Impaired T cell function in argininosuccinate synthetase deficiency

Abstract

ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In human and mouse tissues, ASS1 protein is found in several components of the immune system, including the thymus and T cells. However, the role of ASS1 in these tissues remains to be defined. Considerable attention has been focused recently on the role of metabolism in T cell differentiation and function. Based on the expression of ASS1 in the immune system, we hypothesized that ASS1 deficiency would result in T cell defects. To evaluate this question, we characterized immune function in hypomorphic fold/fold mice. Analysis of splenic T cells by flow cytometry showed a marked reduction in T cell numbers with normal expression of activation surface markers. Gene therapy correction of liver ASS1 to enhance survival resulted in a partial recovery of splenic T cells for characterization. In vitro and in vivo studies demonstrated the persistence of the ASS1 enzyme defect in T cells and abnormal T cell differentiation and function. Overall, our work suggests that ASS1 plays a role in T cell function, and deficiency produces primary immune dysfunction. In addition, these data suggest that patients with ASS1 deficiency (citrullinemia type I) may have T cell dysfunction.

Keywords: arginine; differentiation; immunity; metabolism.

Figure 1.. ASS1 in the mammalian immune system.

(A) Proposed model of ASS1 activity in T cells. (B) Mouse immune organs contain mouse ASS1 (mASS1) protein, and protein homogenates from various WT mouse organs were probed by immunoblot for mouse ASS1. (C) Human immune organs contain hASS1. Protein homogenates from various human immune organs were probed by immunoblot for hASS1. (D) hASS1 in peripheral blood. PBMCs and naïve T cells from healthy volunteers (n = 5) were stained for hASS1 with confirmation by immunoblot. Red, Control antibody; all other colors, hASS1. Representative figure for flow cytometry of naïve T cells and immunoblot confirmation (3 separate experiments). (E) Mouse T cells can proliferate with citrulline supplementation. Mouse splenocytes (n = 4) were stimulated, and proliferation was measured in arginine-free media, supplemented with citrulline or ASA or arginine or ornithine. Cit, Citrulline; Arg, arginine; Orn, ornithine. Error bars indicate sem. The data are representative of 3 independent experiments with similar results. (F) A subset of human T cells can proliferate with citrulline supplementation. Human PBMCs (n = 3) were stimulated with PHA-L in arginine-free media, supplemented with 1 mM arginine or 1 mM citrulline. **P < 0.01. Error bars indicate sem. The data are representative of 3 independent experiments with similar results.

Figure 2.. Improvement of T cell numbers following gene therapy-treated fold/fold mice.

(A) Increase in splenocyte numbers in treated fold/fold mice. fold/fold mice (n = 8) treated with liver-directed gene therapy [18] to improve survival resulted in increased spleen size and splenocyte numbers. (B) Splenic T cells in treated fold/fold. Splenocytes from treated fold/fold mice showed comparable CD4 and CD8 T cell ratios by flow cytometry. (C) Thymic T cells in treated fold/fold. Reduced thymocytes from treated fold/fold mice (lower) showed comparable ratios of double-positive T cells (upper) by flow cytometry. (D) Cell surface markers on treated fold/fold T cells. Cell surface markers indicative of activation (CD44, CD69) and cellular arginine status (CD247) were comparable with WT. *P < 0.05. Error bars indicate sem. The data are representative of 3 independent experiments with similar results.

Figure 3.. Persistence of ASS1 deficiency in fold/fold T cells.

(A) hASS1 expression is absent in fold/fold T cells. Gene therapy in fold/fold mutants resulted in liver-specific expression on hASS1 (n = 3/condition). (B) T cells from fold/fold do not proliferate in arginine-free, citrulline-supplemented media. Splenic T cells (n = 3) from WT (n = 3) and treated fold/fold mice were stimulated in arginine-free media with 1 mM citrulline. Error bars indicate sem. The data are representative of 3 independent experiments with similar results. ***P < 0.001.

Figure 4.. Abnormal differentiation of fold/fold T cells.

(A) Sorted, naïve CD4 T cells (CD4⁺CD62L^highCD44^lowCD25^neg) were differentiated under (B) Th1 and Th17 conditions in the presence of T-depleted splenocytes as APCs for 3 d. Cells were restimulated with PMA + ionomycin and Golgi stop for 4 h and stained for different intracellular cytokines. The data are representative of 3 independent experiments with similar results. *P < 0.05.

Figure 5.. Abnormal fold/fold T cell response to influenza.

Bone marrow from WT (n = 5) and treated fold/fold (n = 5) mice was transplanted into WT mice. To isolate the T cell response, infection was performed initially with X31, followed by PR8 at 30 d. Tetramer-positive CD4⁺ T cells for (A) NP and (B) PA peptides were detected by flow cytometry. C) Splenic weights following radiation and bone marrow reconstitution. *P < 0.05; **P < 0.01.