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. 2020 Jan 28:11:52.
doi: 10.3389/fimmu.2020.00052. eCollection 2020.

PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion

Johanna A Knipper  1 , David Wright  1 , Andrew P Cope  2 , Bernard Malissen  3   4 , Rose Zamoyska  1
Affiliations

PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion

Johanna A Knipper et al. Front Immunol. .

Abstract

Lymphopenic insult has been shown to precipitate the initiation of autoimmune disease in murine models such as the Non-obese diabetic mouse. Similarly, in man lymphopenia induced by mAb therapy, for instance Alemtuzumab as treatment for Multiple Sclerosis, can precipitate development of secondary autoimmune disease in up to 30 % of patients. We asked whether an identified autoimmune susceptibility locus might increase the risk of developing autoimmunity in the context of mAb-induced lymphopenia in a mouse model. A single nucleotide polymorphism (SNP) in the gene encoding the tyrosine phosphatase PTPN22 (R620W) is associated with multiple human autoimmune diseases, and PTPN22 has been shown to modulate T cell responses, particularly to weak antigens. In keeping with this, PTPN22-deficient or PTPN22 R619W mutant murine T cells adoptively transferred into immunodeficient lymphopenic hosts showed a higher lymphopenia-induced proliferation rate than WT cells. We induced lymphopenia by treating wild-type or PTPN22 knock-out mice with T cell depleting antibodies and monitored reconstitution of the T cell pool. We found that PTPN22 deficient T cells acquired a more activated effector phenotype, with significantly more IFNγ producing cells. This resulted from expansion driven by self-peptide MHC, as it was evident when the contribution of IL-7 to lymphopenic expansion was blocked with IL-7R Ab. Interestingly, Foxp3+ Tregs were also considerably expanded in PTPN22-deficient and PTPN22 R619W mice, as was the frequency of both CD25+ and CD25- CD4 T cells that produce IL-10. Using bone marrow chimeric mice, we showed that PTPN22 influenced development of both regulatory and effector T cell functions in a cell-intrinsic manner. Overall the expansion of Tregs is likely to keep the expanded T effector populations in check and sparing Treg during therapeutic mAb depletion may be a useful strategy to prevent occurrence of secondary autoimmunity.

Keywords: PTPN22; autoimmunity; interleukin-7; lymphopenia; regulatory T cells.

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Figures

Figure 1
Figure 1
PTPN22 controls T cell effector phenotype but does not affect T cell proliferation rate in response Ab-mediated lymphopenia. (A) Scheme of experimental set-up. T cell expansion was assessed by measuring CD4 and CD8 T cell frequencies in blood before T cell depletion throughout the length of the experiment. Two-way ANOVA with Sidak's multiple comparison test. N = 4–6 animals per group, one representative experiment out of three independent experiments in total. Absolute T cell numbers and T cell phenotype analysis in Spleen 2 weeks post depletion (B) and 8 weeks post depletion (C). Each dot represents one animal, data pooled of two independent experiments in (B) with N = 6–10 animals per group. Data in (A,C) shows one representative experiment out of three independent experiments in total with N = 4–6 animals per group. Two-way ANOVA with Sidak's multiple comparison test. n.s. = non-significant. A p < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
PTPN22 controls T cell proliferation when IL-7 availability is limited. (A) Scheme of experimental set-up. (B) CD4 and CD8 T cell frequency in blood 3 and 10 days post T cell depletion in combination with IL-7Rα blockade. Data means with SD shown, N = 7 animals per genotype and time point. Two-way ANOVA with Sidak's multiple comparison test. (C) Analysis of T cells number and IFNγ expression in spleen 2 weeks post T cell depletion or T cell depletion in combination with IL-7 Rα blockade. Each dot represents one animal, N = 7–8 animals per group, shown data is pooled of two independent experiments. Two-way ANOVA with Sidak's multiple comparison test. A p < 0.05 was considered significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 3
Figure 3
Foxp3+ Tregs and IL-10 production are cell-intrinsically controlled by PTPN22 in steady state and LIP. (A) Ab-mediated depletion efficacy of CD4+Foxp3+ Tregs and CD4+Foxp3− conventional CD4 T cells 3 days post anti-CD4 and anti-CD8 injection. Data shown as mean with SD with N = 7 animals per group. Pooled data of two independent experiments. (B) Proportion and absolute numbers of regulatory T cells 2 weeks after T cell depletion in combination with IL-7R blockade and (C) 8 weeks post depletion. Each dot represents one animal. In (B) N = 7–8 animals per group, shown data is pooled of two independent experiments. In (C) N = 4–6 animals per group, one representative experiment out of three independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (D) IL-10 production by CD4 T cells, CD4+CD25+ Tregs and CD4+CD25− conventional T cells 2 weeks post depletion. To analyze IL-10 production cells were re-stimulated in presence of Brefeldin A ex vivo. Each dot represents one animal. Each dot represents one animal. N = 6–8 animals per group, shown data is pooled of two independent experiments. Two-way ANOVA with Sidak's multiple comparison test. (E) Bone marrow from CD45.1 PTPN22 WT and CD45.2 PTPN22 KO donor animals was mixed 1:1 and transplanted in into fully irradiated CD45.1/2 PTPN22 WT recipient mice. The Foxp3+ frequency within PTPN22 WT CD45.1+CD4+ or PTPN22 KO CD45.2+CD4+ cell pool was assessed 15 weeks post transfer. Each dot represents one animal (N = 11), data is pooled of two independent experiments. A paired t-test was performed to compare PTPN22 WT and KO cells within the same recipient. (F,G) Phenotypical analysis of Foxp3+ expressing PTPN22 WT CD45.1+CD4+ and PTPN22 KO CD45.2+CD4+ cells from the experiment outlined in (E). Each dot represents one animal (N = 11), data is pooled of two independent experiments. A paired t-test was performed to compare PTPN22 WT and KO cells within the same recipient. A p < 0.05 was considered significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 4
Figure 4
CD4 PTPN22 KO cells outnumber WT when co-transferred into lymphopenic host. (A) Adoptive transfer of naive CD4+CD45.1+ PTPN22 WT and CD4+CD45.2+ PTPN22 KO cells into lethally irradiated CD45.1/2 WT hosts in a 1:1 ratio. Ratio of transferred cells was assessed 11 days post transfer in spleen and lymph nodes. (B) Analysis of transferred cells 11 days post transfer for expression of CD44, Ki67 and (C) cytokines IL-10 and IFNγ. (D) Expression of CD25 before transfer and expression of Foxp3 of transferred CD4 T cell population post transfer. (E) Frequency of Foxp3+ expressing cells within the CD4+CD45.1+ PTPN22 WT cell population in comparison to Foxp3 expressing cells within the CD4+CD45.2+ PTPN22 KO cell population. Data shown in (A–E) is from one representative experiment of two independent experiments is shown with N = 6 animals. Each dot represents one animal. CD45.1+ PTPN22 WT and CD45.2+ PTPN22 KO cells obtained from the same host were compared by a paired t-test in (A–E). A p < 0.05 was considered significant *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
PTPN22 R619W negatively regulates T cell proliferation in response to lymphopenia. (A) Adoptive transfer of CD45.1 PTPN22 WT, CD45.1/2 PTPN22 R619W and CD45.2 PTPN22 KO CD8 OT-1 cells in a 1:1:1 ratio into lymphopenic Rag-1 KO hosts. Frequency of CD8 T cells from the different genotypes were analyzed 4 weeks after transfer. (B) Analysis of CD44 expression and (C) IFNg expression in response to re-stimulation with T4 peptide 4 weeks post transfer. Data shown in (A–C) is from one representative experiment out of two independent experiments with N = 5 recipient animals, each dot represents one animal. PTPN22 WT, R619W and KO cells were compared by paired one-way ANOVA. (D) CD45.1 PTPN22 WT and CD45.2 PTPN22 R619W bone marrow was mixed 1:1 and transplanted in into fully irradiated CD45.2 PTPN22 R619W recipient mice. The Foxp3+ frequency within PTPN22 WT CD45.1+CD4+ or PTPN22 R169W CD45.2+CD4+ cell pool was assessed 15 weeks post transfer. Each dot represents one animal (N = 13), data is pooled of two independent experiments. A paired t-test was performed to compare PTPN22 WT and R619W cells within the same recipient. (E) Phenotypical analysis of Foxp3+ expressing PTPN22 WT CD45.1+CD4+ and PTPN22 KO CD45.2+CD4+ cells from the experiment outlined in (D). Each dot represents one animal (N = 13), data is pooled of two independent experiments. A paired t-test was performed to compare PTPN22 WT and R619W cells within the same recipient. n.s., non-significant. A p < 0.05 was considered significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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