C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/−) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/− mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/− mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/− T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/− mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.


Mice
The D409 V/null mice (9 V/null; Gba1 9V/− ) and WT control were of the mixed background FVB/C57BL/6J/129SvEvBrd (50:25:25) [4] and were 12 weeks of age. The new nomenclature for D409V includes the 39 amino acid leader sequence and would then be Asp448Val or p.D448V. Mice were maintained under pathogen-free conditions. All mice were housed under pathogen-free conditions in the barrier animal facility according to IACUC-approved protocol (IACUC2020-0052) at Cincinnati Children's Hospital Research Foundation (CCHRF).

Cell Preparation
Lung, spleen, blood, and peritoneal lavage from WT and Gba1 9V/− mice were removed aseptically. Single cell suspensions prepared from lung were obtained from minced pieces that were treated with Liberase Cl (0.5 mg/mL) and DNase (0.5 mg/mL) in RPMI (45 min, 37 • C) and spleen by direct grinding. Blood mononuclear cells were obtained after red blood cell (RBC) lysis (155 mM NH 4 Cl, 10 mM NaHCO 3 , 0.1 mM EDTA). Single cell suspensions prepared from lung, spleen, and the peritoneal lavage were filtered through a 70-micron cell strainer followed by RBC lysis, passage through a strainer, and pelleted by centrifugation at 350 g. Viable cells were counted using a Neubauer chamber and trypan blue exclusion. Mφs, DCs, CD4 + T lymphocytes, and CD8 + T lymphocytes were purified from single cell suspensions of lung and spleen using CD11c, CD11b, CD4 (L3T4), and CD8a (Ly2) microbeads according to the manufacturer's protocol.

Flow Cytometry
FACS staining was performed for characterization of immunological cell types in lung, spleen, blood, and peritoneal lavage and the chemotactic cells obtained after their migration. These cells were washed with PBS containing 1% BSA. After incubation for 15 min at 4 • C with the blocking antibody 2.4G2 (anti-FcγRII and III), all cells were stained at 4 • C for 45 min with the appropriate labeled antibodies for different cell types, i.e., antimouse CD11b and anti-mouse F4/80 antibodies for Mφs, anti-mouse CD11b, anti-mouse CD11c antibodies for DCs, anti-mouse CD3, CD4, and CD8, antibodies for T cells, and anti-mouse B220 antibodies for B cells. In separate batches, the cells were stained with the respective isotypes. Flow cytometric analyses were performed, where Mφs were gated first by their typical FSC/SSC pattern based on monocyte gated cells and their F4/80 positivity and double stained for F4/80 and CD11b. For DCs, monocyte gated cells from FSC/SSC pattern were gated for CD11c positivity and double stained for CD11c and CD11b. Purified Mφs and DCs were used to perform intracellular cytokine staining for CXCL9 and its isotype, (e.g., Armenian Hamster IgG). Flow cytometric analyses of T lymphocytes were generated after gating lymphocytes from forward and side scatter and then identifying the CD3 + , B220 − CD3 + CD4 + , and B220 − CD3 + CD8 + T lymphocytes. Mononuclear cells prepared from blood as well as purified CD4 + T and CD8 + T cells were used to perform surface staining for CXCR3 and its isotype, (e.g., Armenian Hamster IgG). In an addition experiment, purified CD4 + T cells and CD8 + T cells were used for chemotaxis assays. Flow cytometric analyses were performed on a LSR II, and FCS Express software.

T Cell Chemotaxis
CD4 + T cells prepared from spleen of WT and Gba1 9V/− mice were suspended in chemotaxis medium (GBSS containing 2% BSA) at a density of 5 × 10 6 cells/mL. The different concentration of CXCL9, (e.g., 0, 2, 4, 8, 16, and 32 nM) in chemotaxis medium, placed in the bottom wells of a micro-Boyden chambers and overlaid with a 3 µm polycarbonate membrane. Then, 50 µL of the cells were placed in the top wells and incubated for 45 min at 37 • C. Subsequently, the membranes were removed and the cells on the bottom side of the membrane were stained with Diff-Quick. The numbers of migrated cells in five high-power fields were counted and the number of cells per mm 2 was calculated by computer assisted light microscopy. Results are expressed as the mean value of triplicate samples.

Ex Vivo Blocking of CXCR3 and T Cells Chemotaxis
To examine whether ex vivo blocking of CXCR3 using mouse anti-CXCR3 antibodies can reduce CXCL9 mediated chemotaxis of T cell subsets in Gba1 9V/− GD model, spleen-derived CD4 + T cells and CD8 + T cells (5 × 10 6 cells/mL) prepared from WT and Gba1 9V/− mice were treated in the presence and absence of antibodies to mouse CXCR3 (10 µg/mL) at 4 • C for 30 min. These cells were applied to subsequent top wells of Boyden chemotaxis chamber and chemotaxis was performed towards CXCL9 (16 nM) at 37 • C and 5% CO 2 for 45 min. The membrane was removed, and cells were scraped off using a vertical glass slide on the top of 50 mL falcon tube. These cells were stained with antibodies to specific cell phenotypes as discussed above.

In Vivo Blocking of CXCR3 and T Cells Chemotaxis
To examine whether in vivo blocking of CXCR3 alters CXCL9-mediated increased tissue recruitment of T cells in Gaucher disease, WT (n = 5) and Gba1 9V/− mice (n = 5) were injected intraperitoneally (IP) with CXCL9 (200 nM:100 µL) and their vehicle (PBS). In some experiments, mice were pretreated intravenously (IV) with mouse anti-CXCR3 antibodies (1.0 mg/kg body weight) prior to IP injections of CXCL9 or vehicle (PBS). After 6 h, mice were killed, and the peritoneal cavity was lavage with 10 mL of PBS. Peritoneal cells were washed once with PBS, and 10 5 cells in 200 µL of PBS were used for performing FACS staining with antibodies to mouse CD3, CD4, and CD8s.

Determination of CXCL9 Production
Mφs and DCs purified from lung of the strain-matched Gba1 9V/− and WT mice were cultured (10 6 cells/200 µL of complete RPMI media) for 48 h. CXCL9 concentrations were determined in the cell supernatants by commercial ELISA kits according to the manufacturer's instructions.

Figure 1.
Immune phagocytes that cause increased amounts of CXCL9 in Gba1 9V/-mouse tissues. CXCL9 amounts in monocyte gated F4/80 hi CD11b + Mɸs (a-e) and CD11c hi CD11b + DCs (f-j) from lung of strain-matched Gba1 9V/-and WT mice (n = 5/group). Delta Mean Fluorescence Intensity (δ MFI): CXCL9 MFI-isotype MFI. In the histograms of isotypes (c-h), specific antibodies (d-i), and the bar diagrams, the black lines/columns correspond to WT and the maroon lines/columns to Gba1 9V/-cell. Values in d-h are the means ± SD. and asterisks show significant differences between WT and Gba1 9V/-mice (**** p < 0.0001). Three independent experiments were conducted, and groups were compared using student's t-tests.

Identification of CXCR3 Positive T Cells in Gba1 9V/-Mice
The single cell suspensions prepared from blood of WT and Gba1 9V/-mice were analyzed for CXCR3 + CD3 + T cells. Compared to WT mouse samples, those from Gba1 9V/-mice CD3 + T cells had elevated amounts of CXCR3 (Supplementary Figure S1a-d; p < 0.0001). An additional experiment CD4 + T cells and CD8 + T cells purified from lung of WT and Gba1 9V/-mice were analyzed for CXCR3. Compared to WT mice, Gba1 9V/-mouse CD4 + T cells had elevated CXCR3 (  Immune phagocytes that cause increased amounts of CXCL9 in Gba1 9V/− mouse tissues. CXCL9 amounts in monocyte gated F4/80 hi CD11b + Mφs (a-e) and CD11c hi CD11b + DCs (f-j) from lung of strain-matched Gba1 9V/− and WT mice (n = 5/group). Delta Mean Fluorescence Intensity (δ MFI): CXCL9 MFI-isotype MFI. In the histograms of isotypes (c-h), specific antibodies (d-i), and the bar diagrams, the black lines/columns correspond to WT and the maroon lines/columns to Gba1 9V/− cell. Values in d-h are the means ± SD. and asterisks show significant differences between WT and Gba1 9V/− mice (**** p < 0.0001). Three independent experiments were conducted, and groups were compared using student's t-tests.

Identification of CXCR3 Positive T Cells in Gba1 9V/− Mice
The single cell suspensions prepared from blood of WT and Gba1 9V/mice were analyzed for CXCR3 + CD3 + T cells. Compared to WT mouse samples, those from Gba1 9V/− mice CD3 + T cells had elevated amounts of CXCR3 (Supplementary Figure S1a-d; p < 0.0001). An additional experiment CD4 + T cells and CD8 + T cells purified from lung of WT and Gba1 9V/− mice were analyzed for CXCR3. Compared to WT mice, Gba1 9V/− mouse CD4 + T cells had elevated CXCR3 (Figure 2a   CXCR3 surface expression in pulmonary T cell subsets from Gba1 9V/-mice. CXCR3 expression in FACS-sorted CD3 + CD4 + T cells (a-d) and CD3 + CD8 + T cells (e-h) from lung of strain-matched Gba1 9V/-and WT mice (n = 5/group). δ MFI: CXCR3 MFI-isotype MFI. In the dot plots of isotypes and specific antibodies (a,e), in the histograms of isotypes (b,f), specific antibodies (c,g), and the bar diagrams (d,h), the black lines/columns correspond to WT and the maroon lines/columns to Gba1 9V/-mice. Values in d and h are the means ± SD. and asterisks show significant differences between WT and Gba1 9V/-mice (**** p < 0.0001). Three independent experiments were conducted, and groups were compared using student's t-tests.

Effect of CXCL9 in Ex Vivo Chemotaxis of T Cells in Gba1 9V/-Mice
Gba1 9V/-mice immune phagocytes, (e.g., Mɸs and DCs) showed increased amounts of CXCL9 and their receptor CXCR3 on T cell subsets when compared to WT. These data suggested a potential role of the CXCL9-CXCR3 pathway for increased numbers of T cells in Gba1 9V/-mouse tissues. To confirm this, several concentrations of CXCL9 (0, 2, 4, 8, and 16 nM) were used to generate dose response curves for ex vivo chemotaxis of WT and Gba1 9V/-mouse spleen-derived CD4 + T cells. CXCL9 caused dose-depended increase in chemotaxis of CD4 + T cells in WT and Gba1 9V/-mice; compared to WT, such effects were more pronounced in Gba1 9V/-mice (Figure 3a-c; p < 0.01; p < 0.0001). δ MFI: CXCR3 MFI-isotype MFI. In the dot plots of isotypes and specific antibodies (a,e), in the histograms of isotypes (b,f), specific antibodies (c,g), and the bar diagrams (d,h), the black lines/columns correspond to WT and the maroon lines/columns to Gba1 9V/− mice. Values in d and h are the means ± SD. and asterisks show significant differences between WT and Gba1 9V/− mice (**** p < 0.0001). Three independent experiments were conducted, and groups were compared using student's t-tests. Figure 2. CXCR3 surface expression in pulmonary T cell subsets from Gba1 9V/-mice. CXCR3 expression in FACS-sorted CD3 + CD4 + T cells (a-d) and CD3 + CD8 + T cells (e-h) from lung of strain-matched Gba1 9V/-and WT mice (n = 5/group). δ MFI: CXCR3 MFI-isotype MFI. In the dot plots of isotypes and specific antibodies (a,e), in the histograms of isotypes (b,f), specific antibodies (c,g), and the bar diagrams (d,h), the black lines/columns correspond to WT and the maroon lines/columns to Gba1 9V/-mice. Values in d and h are the means ± SD. and asterisks show significant differences between WT and Gba1 9V/-mice (**** p < 0.0001). Three independent experiments were conducted, and groups were compared using student's t-tests.

Pharmaceutical Targeting of CXCR3 Leads to the Reduction of CXCL9 Mediated Ex Vivo T Cells Chemotaxis in Gba1 9V/− Mice
Pharmaceutical blocking of CXCR3 confirmed the altered CXCL9-mediated ex vivo T cell chemotaxis in Gba1 9V/− mice. Mouse anti-CXCR3 antibodies or vehicle (PBS) treated WT and Gba1 9V/− mouse spleen-derived CD4 + T cells were used for assessing their chemotaxis towards CXCL9 (Figure 4a-d). Similarly, mouse anti-CXCR3 antibodies or vehicle (PBS) treated WT and Gba1 9V/− mouse spleen-derived CD8 + T cells were used for assessing their chemotaxis towards CXCL9 (Figure 5a-d).

Pharmaceutical Targeting of CXCR3 Leads to the Reduction of CXCL9 Mediated Ex Vivo T Cells Chemotaxis in Gba1 9V/-Mice
Pharmaceutical blocking of CXCR3 confirmed the altered CXCL9-mediated ex vivo T cell chemotaxis in Gba1 9V/-mice. Mouse anti-CXCR3 antibodies or vehicle (PBS) treated WT and Gba1 9V/-mouse spleen-derived CD4 + T cells were used for assessing their chemotaxis towards CXCL9 (Figure 4a-d). Similarly, mouse anti-CXCR3 antibodies or vehicle (PBS) treated WT and Gba1 9V/-mouse spleen-derived CD8 + T cells were used for assessing their chemotaxis towards CXCL9 (Figure 5a-d).
In additional experiments, anti-CXCR3 antibodies and vehicle (PBS) were used to treat WT and Gba1 9V/-mice spleen-derived CD4 + T and CD8 + T cells. These   In additional experiments, anti-CXCR3 antibodies and vehicle (PBS) were used to treat WT and Gba1 9V/− mice spleen-derived CD4 + T and CD8 + T cells. These CD4 + T cells.

Pharmaceutical Targeting of CXCR3 Causes the Reduction of CXCL9 Mediated In Vivo T Cells Chemotaxis in Gba1 9V/-Mice
To confirm if in vivo administration of mouse anti-CXCR3 antibodies decrease the CXCL9-mediated chemotaxis of T cell subsets in GD, WT and Gba1 9V/-mice were treated with CXCL9 and its vehicle (PBS) in the presence and absence of mouse anti-CXCR3 antibodies The peritoneal cells were analysed for total cell infiltrates as well as the CD3 + CD4 + T cells and CD3 + CD8 + T cells (see Methods). Compared to vehicle (PBS) or mouse anti-CXCR3 antibodies, administered CXCL9 to WT mice showed increased peritoneal cell recruitment (Supplementary Figure S4a-c,e; p < 0.01). Compared to administered CXCL9, Three independent experiments were conducted (**** p < 0.0001).

Pharmaceutical Targeting of CXCR3 Causes the Reduction of CXCL9 Mediated In Vivo T Cells Chemotaxis in Gba1 9V/− Mice
To confirm if in vivo administration of mouse anti-CXCR3 antibodies decrease the CXCL9-mediated chemotaxis of T cell subsets in GD, WT and Gba1 9V/− mice were treated with CXCL9 and its vehicle (PBS) in the presence and absence of mouse anti-CXCR3 antibodies The peritoneal cells were analysed for total cell infiltrates as well as the CD3 + CD4 + T cells and CD3 + CD8 + T cells (see Methods). Compared to vehicle (PBS) or mouse anti-CXCR3 antibodies, administered CXCL9 to WT mice showed increased peritoneal cell recruitment (Supplementary Figure S4a These findings were also obtained for CD4 + T cells (Figure 6e-i; p < 0.0001, p < 0.001). In WT mice, the CD4 + T cell differences were not significant when compared with vehicle, mouse anti-CXCR3 antibodies, CXCL9 and mouse anti-CXCR3 antibodies administered prior to CXCL9 (Figure 6a-e; ns). mouse anti-CXCR3 antibodies given prior to CXCL9 injection abrogated the CXCL9 mediated increased recruitment of peritoneal cells in WT mice (Supplementary Figure S4ce; p < 0.01). As compared to vehicle or mouse anti-CXCR3 antibodies administration, CXCL9 injected Gba1 9V/-mice showed more pronounced peritoneal cell infiltrates (Supplementary Figure S4f-h, and e; p < 0.0001). Compared to administered CXCL9, mouse anti-CXCR3 antibodies given prior to CXCL9 injection caused marked reductions in the increased recruitment of peritoneal cells in Gba1 9V/-mice (Supplementary Figure S4e,h,i; p < 0.0001). These findings were also obtained for CD4 + T cells (Figure 6e-i; p < 0.0001, p < 0.001). In WT mice, the CD4 + T cell differences were not significant when compared with vehicle, mouse anti-CXCR3 antibodies, CXCL9 and mouse anti-CXCR3 antibodies administered prior to CXCL9 (Figure 6a-e; ns). Figure 6. In vivo blocking of CXCR3 alters the CXCL9-mediated CD4 + T cells chemotaxis in Gba1 9V/-mice. WT and Gba1 9V/mice were injected with intraperitoneal administration of CXCL9 and its vehicle as described in the method. In additional experiments, these mice were injected with intravenous injection of mouse anti-CXCR3 antibodies prior to intraperitoneal injection of CXCL9 or vehicle and the peritoneal cells were collected and analyzed by FACS. The dot plots and the corresponding bar diagrams represent the percentage of migrated CD3 + CD4 + T cells in WT (a-e) and Gba1 9V/-mice (e-i). WT (black columns), Gba1 9V/-(maroon columns) and the values shown are the mean ± SD. and group comparison were performed with ANOVA. Three independent experiments were conducted (ns, not significant; ***, p < 0.001, ****, p < 0.0001).
As compared to PBS or mouse anti-CXCR3 antibodies treated mice, CXCL9 injected Gba1 9V/-mice showed elevated recruitment of peritoneal CD8 + T cells (Figure 7e  In additional experiments, these mice were injected with intravenous injection of mouse anti-CXCR3 antibodies prior to intraperitoneal injection of CXCL9 or vehicle and the peritoneal cells were collected and analyzed by FACS. The dot plots and the corresponding bar diagrams represent the percentage of migrated CD3 + CD4 + T cells in WT (a-e) and Gba1 9V/− mice (e-i). WT (black columns), Gba1 9V/− (maroon columns) and the values shown are the mean ± SD. and group comparison were performed with ANOVA. Three independent experiments were conducted (ns, not significant; ***, p < 0.001, ****, p < 0.0001).
As compared to PBS or mouse anti-CXCR3 antibodies treated mice, CXCL9 injected Gba1 9V/− mice showed elevated recruitment of peritoneal CD8 + T cells (  In vivo blocking of CXCR3 alters the CXCL9-mediated CD8 + T cells chemotaxis in Gba1 9V/-mice. WT and Gba1 9V/mice were injected with intraperitoneal administration of CXCL9 and its vehicle as described in the method. In additional experiments, these mice were injected with intravenous injection of mouse anti-CXCR3 antibodies prior to intraperitoneal injection of CXCL9 or vehicle and the peritoneal cells were collected and analyzed by FACS. The dot plots and the corresponding bar diagrams represent the percentage of migrated CD3 + CD8 + T cells in WT (a-e) and Gba1 9V/-mice (e-i). WT (black columns), Gba1 9V/-(maroon columns), and the values shown are the mean ± SD. and group comparison were performed with ANOVA. Three independent experiments were conducted (ns, not significant; * p < 0.05; **** p < 0.0001).

Discussion
Here, Mɸs and DCs have been recognized as the sources of local increases of CXCL9 in Gba1 9V/-mice. Furthermore, CD4 + T cells and the CD8 + T cells from Gba1 9V/-mice had increased amounts of CXCR3. Although not explicitly tested, these findings implicate mutant Gba1 and the resultant excess tissue accumulation of GC in increasing the production/expression of CXCL9 in GD. Such increased CXCL9 directed the chemotaxis of CXCR3 expressing T cell subsets in the Gba1 9V/-mouse model of GD. This is supported by the lung-derived Gba1 9V/-Mɸs and DCs having increased amounts of CXCL9 and the T cell subsets with increased amounts of CXCR3.
In addition, these findings highlight the importance of the CXCL9-CXCR3 axis in the induction of ex vivo and in vivo chemotaxis of CD4 + and CD8 + T cells in the Gba1 9V/-mouse.
In certain tissues, e.g., lung, intestine, and tumor, cause downregulation of chemokine receptor expression once the infiltrating cells reside in the tissues and are exposed to high concentrations of ligands and/or interact with abnormal local production of pro-in- Figure 7. In vivo blocking of CXCR3 alters the CXCL9-mediated CD8 + T cells chemotaxis in Gba1 9V/− mice. WT and Gba1 9V/− mice were injected with intraperitoneal administration of CXCL9 and its vehicle as described in the method. In additional experiments, these mice were injected with intravenous injection of mouse anti-CXCR3 antibodies prior to intraperitoneal injection of CXCL9 or vehicle and the peritoneal cells were collected and analyzed by FACS. The dot plots and the corresponding bar diagrams represent the percentage of migrated CD3 + CD8 + T cells in WT (a-e) and Gba1 9V/− mice (e-i). WT (black columns), Gba1 9V/− (maroon columns), and the values shown are the mean ± SD. and group comparison were performed with ANOVA. Three independent experiments were conducted (ns, not significant; * p < 0.05; **** p < 0.0001).

Discussion
Here, Mφs and DCs have been recognized as the sources of local increases of CXCL9 in Gba1 9V/− mice. Furthermore, CD4 + T cells and the CD8 + T cells from Gba1 9V/− mice had increased amounts of CXCR3. Although not explicitly tested, these findings implicate mutant Gba1 and the resultant excess tissue accumulation of GC in increasing the production/expression of CXCL9 in GD. Such increased CXCL9 directed the chemotaxis of CXCR3 expressing T cell subsets in the Gba1 9V/− mouse model of GD. This is supported by the lung-derived Gba1 9V/− Mφs and DCs having increased amounts of CXCL9 and the T cell subsets with increased amounts of CXCR3.
In addition, these findings highlight the importance of the CXCL9-CXCR3 axis in the induction of ex vivo and in vivo chemotaxis of CD4 + and CD8 + T cells in the Gba1 9V/− mouse.
In certain tissues, e.g., lung, intestine, and tumor, cause downregulation of chemokine receptor expression once the infiltrating cells reside in the tissues and are exposed to high concentrations of ligands and/or interact with abnormal local production of proinflammatory cytokines [86][87][88][89]. To avoid this limitation, the current study used spleen derived T cells for testing CXCL9-mediated ex-vivo chemotaxis in the Gba1 9V/− mouse. This study identified a direct role of the CXCL9-CXCR3 axis in aiming the excess tissue recruitment of T cells in the Gba1 9V/− mouse. CXCR3 is an attractive therapeutic target for treating T cell-mediated inflammatory diseases [83,84,[90][91][92][93][94][95][96]. CXCL9-mediated ex vivo and in vivo chemotaxis of T cell subsets (i.e., CD4 + T and CD8 + T cells) in the presence or absence of mouse anti-CXCR3 antibodies showed that targeting CXCR3 caused marked reduction in CXCL9-mediated enhanced tissue recruitment of T cell subsets in GD.
Mice express a single isoform of CXCR3 that exclusively binds to CXCL9, CXCL10, and CXCL11. CXCR3a, b and alt isoforms exist in humans [97]. Human CXCR3a is equivalent to mouse CXCR3 and binds CXCL9, CXCL10, and CXCL11. Human CXCR3b binds to CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. Human CXCR3alt binds specifically to CXCL11 [97]. The translational potential of this research could be challenging as in contrast to murine CXCL9 and CXCL11, human CXCL9-11 are inactivated rapidly in the presence of physiological concentrations of dipeptidyl peptidase IV/CD26 [98][99][100]. Despite of these complexities, the current study invites investigation into the different isoforms of CXCR3 and their ligands, i.e., CXCL9-11, as well as their up and/or downstream signaling that enhance T cell trafficking in GD. These findings open new areas of research that may identify CXCR3 and several of their ligands as interesting drug targets for modulation of immune cell function that fuel tissue inflammation in GD and other lysosomal storage sicknesses.