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. 2019 Aug 15;203(4):789-794.
doi: 10.4049/jimmunol.1800931. Epub 2019 Jun 28.

Cutting Edge: CXCR3 Escapes X Chromosome Inactivation in T Cells during Infection: Potential Implications for Sex Differences in Immune Responses

Steve Oghumu  1 , Sanjay Varikuti  2 , James C Stock  2 , Greta Volpedo  2 , Noushin Saljoughian  2 , Cesar A Terrazas  2 , Abhay R Satoskar  1
Affiliations

Cutting Edge: CXCR3 Escapes X Chromosome Inactivation in T Cells during Infection: Potential Implications for Sex Differences in Immune Responses

Steve Oghumu et al. J Immunol. .

Abstract

CXCR3, an X-linked gene, is subject to X chromosome inactivation (XCI), but it is unclear whether CXCR3 escapes XCI in immune cells. We determined whether CXCR3 escapes XCI in vivo, evaluated the contribution of allelic CXCR3 expression to the phenotypic properties of T cells during experimental infection with Leishmania, and examined the potential implications to sex differences in immune responses. We used a bicistronic CXCR3 dual-reporter mouse, with each CXCR3 allele linked to a green or red fluorescent reporter without affecting endogenous CXCR3 expression. Our results show that CXCR3 escapes XCI, biallelic CXCR3-expressing T cells produce more CXCR3 protein than monoallelic CXCR3-expressing cells, and biallelic CXCR3-expressing T cells produce more IFN-γ, IL-2, and CD69 compared with T cells that express CXCR3 from one allele during Leishmania mexicana infection. These results demonstrate that XCI escape by CXCR3 potentially contributes to the sex-associated bias observed during infection.

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Figures

Figure 1:
Figure 1:. Generation, characterization and validation of CXCR3RFP reporter and CXCR3GFP/RFP dual reporter mouse.
(A) Targeting vector for generation of CXCR3RFP reporter mouse and strategy for homologous integration onto the CXCR3 gene locus. Southern blot strategy for detection of wild type and mutated gene in electroporated ES cells. A similar strategy was used for confirming germ line transmission in F1 mice. p – probe; b – BamH1 site; tk – Thymidine kinase gene cassette; E1 and E2 – exons 1 and 2. (B) Southern blot screening of genomic DNA extracted from embryonic stem cell clones transfected with CXCR3RFP targeting vector. (C) Genotyping of wild type, homozygous and heterozygous CXCR3RFP mice by PCR is also shown. (D) Assessment of CXCR3 production in CXCR3GFP, CXCR3RFP and CXCR3WT mice by flow cytometry. Splenoctyes were activated in vitro with anti CD3 and CD28 antibodies according to a protocol previously described (22). CXCR3 was detected by staining with PE-Cy7 conjugated anti CXCR3 antibodies (CXCR3GFP, CXCR3RFP and CXCR3WT mice), GFP fluorescence (CXCR3GFP mice), and RFP fluorescence (CXCR3RFP mice). (E) Generation of female CXCR3GFP/RFP dual reporter mice by breeding CXCR3GFP and CXCR3RFP mice. In these female mice, one CXCR3 allele is tagged with a GFP reporter while the other allele is tagged with an RFP reporter.
Figure 2:
Figure 2:. CXCR3 gene escapes X-chromosome inactivation in T cells.
(A) T cells were isolated from spleens of CXCR3GFP/RFP mice and were activated in vitro. Allelic expression of CXCR3 was determined by GFP and RFP fluorescence using flow cytometry. WT, CXCR3GFP and CXCR3RFP mice were used as controls. Representative plots of bi-allelic expression of CXCR3 in CXCR3GFP/RFP mice are shown in the CD3 gated double positive (upper right) quadrant of CXCR3GFP/RFP mice. Graph showing quantification of GFP, RFP, and GFP+RFP expressing cells over three independent experiments. (B) Determination of bi-allelic CXCR3 gene expression in CXCR3GFP/RFP mice by imaging flow cytometry. Single cells were captured and visualized for CXCR3-GFP, CXCR3-RFP and CD3 expression. Dot plots and images of selected mono-allelic and bi-allelic CXCR3 expressing T cells are shown. Yellow arrows indicate images of bi-allelic CXCR3 expressing CD3+ T cells. (C) Mono-allelic, and bi-allelic CXCR3 expressing cells in female CXCR3GFP/RFP mice were activated and stained for CXCR3 protein expression using PE-Cy7 conjugated anti-CXCR3 antibodies. A dot plot of gating used to determine the levels of CXCR3 expression represented by mean fluorescence intensities (MFI) of the respective T cell populations, are shown on the left. MFI are shown as a bar graph. The negative control T cell population was obtained from CXCR3 knock out mice. *p value < 0.05. Error bars represents SEM.
Figure 3:
Figure 3:. Bi-allelic CXCR3 expressing T cells are increased during in vivo infection with Leishmania mexicana.
(A) Representative ear lesions in of CXCR3−/−, CXCR3+/− and CXCR3WT mice infected with L. mexicana. (B) Lesion thickness, circumference and parasitic burdens of CXCR3−/−, CXCR3+/− and CXCR3WT mice infected with L. mexicana. (C-E) Flow cytometric analysis of allelic CXCR3 expression in CD3+ gated cells of the (C) draining popliteal lymph node, (D) spleen and (E) ears of L. mexicana infected and uninfected CXCR3GFP/RFP dual reporter mouse. Graphs showing percentages of mono-allelic and bi-allelic CXCR3 expressing T cells are also shown. *p value < 0.05, **p value < 0.01. Error bars represents SEM. All experiments were performed at 8 weeks post infection.
Figure 4:
Figure 4:. Bi-allelic CXCR3 expressing T cells in female mice display a more activated phenotype upon activation during in vivo infection with Leishmania mexicana.
(A) parasitic loads in footpads of L. mexicana infected homozygous CXCR3GFP female and hemizygous CXCR3GFP male reporter mice (n=3 per group). (B) IFN-γ production and total cell count in sub-optimally re-stimulated CD3+ T cells from lymph nodes of L mexicana infected male and female CXCR3GFP reporter mice and negative control as determined by flow cytometry (n=3 per group). CD3+ T cells were gated. (C) Real-time PCR analysis of IFN-γ, IL-2. IL-4, CD69, CD25, T-bet, Gata3, and IRF-1 in mono-allelic, bi-allelic and non-CXCR3 expressing T cells in spleens of L. mexicana infected CXCR3GFP/RFP dual reporter mice. GFP–/RFP –, GFP+/RFP– GFP–/RFP+ and GFP+ /RFP+ T cells were sorted from infected CXCR3GFP/RFP mice for gene expression analysis. *p value < 0.05. Error bars represents SEM. (D) Percentage of GFP+/RFP– GFP–/RFP+ and GFP+/RFP+ splenic T cells producing IFN-γ after restimulation. (E-F) Expression of CD62L (E) and CD44 (F) in the spleen of L. mexicana infected CXCR3GFP/RFP dual reporter mice. *p value < 0.05. Error bars represents SEM.
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