either complete RPMI (RPMI) media, Asn-deficient RPMI or RPMI with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at 0, 12, 24, 36 or 48 hours. On day 3 cells were restimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin for intracellular staining. (B) Representative flow cytometry contour plots depicting intracellular staining of IFN-γ in TH1 differentiation conditions in RPMI or RPMI without Asn (C) Quantification of the proportions of IFNγ-producing CD4+ T cells under TH1 differentiation conditions. (D) Quantification of IFN-γ gMFI as shown in C. (E) Representative flow cytometry contour plots depicting intracellular staining of IL-17A in npTH17 differentiation conditions in RPMI or RPMI without Asn. (F) Quantification of the proportions of IL-17A-producing CD4+ T cells under npTH17 differentiation conditions. (G) Quantification of IL-17A gMFI as shown in F. (H) Representative flow cytometry contour plots depicting intracellular staining of IL-17A in pTH17 differentiation conditions in RPMI or RPMI without Asn. (I) Quantification of the proportions of IL-17A-producing CD4+ T cells under pTH17 differentiation conditions. (J) Quantification of IL-17A gMFI as shown in I. Each dot in panels C-D, F-G, and I-J represents cells obtained from an individual animal. Results are shown as mean ± SD and are representative of at least 2 independent experiments. non-significant (n.s), *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test. Schematics were created using BioRender.com.

Asparagine deficiency ameliorates experimental autoimmune encephalomyelitis (EAE).

(A) Mice were treated with a single dose of 25 IUs of PEGylated-asparaginase (PEG-AsnASE) or PBS i.p. one day prior to immunization with MOG35-55/CFA and pertussis toxin (PTX) to induce EAE and monitored daily for signs of disease. (B) Quantification of the average maximal EAE scores in PBS vs. PEG-AsnASE treated mice. (C) Quantification of the mean day of onset in PBS vs. PEG-AsnASE treated mice. (D) EAE was induced by immunization with MOG35-55/CFA and pertussis toxin (PTX) and scored daily for disease. Mice were treated with a single dose of 25 IUs of PEG-AsnASE or PBS i.p on day 8 of active EAE. (E) Quantification of the average maximal EAE scores in PBS vs. PEG-AsnASE treated mice. (F) Quantification of the mean day of onset in PBS vs. day 8 PEG-AsnASE treated mice. (G) Schematic of experimental design. Pathogenic T helper 17 (pTH17) cells were differentiated from naive CD4+ FoxP3− T cells from 2D2 TCR transgenic mice in RPMI media with or without Asn and viable 2D2 cells were adoptively transferred (4×106/mouse) into 10-week-old C57BL/6J female recipients to induce EAE. Mice were monitored daily for disease. (H) Representative flow plot displaying percentage of viable pTH17 polarized 2D2 cells in sufficient and deficient conditions prior to transfer. (I) Daily EAE scores. (J) Quantification of the average maximal EAE scores in mice receiving pTH17 cells generated in the presence or absence of Asn. (K) Quantification of the proportions of CNS-infiltrating Vβ11+Vα3.2+ 2D2 pTH17 cells at peak of EAE (pTH17 RPMI n=16, pTH17 Asn-deficient RPMI n=12). (L) Quantification of the proportions of Vβ11+Vα3.2+ 2D2 pTH17 cells actively undergoing apoptosis (Annexin-V+PI−) in the CNS and inguinal lymph node at peak of EAE. (M) Quantification of the proportions of TMRM/MTG low 2D2 pTH17 cells in the CNS and inguinal lymph node at peak of EAE. (N) Quantification of OPP gMFI in 2D2 pTH17 cells in the CNS and inguinal lymph node at peak of EAE. (O) Quantification of the absolute numbers of the indicated cytokines expressed by 2D2 pTH17 cells in the CNS at peak of EAE. Results are shown as mean ± SEM (A, D, I) or mean ± SD (B-C, E-F, J-O) and are pooled (A-F, I-K, M, O) or a representative of at least two independent experiments (H, L, N). Each dot represents an individual mouse (B-C, E-F, J-O) *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001 two-way ANOVA (A, D & I) and Student’s t test (B-C, E-F, and J-O).

Extracellular asparagine is essential for CD4+ T cell activation.

(A) Expression levels of the activation marker PD-1 following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs in RPMI lacking the indicated individual amino acids shown in red. Non-essential amino acids (NEAA) include asparagine (Asn), aspartate (Asp), glutamate (Glu), proline (Pro), arginine (Arg), glutamine (Gln), glycine (Gly), serine (Ser), and tyrosine (Tyr). Quantification of the proportions of CD4+ T cells expressing cell surface activation markers CD44 (B), CD25 (C), CD69 (D), and PD-1 (F) as well as expression levels of CD69 (E) and PD-1 (G) on a per cell basis following 24 hours of stimulation with plate-bound anti-CD3/CD28 mAbs in complete RPMI (RPMI), Asn-deficient RPMI or RPMI with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at the start of culture. (H) Quantification of the proportions of viable CD4+ T cells in each respective culture condition after 3 days of stimulation with plate-bound anti-CD3/CD28 mAbs in RPMI, Asn-deficient RPMI or RPMI with 10 IUs/L PEG-AsnASE added at the start of culture. (I) Representative flow cytometry contour plots depicting Annexin-V and propidium iodide staining in naive CD4+ T cell following 2 days of stimulation with plate-bound anti-CD3/CD28 mAbs in RPMI, Asn-deficient RPMI or RPMI with 10 IUs/L PEG-AsnASE added from the initiation of culture. Upper right quadrants depict the proportions of CD4+ T cells that have undergone apoptosis in each respective culture condition. (J) Quantification of the proportion of Annexin-V propidium iodide double-positive CD4+ T cells as shown in I. Each dot represents cells obtained from an individual animal (B-H, J). Results are shown as mean ± SD and are pooled from 2 independent experiments (B-H, J) or a representative of 2 independent experiments (A, I). non-significant (n.s), *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Turkey’s multiple comparison test (B-H, J) or Dunnet’s multiple comparison test (A).

Expression of CD4+ T cell activation proteins in asparagine sufficient and deficient conditions.

(A-C) Quantification of the proportions of CD4+ T cells expressing CD25 (A), PD-1 (B), and CD25 (C) following 2 days of stimulation with plate-bound anti-CD3/CD28 mAbs in complete RPMI, Asn-deficient RPMI or RPMI with 10 IUs/L PEGylated-asparaginase added at 0, 6, 12, 24, or 36 hours. Each dot represents cells obtained from an individual animal (A-C). Results are shown as mean ± SD and are representative of 2 independent experiments non-significant (n.s), *p < 0.05, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test.

Asparagine deprivation impairs mitochondrial function in CD4+ T cells.

(A-B) Naive CD4+ T cells were stimulated for 48 hours with plate-bound anti-CD3/CD28 mAbs in either complete RPMI media (RPMI), Asn-deficient RPMI or RPMI treated with 10 IUs/L PEGylated-asparaginase (PEG-AsnASE) added at 0, 6, 12, 24, or 36 hours. (A) Quantification of mitotracker green (MTG) gMFI. (B) Quantification of tetramethyl rhodamine methyl ester (TMRM) gMFI. Results are shown as mean ± SD and are representative of at least 2 independent experiments. non-significant (n.s), **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnet’s multiple comparison test.

Asparagine deprivation impairs pTH17 function in a model of induced EAE.

(A) Pathogenic T helper 17 (pTH17) cells were differentiated from naive CD4+ FoxP3− T cells from 2D2 TCR transgenic mice in RPMI media with or without Asn. Viable pTH17 polarized 2D2 cells were adoptively transferred (4×106/mouse) into 10-week-old C57BL/6J female recipients to induce EAE. Mice were scored daily for signs of disease. (B) At peak EAE (Day 16), 2D2 cells were isolated from the CNS and inguinal lymph (iLN) node and analyzed. Quantification of the absolute numbers of central nervous system (CNS)-infiltrating Vβ11+Vα3.2+ 2D2 pTH17 cells at peak of EAE (pTH17 RPMI n=16, pTH17 Asn-deficient RPMI n=12). (C) Quantification of the proportions of Vβ11+Vα3.2+ 2D2 pTH17 cells that have undergone apoptosis (Annexin-V+PI+) in the CNS and iLN node at peak of EAE. (D-E) Mitotracker green (MTG) and tetramethyl rhodamine methyl ester (TMRM) gMFI in iLN and CNS infiltrating TCRVβ11+TCRVα3.2+ CD4+ T cells at peak of EAE. Each dot represents an individual mouse (B-E). Results are shown as mean ± SD and are pooled (A-B) or a representative of at least 2 independent experiments (C-E). non-significant (n.s), *p < 0.05 **p < 0.01, ****p < 0.0001, two-way ANOVA (A), and Student’s t-test (B-E).