
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)02055-8
10.1016/j.isci.2024.110830
110830
Article
CXCR4-enriched T regulatory cells preferentially home to bone marrow and resolve inflammation
Huang Meixian 1
Ke Zeng 1
Lyu Mi-Ae 1
Masarova Lucia 2
Sadeghi Tara 3
Flowers Christopher R. 1
Parmar Simrit simrit.parmar@gmail.com
14∗
1 Department of Lymphoma/ Myeloma, MD Anderson Cancer Center, Houston, TX, USA
2 Department of Leukemia, MD Anderson Cancer Center, Houston, TX, USA
3 Cellenkos Inc., Houston, TX, USA
∗ Corresponding author simrit.parmar@gmail.com
4 Lead contact

27 8 2024
20 9 2024
27 8 2024
27 9 11083012 4 2024
15 6 2024
23 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

CXCR4 cell surface expression is critical for the homing of T regulatory (Treg) cells to the bone marrow (BM). We hypothesize that CXCR4 enrichment on Tregs cell surface may abbreviate their transit time to reach BM. Umbilical cord-blood CD25+ Tregs underwent CXCR4 dual enrichment and ex vivo expansion using the CRANE process to generate CXCR4-enriched Tregs (TregCXCR4) cells, which showed a faster migration across the Transwell membrane toward CXCL12/stromal cell-derived factor 1α (SDF1α) at 15, 30, and 60 min, when compared to unmanipulated Tregcontrol cells (p < 0.0001). TregCXCR4 exhibited preferential homing to BM in vivo at 12 and 24 h. Metacluster analysis of BM showed a decrease in CD8+ and an increase in CD39 and CD73 and CXCR5 when compared to Tregcontrol. TregCXCR4 decreased plasma TGF-β1/β2 and IFN-γ levels. When compared to control, TregCXCR4 cells decreased in CD8+ T cell, IFN-γ, and TNF-α expression in BM. We conclude that TregCXCR4 show enhanced migration toward CXCL12/SDF1α and a preferential homing to BM resulting in resolution of inflammation.

Graphical abstract

Highlights

• CXCR4-enriched Tregs (TregCXCR4) exhibit upregulation of FoxP3, Helios, and IL-10

• TregCXCR4 transit faster toward CXCL12/SDF-1α ligand compared to Tregcontrol

• TregCXCR4 preferentially home to bone marrow to decrease CD8, IFN-γ, and TNF-α expression

• TregCXCR4 decrease TGF-β1/β2 and IFN-γ levels in plasma

Immunology; Immunity; Cell biology

Subject areas

Immunology
Immunity
Cell biology
Published: August 27, 2024
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pmcIntroduction

Adoptive therapy with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) can resolve lung inflammation,1,2,3 prevent graft vs. host disease,4,5,6 treat bone marrow (BM) failure,7 and improve albuminuria in lupus nephritis.8 Treg cell migration requires tissue-specific cues involving integrins, chemokine, and other G protein-coupled receptors.9 Specifically, CXCR4 signaling has been shown to play a critical role in the homing of Tregs to the bone marrow, where blocking CXCR4 decreases human Treg bone marrow trafficking,10 and ablation of CXCR4 in Treg cells in vivo resulted in their moderate but statistically significant decrease in the bone marrow.11 Therefore, we hypothesize that CXCR4 enrichment would generate a relatively homogenous Treg cell population, which would result in a relatively quantified payload delivery of these cells. Especially in diseases such as myelofibrosis, where the CXCR4/CXCl12 axis plays a major role in the disease pathogenesis,12 with a larger role for uncontrolled inflammation,13 CXCR4-enriched Tregs would be an ideal therapeutic candidate.

We also evaluated whether the regulatory/suppressor function of the CXCR4-enriched Treg cells would be different from the control Treg cells based on their differences in the migratory capacity.14

Results

Dual enrichment of UCB Treg cells leads to higher CXCR4 cell surface expression and faster homing to its ligand SDF1α

Post CD25 enrichment,1 the cell surface expression of CD4+25hi127lo increased from 34.8% to 80.2%, and the co-expression of intracellular FoxP3 and Helios increased from 10.6% to 76.8% when gated on CD4+ T cells, 17.6%–91.9% when gated on CD4+25 hi127lo Tregs, and remained unchanged at 6.3%–10.1% when gated on CD4+25− conventional T cell (Tcon), compared to their respective levels prior to CD25 enrichment (Figure 1A; Table S1). A second CXCR4 enrichment of day 3 or 4 cultured cells led to a significant increase in the CXCR4hi cell population (Figures 1B and 1C) from 73.8% ± 11.4% to 95.1% ± 2.8% (Figure 1D). The enrichment process led to an increase in median fluorescence intensity (MFI) of CXCR4 from 25,945 ± 7,320 to 134,065 ± 11,950 (n = 3, p < 0.001, mean ± SD). After 14 days of culture, CXCR4-enriched Treg cells (TregCXCR4) and control Treg cells (Tregcontrol) showed no differences in CD4+8+ (Figure 1E) or CD4+25 + 127lo cell populations (Figure 1F). These populations are shown in Figure 1G. The percent of FOXP3 demethylation in Tcon, Tregcontrol, and TregCXCR4 was 3.9 ± 0.8, 52.5 ± 14.4, and 60.4 ± 19.5, respectively (Figure 1H, p < 0.0001). Additionally, when compared to TregCXCR4negative, the TregCXCR4 cells showed a higher percent of FOXP3 demethylation (41.0 ± 25.9 vs. 72.3 ± 10.3, p = 0.03, Figure 1I). In order to examine the impact of CXCR4 enrichment on their function, TregCXCR4 and Tregcontrol cells were activated with CD3/CD28 beads and IL-2, revealing no discernible disparities in the secretion of suppressor cytokine, IL-10 (Figure 1J), whereas a significantly higher suppression of Tcon cell proliferation by TregCXCR4 (80.1 ± 11.3%) was observed at a 1:1 ratio when compared to Tregcontrol cells (60% ± 8.4%) (p < 0.001, Figure 1K).Figure 1 CXCR4-enriched Tregs home faster to SDF1α

(A) Fluorescence-activated cell sorting (FACS) plots were utilized to compare the percentages of CD4+CD25hiCD127lo cells and the co-expression of FoxP3 and Helios in Treg cells before and after CD25 enrichment in day 0 UCB mononuclear cells (MNCs). Upper: the identification of CD4+CD25hiCD127lo Tregs from CD3+CD4+ T cells was shown, lower: while the co-expression of FoxP3 and Helios in the indicated CD4+ T cells, CD4+CD25hiCD127lo Tregs, or CD3+CD4+CD25− Tcon gates was depicted.

(B) Dot plots and (C) histograms illustrating the expression of CXCR4 in day 4 Treg cells before and after CXCR4 enrichment (Tregcontrol and TregCXCR4).

(D) The percentage of CXCR4hi cells among day 3 or day 4 Tregcontrol and TregCXCR4 cells was reported. Data were presented as mean ± standard deviation (SD); n = 16; ∗∗∗∗p < 0.0001; unpaired t test.

(E) CD4+CD8+ co-expression and (F) CD127+ expression (n = 3) and (G) t-SNE plot visualization of CD4+CD8+ (green) and CD127+ (orange) subpopulations overlaid on day 14 expansion pre-and post- CXCR4 Tregs (purple).

(H) The demethylation status of FoxP3, located in intron 1, was significantly higher in expanding Tregcontrol and TregCXCR4 compared to Tcons. Data were presented as mean ± SD; Tcons, n = 6 to 12; ∗∗∗∗p < 0.0001; one-way ANOVA.

(I) In comparison to TregCXCR4neg, the FoxP3 gene exhibited higher levels of demethylation in TregCXCR4. Data were analyzed as mean ± SD; n = 6 to 8; ∗p < 0.05; unpaired t test.

(J) Tregcontrol and TregCXCR4 cells were activated with CD3/CD28 beads and IL-2. Following a 24-h incubation, IL-10 levels in the supernatant of the cell cultures were quantified using ELISA. The data were presented as mean ± SD; n = 2; p = ns; unpaired t test.

(K) The average percentage of inhibition of Tcon proliferation by Tregcontrol and TregCXCR4 cells at various Treg:Tcon ratios was determined. At a 1:1 ratio, TregCXCR4 cells demonstrated a greater inhibition compared to Tregcontrol cells. ∗∗∗p < 0.001; n = 12; two-way ANOVA tests; mean ± SD.

(L) Diagrammatic representation of Transwell system.

(M) TregCXCR4 cells show enhanced migration to CXCL12/SDF1α compared to Tregcontrol, which is inhibited by CXCR4 antagonist. Upper chamber was plated with 1 × 106 TregCXCR4 or Tregcontrol cells. Either PBS, CXCL12/SDF1α, or CXCL12/SDF1α+AMD3100 was added to the bottom chamber. At 15 min post plating of the upper chamber, the number of cell migrated to the bottom chamber was counted and recorded (n = 3, p < 0.001).

(N) Representative images were captured of migrated Tregcontrol and TregCXCR4 cells (purple stained) in response to CXCL12/SDF1α in the presence or absence of AMD3100, on insert membranes at 15 min post plating.

(O) The absolute number of migrated cells on the membranes was quantified. TregCXCR4 cells demonstrated heightened migration toward SDF-1 in comparison to Tregcontrol cells, as evidenced by cell counts of 2,240 ± 241 vs. 871 ± 113 at 15 min, 3,708 ± 924 vs. 922 ± 263 at 30 min, and 2,286 ± 400 vs. 1,325 ± 37 at 60 min. Statistical significance was indicated by ∗p < 0.05 and ∗∗p < 0.01; two-way ANOVA test; mean ± SD.

CXCL12, also known as stromal cell-derived factor 1 (SDF-1), is a ligand for CXCR4 and a chemoattractant in the bone marrow.15 Transwell membrane experiment was set as described earlier to examine the egress of TregCXCR4 vs. Tregcontrol across the membrane and toward the chemoattractant, CXCL12/SDF1α (Figure 1L). As shown in Figure 1M, when compared to Tregcontrol cells, as early at 15 min time point, a significantly higher number of TregCXCR4 cells migrated toward CXCL12/SDF1α, across the Transwell membrane into the lower chamber (n = 3, p = 0.0019, Figure S1). This migration effect was predictably blocked by the addition of CXCR4 antagonist, AMD3100.10 Photomicrograph of representative experiment is shown in Figure 1N. Migration advantage of TregCXCR4 cells over Tregcontrol cells was maintained at 15, 30, and 60 min (Figures 1O and S2).

Functional and immunophenotypic diversity for Tregcontrol and TregCXCR4 revealed by viSNE visualization

T helper (Th)-like Treg subsets, also referred to as polarized Treg subsets, demonstrate functional diversity, facilitated by their shared homing receptor distribution, which enables effective colocalization. To assess the impact of CXCR4 enrichment on Th-like Treg subsets within Tregcontrol and TregCXCR4 populations, viSNE was utilized for visualization of high-dimensional single-cell data. Figure 2A illustrated that an increase in CXCR4 expression was accompanied by a reduction in chemokine receptors such as CXCR5 (Tf-Treg), CXCR3 (Th1-Treg), CCR4 (Th2-Treg), and CCR6 (Th-17 Treg). In Th17-like Treg cells, a decrease in the MFI of CCR6 expression was noted in TregCXCR4 (492 ± 142) compared to Tregcontrol (633 ± 46) cells, along with a decrease in IL-17A secretion (0.9 ± 0.3 vs. 3.7 ± 2.3 pg/mL, n = 4) (Figure 2B). Consistent interrelationships among chemokine receptors, transcriptional regulators, and cytokine production were observed. viSNE plots revealed an upregulation of suppression markers FoxP3, Helios, IL-10, and pSTAT5 in TregCXCR4, while a downregulation was observed in T-bet and IFN-γ (Th1-Treg), IL-4 (Th2-Treg), and IL-17 (Th17-Treg) compared to Tregcontrol (Figure 2C). Manual gating on the CD4+CD25+CD127− Treg cell population confirmed these findings, defining four subsets of Th-like Tregs as Th1-like (CD45RO+CXCR3+), Th2-like (CD45RO+CXCR3−CCR6−CCR4+), Th17-like (CD45RO+CXCR3−CCR6+), and Tf-Tregs (CXCR5+). These subsets depicted on the viSNE map were color-coded as follows: CXCR4 Treg in purple, Th1-Treg in yellow, Th2-Treg in orange, Tf-Treg in light blue, and Th17-Treg in green (Figure 2D). The percentages of Th1 and Th2-Tregs (ranging from 4.3% to 6.8%) were found to be decreased in TregCXCR4 compared to Tregcontrol, while remaining stable in Th17-Tregs and Tf-Tregs (ranging from 0.3% to 2.6%) (Figure 2E; Tables S2–S4). Therefore, the high CXCR4 expression on TregCXCR4 cells, accompanied by a relative decrease in other homing receptors including CXCR5, CXCR3, CCR4, and CCR6, supports their preferential homing to its ligand CXCL12/SDF-1α expressed in bone marrow and other tissues.Figure 2 Functional and immunophenotypic diversity for Tregcontrol and TregCXCR4 revealed by viSNE visualization

(A) viSNE visualization was used to illustrate the varying expression intensity of chemokine receptors (CXCR5, CXCR3, CCR6, and CCR4) among different Treg subtypes (Tf-Treg, Th1-like Treg, Th17-like Treg, and Th2-like Tregs) in Tregcontrol and TregCXCR4, revealing the identification of distinct immunophenotypic classifications.

(B) Comparative analysis of CCR6 expression and IL-17 levels between Tregcontrol and TregCXCR4 revealed that TregCXCR4 displayed lower CCR6 expression and reduced IL-17 levels in the cell culture supernatant (mean ± SD; n = 4; unpaired t test).

(C) viSNE plots characterizing representative transcriptional regulators for Tregcontrol and TregCXCR4 with upregulation of FoxP3, Helios, IL-10, and pSTAT5 in TregCXCR4 and a downregulation of T-bet and IFN-γ (Th1-like Treg subset), IL-4 (Th2-like Treg), and IL-17 (Th17-like Treg) compared to Tregcontrol.

(D) The subsets on the viSNE map were color-coded as follows: CXCR4Treg in purple, Th1-like Treg in yellow, Th2-like Treg in orange, Tf-Treg in light blue, and Th17-like Treg in green (n = 4; Tables S2–S4).

(E) Comparison of percentages of Th-like Tregs between Tregcontrol and TregCXCR4 revealed that Th-like Tregs and Tf-Treg subsets decreased in TregCXCR4 compared to Tregcontrol (n = 5, mean ± SD, Tables S2–S4).

(F) viSNE map visualization illustrating the varying expression levels of Treg-associated marker CD25, the TNF receptor superfamily protein GITR, antiapoptotic protein BCL-2. Each marker was depicted with a unique color gradient, depicting their distribution in Tregcontrol and TregCXCR4.

(G) viSNE plot effectively demonstrated the differentiation of Tregcontrol and TregCXCR4, emphasizing specific markers associated with CD62LhiCD45RAhi central Treg (cTreg), CD45RAloCD45ROhi memory Treg (mTreg), CD45RAloCD45ROhiHLA-DRhi effector Treg (eTreg) cells. Additionally, distinct cell clustering was observed among adenosine signaling markers (CD39 and CD73), inhibitory receptor PD-1, and effector markers (HLA-DR and CD95), leading to the identification of a small proportion of heterogeneous Treg subsets characterized by the expression level of CD39hi, PD-1hi, and HLA-DRhi.

(H) These findings were further supported by heatmap analysis.

(I) The heatmap showed expression patterns on the viSNE landscape with clear gating strategies, assigning colors based on marker intensity for detailed analysis of phenotypic complexity and comparison between Tregcontrol and TregCXCR4.

The visualization of viSNE was utilized to assess the immunophenotypic diversity of regulatory mechanisms for Tregcontrol and TregCXCR4. Our findings revealed that TregCXCR4 exhibited elevated expression levels of the Treg-associated marker CD25 and TNF receptor superfamily protein GITR compared to Tregcontrol. Conversely, there was a downregulation of the antiapoptotic protein BCL-2 in TregCXCR4 relative to Tregcontrol (Figure 2F). To investigate the homeostasis and functional capabilities of Treg cells, a viSNE plot was employed to visualize the differentiation and activation markers. Our findings highlighted distinct Treg subsets, including central Treg, memory Treg, and effector Treg within Tregcontrol and TregCXCR4. An upregulation of CD62L, CD45RA/RO, CD39, and HLA-DR was observed in TregCXCR4 (Figure 2G). Furthermore, cell clustering revealed a small proportion of functional heterogeneity within Treg cell subsets, characterized by high expression levels of CD39, HLA-DR, and PD-1. Specifically, the CD39hi phenotype in TregCXCR4 displayed a significantly higher CD39 MFI of 38,527 vs. 29,762 in Tregcontrol, while the expression of CD73 increased from undetectable levels to an MFI of 4,450 in TregCXCR4, suggesting a potentially enhanced suppressive capacity in this subset. Similarly, HLA-DRhi cell populations increased in TregCXCR4, rising from MFI of 93,302 in Tregcontrol to MFI of 95,152 in TregCXCR4. These characteristics were visualized on a viSNE landscape, where individual cells were color-coded to represent the level of marker expression (Figures 2G and 2H). The gating strategies were clearly outlined, and the accompanying heatmap enabled a thorough evaluation of the phenotypic changes in Treg cells, facilitating a direct comparison of expression profiles between Tregcontrol and TregCXCR4 (Figures 2H and 2I).

TregCXCR4 preferentially home to bone marrow via the CXCR4/CXCL12 axis in vivo

To investigate the in vivo migration of Tregs enriched on CXCR4 and determine the potential role of CXCL12 in conferring tissue-specific functional properties to Treg cells, a xenogeneic NSG mouse model was employed. Twelve and 24 h after tail vein injection of CellTrace Violet (CTV)-labeled TregCXCR4 or Tregcontrol cells, mice were euthanized and different tissues including BM, peripheral blood (PB), liver, and spleen were analyzed (Figure 3A). At the 12- and 24-h (Figure 3B) interval, TregCXCR4 cells exhibited a significantly superior homing to BM compared to Tregcontrol (Figure 3C; p = 0.0319, n = 4 and p = 0.0362, n = 5). While an increase in CTV-labeled TregCXCR4 cells was observed in the spleen (p = 0.0356) and liver (p = 0.0304), such difference was not detected at 24 h in the spleen (p = 0.9) and was, in fact, reversed in the liver (p = 0.008). In stark contrast, TregCXCR4 cells were significantly less prevalent in PB at 12 (p = 0.0009) and 24 h (p = 0.0079) compared to Tregcontrol. In BM, the Treg homing index,16 calculated as a measure of the relative abundance of TregCXCR4 to Tregcontrol, rose significantly from their initial measurement to 12 h (p = 0.0006), a trend that, while attenuated, remained significant at 24 h (p = 0.0263) (Figure 3D), with a corresponding decline in PB at the same time points (p = 0.0186 and p = 0.0028, respectively). While the Treg homing index for TregCXCR4 when compared to Tregcontrol increased markedly at 12 h in the spleen (p = 0.0003), it returned to its initial levels by 24 h (p = ns). The findings on the migration of TregCXCR4 cells may play a role in shaping their tissue-specific functional characteristics, with a focus on their tendency to migrate preferentially to CXCL12-rich environments in the bone marrow and spleen, facilitated by the CXCL12-CXCR4 axis (Figures 1L–1O).Figure 3 CXCR4 determines preferential homing of TregCXCR4 cells to the BM and spleen when compared to Tregcontrol cells

(A) In vivo homing experimental design schematic. NSG mice were injected through tail vein with 1 × 107 TregCXCR4 or TregControl cells and were euthanized at 12 and 24 h to harvest organs including bone marrow (BM), spleen (SP), liver (LV), and peripheral blood (PB).

(B) Representative plots of CellTrace Violet (CTV)-labeled human TregCXCR4 (right column, blue gating) versus TregControl cells (left column, green gating) in the BM, PB, LV, and SP at 12 and 24 h post injection.

(C) Quantitative analysis of TregCXCR4 versus TregControl cells at 12 (n = 4) and 24 h (n = 5) in BM, PB, liver, and spleen; p = 0.03, 0.02, 0.03, and 0.03, respectively for 12 h, and p = 0.03, 0.0009, 0.008, and 0.9, respectively.

(D) Homing index calculated as ratio of TregCXCR4 to TregControl in BM, PB, liver, and spleen at 12 (left column) and 24 h (right column) post-cell infusion. The homing index was derived from the ratio of TregCXCR4 to TregControl cells within each organ. Percentages denoted the proportion of cells that have migrated to each tissue at the specified time intervals. Data were presented as mean ± SD for a sample size of n = 4 mice per group at 12 h, and n = 8 for BM and spleen, n = 5 for PB and liver at 24 h. Statistical evaluations were performed using unpaired t tests and one-way ANOVA to determine significance levels (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

(E) Quantitative analysis of CXCR4 mean fluorescence intensity (MFI) on human TregCXCR4 and TregControl cells derived from murine bone marrow and spleen.

(F) The levels of human and murine cross-reactive CXCL12 expression in stromal cells from murine bone marrow and spleen were compared between TregControl and TregCXCR4 groups at 24 h. Values for (E) and (F), presented as mean ± SD for n = 3, Student’s t test, ∗p < 0.05 ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

(G) viSNE plotted 12,500 cells from mock-PBS and Treg-infusion murine BM (top), distinguishing hematopoietic and stromal and endothelial cells (middle to bottom). Top: differentiated between hematopoietic cells and stromal cells in the bone marrow using a color gradient based on mouse CD45 and cross-reactive CXCL12. Cells were colored by cell type and expression of CD25 (upper middle). The viSNE map also displayed the expression of cross-reactive CXCL12 in murine BM stromal cells (lower middle) and the level of human CXCR4 expression (bottom panel) in the TregControl (left) and TregCXCR4 groups (right).

To further elucidate the involvement of the CXCL12-CXCR4 axis in Treg cell homing, we conducted a comparative analysis of CXCR4 expression levels on human Tregcontrol cells and TregCXCR4 cells, as well as the human and murine cross-reactive CXCL12 on murine bone marrow and spleen stromal cells following Treg infusion at 24 h. Consistent patterns in the levels of the CXCL12-CXCR4 axis and the homing index were observed in both BM and spleen (Figure 3D). Our study revealed a significantly higher expression of MFI CXCR4 on human TregCXCR4 cells (11.5 × 104) compared to Tregcontrol cells (9.4 × 104) in the bone marrow (Figure 3E). Notably, there was a significant increase in the TregCXCR4 group in terms of cross-reactive CXCL12 expression on murine BM stromal cells compared to Tregcontrol group (p = 0.011), while no significant changes were noted in the murine spleen (p = ns) (Figure 3F).

Our recent clinical trial17 results demonstrate an increase in FOXP3+CD4+ T cells in the bone marrow biopsy of a patient with aplastic anemia after treatment with CK0801 (Tregcontrol) on day +30, indicating a potential correlation with improved transfusion requirements.7 Additionally, a viSNE plot was utilized to visualize the localization of human Tregcontrol and TregCXCR4 cells in the bone marrow of NSG murine recipients, distinguishing murine hematopoietic (mCD45+) and BM stromal and endothelial cells (CD45−CXCL12+). The increased expression of CXCL12 in murine bone marrow stromal and endosteal regions, particularly in the TregCXCR4 group, was associated with higher levels of CXCR4-positive expression in human TregCXCR4 compared to Tregcontrol (Figure 3G). This homing pattern suggested that TregCXCR4 preferentially home to the bone marrow via the CXCR4/CXCL12 axis in vivo, potentially resolving inflammation in the bone marrow.

Immune assessment of bone marrow Tregs

Bone marrow samples were analyzed using t-SNE to visualize metaclusters 6, 7, and 10, which exhibited a higher presence in TregCXCR4 recipients compared to Tregcontrol recipients (n = 3 p = 0.044, ∗p = 0.016, and p = 0.663, respectively, as shown in Figures 4A, 4B, and 4C). The distribution of cell populations between TregCXCR4 and Tregcontrol recipients within these metaclusters revealed an overall increase in CD4+, a decrease in CD8+, and an increase in the expression of CD39, CD73, and CXCR5. While an increase in CCR6 expression was noted in metacluster 6 and a decrease in metacluster 7, these differences were not significant. Additionally, the distribution of CD45RA across the three metaclusters did not show statistically significant differences. In metacluster 7, there was an observed increase in co-expression for CD39, CD73, and CXCR5, while a decrease was noted for CD45RA, CD95, HLA-DR, CD8, and CD127 in TregCXCR4 compared to Tregcontrol recipients (Figures 4D and 4E). This finding was further supported by the heatmap illustrating their expression profile (Figure 4F). Additionally, human TregCXCR4 cells in murine BM recipients showed a stronger correlation with CD39 and a weaker correlation with CD95, HLA-DR, and CCR6 within the metacluster 7, as compared to Tregcontrol recipients (Figure 4G).Figure 4 Immune assessment of BM Tregs

t-SNE plot of FlowSOM metaclusters analysis of human Tregcontrol and TregCXCR4 samples in mouse bone marrow BM recipients.

(A) Each metacluster identified by a unique color and (B) representative percentage was shown in the pie charts of Tregcontrol (left) and TregCXCR4 (right).

(C) Bar graph showing frequency of each metacluster derived from murine bone marrow of human Tregcontrol and TregCXCR4 cells recipients. Metacluster 6, 7, and 10 had a higher presence of TregCXCR4 compared to Tregcontrol (mean ± SD, for n = 3, Student’s t test, ∗p = 0.044, ∗p = 0.016, and p = 0.663).

(D) Higher expression of CD4, CD39, CD73, and CXCR5 was observed, particularly in metacluster 7.

(E) Quantitative levels of CD4, CD8, CD39, CD73, CCR6, CXCR5, and CD45RA in the TregCXCR4 compared to Tregcontrol within metacluster 7 are presented as mean ± SD for n = 3, Student’s t test, ∗p < 0.05.

(F) Heatmap of FlowSOM metacluster 7 revealed higher expression of suppression markers CD39 and CD73, as well as homing markers CCR4, CXCR5, and CCR7 in TregCXCR4 compared to Tregcontrol. Additionally, TregCXCR4 exhibit lower expression of CD45RA, CD95, HLA-DR, CD8, and CD127.

(G) Mouse bone marrow samples from recipients human TregCXCR4 or Tregcontrol demonstrated an increased correlation of TregCXCR4 administration with the expression of CD39, CD73, CCR4, CXCR5, CCR7, and CD45RA, while a lower correlation was noted with CCR6 and HLA-DR in metacluster 7. The heatmap of FlowSOM metacluster 7 included Spearman’s r coefficients.

TregCXCR4 decrease TGF-β and other plasma cytokines

The plasma levels of TGF-β1 and TGF-β2 were found to be significantly reduced at 24 h in TregCXCR4 recipients compared to Tregcontrol recipients (Figure 5A). Additionally, a heatmap depicting the levels of various plasma cytokines including EGF, Eotaxin, FGF2, FLT-3L, IFN-α2, IL-13, IL-17E/IL-25, IL-27, IP-10, MCP-1, MCP-3, MIP-1β, PDGF AB/BB, and TGF-α at 24 h post-Treg cell injection showed a reduction in their levels among TregCXCR4 recipients compared to Tregcontrol recipients (Figure 5B). Both the TregCXCR4 and Tregcontrol groups showed low levels of other cytokines such as GROα, IL-4, IL-6, IL-8, IL-9, IL-15, IL-17A, IL-18, M-CSF, MDC, MIG/CXCL9, PDGF-AA, RANTES, TNF-β, and VEGF-A (Table S5).Figure 5 TregCXCR4 decrease TGF-β1/β2 and bone marrow inflammation

Mouse plasma was examined for circulating human inflammatory cytokines. TregCXCR4 decreases (A) TGF-β1 and (B) TGF-β2 in vivo. Histogram represents the levels of TGF-β1 and TGF-β2 between TregControl and TregCXCR4 (∗∗p < 0.01, t test, n = 5 each group).

(C) Comparative analysis of heatmap of cytokine normalized expression profile between TregControl and TregCXCR4. The color scale ranges from green (low expression) to red (high expression), indicating the relative abundance of each cytokine. A shift toward the green end of the spectrum denotes decreased expression levels of inflammatory cytokines in the TregCXCR4 group. The y axis categorizes the cytokines, while the X axis separates TregControl and TregCXCR4 (n = 5 mice per group; in vivo homing assay at 24 h post-injection).

(D) TregCXCR4 decrease bone marrow inflammation. Bone marrow biopsy samples from control (PBMC only) and treatment (PBMC+TregCXCR4) were stained using anti-CD8, anti-TNF-α, and anti-IFN-γ antibodies and imaged using a Leica DMi8 inverted microscope. Representative pseudocolor micrograph of bone marrow: control group (left) and treatment group (right). Blue = DAPI (4′,6-diamidino-2-phenylindole) (upper), green = CD8 (second row), red = TNF-α.

(E) TregCXCR4 decrease IFN-γ in bone marrow. Representative pseudocolor micrograph showing IFN-γ secretion (yellow) by human PBMCs and human PBMCs treated with TregCXCR4 in bone marrow from the TregCXCR4 treatment group (PBMCs+TregCXCR4, right graph).

(F) TregCXCR4 decrease IFN-γ secretion. Plasma levels of human IFN-γ were compared between control and treatment arms. Data indicate mean ± SD of two technical replicates for n = 2, Student’s t test, ∗∗∗∗p < 0.0001. Representative intensity of (G) TNF-α and (H) IFN-γ of bone marrow from PBMCs+TregCXCR4 mice (n = 4) was found to be significantly lower compared to PBMCs mice (n = 4) as determined through immunofluorescence staining and quantification using ImageJ software. Values are presented as mean ± SD of two technical replicates for n = 2, Student’s t test (p = 0.0327 and p = 0.0333, ∗p < 0.05).

TregCXCR4 decrease bone marrow inflammation

To better understand the impact of TregCXCR4 on bone marrow inflammation, immunostaining was performed on bone marrow biopsies from control (peripheral blood mononuclear cell [PBMC] only) and treatment arm (PBMC+TregCXCR4), where two longitudinal samples (n = 2, each group) were analyzed. Pseudocolor micrographs were utilized to identify nuclei using DAPI (Figure 5C, upper panel), CD8+ T cells (second row panel), secreted TNF-α (third row panel), and merged images (bottom panel) (Figure 5D). Pseudocolor micrographs were also used to identify IFN-γ (yellow) (Figure 5E) in the treatment group (PBMCs+TregCXCR4) compared to the control group (PBMC only). The corresponding plasma levels of human IFN-γ were significantly decreased in the treatment group (PBMCs+TregCXCR4) compared to the control group (PBMC only) (Figure 5F, Student’s t test, ∗∗∗∗p < 0.0001). Additionally, quantification of the immunofluorescence staining of bone marrow also demonstrated a significant reduction in the levels of TNF-α (Figure 5G, p = 0.032) and IFN-γ (Figure 5H, p = 0.033), respectively.

Discussion

Here, we show that enrichment of Tregs on CXCR4 generates a unique cell population with differential cell surface and intracellular profile as well as a higher expression of suppressor markers. Specifically, our data show that CXCR4 enrichment allows for enhanced transit of TregCXCR4 cells toward chemokine SDF1α in vitro as well as to bone marrow in vivo. Such a preferential homing to BM was associated with a decrease in plasma TGF-β and IFN-γ levels as well as BM CD8+ T cells, IFN-γ, and TNF-α when compared to unmanipulated Tregs.

Preservation of the low expression of PD-1 in both TregCXCR4 and Tregcontrol cells as observed in our data has important implications, since PD-1 negatively regulates the activation and suppressor function of Tregs, where PD-1-deficient Tregs exhibit an activated phenotype and enhanced immunosuppressive function and are potentially associated with better control of autoimmune diseases.18 Furthermore, PD-1-deficient Tregs, revealed down-regulation of genes in the PI3K-AKT-mTOR signaling pathway, in gene set enrichment analyses.19 In fact, even in healthy individuals, high PD-1 expression on human Tregs identifies a dysfunctional, exhausted subset that secretes IFN-γ.20 Additionally, TregCXCR4 cells retained their co-expression of CD45RA/RO, which is unique to the UCB Tregs, and potentially confer gain of effector function (CD45RO), while retaining their ability to proliferate and maintain their naivity (CD45RA).1,8

In the bone marrow, constitutive CXCL12 secretion by stromal cells is crucial for homing and sustaining CXCR4-expressing hematopoietic stem and progenitor cells in their niches.21,22 Our observation of a reciprocal increase in the CXCL12 expression in the mouse bone marrow stromal cells might be explained by mutual reciprocal interactions between CXCR4 and CXCL12.23 The bone marrow endothelial and other stromal cell types highly express both CXCR4 receptor and its ligand CXCL12, implying an autocrine loop mechanism to regulate CXCR4 expression on these cells.24 Acquiring tissue-specific migration as a function of CXCR4 enrichment allowed for increased expression of CD62L, which in turn enabled the homing of Tregs to secondary lymphoid organs.9

CXCR4 enrichment also led to an increase in the co-expression of CD39/CD73 that mediates non-antigen-specific suppressive mechanisms of Tregs by leveraging increased adenosine concentration in the microenvironment to inhibit dendritic cell presentation of antigens and suppression of activated T effectors.25,26 Increased HLA-DR expression in TregCXCR4 support early contact suppressive activity and are essential for Treg suppressor function.27 Clustered increase in the STAT5 and CD73 expression is also suggestive of the spatiotemporal distribution of the TregCXCR4 to be situated closer to the antigen-presenting cell.28 It is also possible that change in the migratory profile of Tregs as a result of CXCR4 enrichment might have also led to differences in T cell receptor specificity and affinity as well as allowed for the expression of additional genes responsible for tissue-specific functioning.26 In vivo, TregCXCR4 maintained the high CD39/73 co-expression but had a relative decrease in HLA-DR along with CD95, which might have been a result of co-localization.29

Decrease in Th17-like (CD45RO+CXCR3−CCR6+) Tregs that secrete inflammatory cytokines including IL-17 and IL-22 and a corresponding decrease in CCR6, on Tregs cell surface30,31 accompanied by a decrease in IL-17 secretion, suggest that CXCR4 enrichment selects for a CCR6-deficient Treg cell population that may exhibit differential suppressor function.32 Additionally, a decrease in Th1-like Treg subset with a corresponding decrease in CXCR3 expression, T-bet33 and IFN-γ secretion, as well as a decrease in Th2-like Tregs with a corresponding decrease in CCR4 expression and IL-4 secretion, in TregCXCR4 cells, is suggestive of decreased plasticity.34 CXCR4 enrichment was also associated with increased expression of the bona fide Treg markers including FoxP3 and Helios,35 IL-10,36 and pSTAT5,37 which might explain the differential impact on the inflammatory cytokines in vivo.

TGF-β, shown to play a pathogenic role in myelofibrosis,38 was decreased to a greater extent by TregCXCR4 cells. The ability of TregCXCR4 to preferentially decrease PDGF AB/BB, where PDGF signaling has been identified to play an important role in pathogenesis of myelofibrosis,39,40 is suggestive of their specific engagement with the culprit pathways engaged in BM inflammation. Additional inflammatory cytokines that are implicated in BM inflammation and fibrosis were also decreased in TregCXCR4 recipients and included (1) EGF and Eotaxin,41 (2) TGF-α, a polypeptide growth factor structurally and functionally related to EGF,42 (3) FGF2, associated with BM fibrosis as well as its correlation with TGF-β in myeloproliferative neoplasm (MPN),43 (4) FLT-3L, which plays an important role in dysmegakaryopoiesis in myelofibrosis,44 (5) IFN-α2 in BM inflammation,45 (6) IL-1RA, pathogenic role in MPN disease progression,46 (7) IL-17E/IL25 in generating reactive oxygen species in BM,47 (8) IP-1048 and MIP1β in mediating TH1 type signaling,49 and (9) MCP-1 and -2, potent chemo-attractant for pathogenic monocytes.50

Myelosuppressive cytokines including TNF-α and IFN-γ have been implicated in driving bone marrow inflammation leading to the death of hematopoietic progenitor cells.51 Additionally, CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction.52 The ability of TregCXCR4 cells to significantly decrease all these three factors compared to the control in a bone marrow inflammation model supports our hypothesis that CXCR4 enrichment of Tregs cells is a viable therapeutic strategy.

In conclusion, we have developed a unique TregCXCR4 cell population that preferentially expresses CD39/73 pathway, is associated with enhanced suppressor function, and homes specifically to the bone marrow to decrease inflammation.

Limitations of the study

Our study has a few limitations including the xenogeneic bone marrow inflammation model, which might not capture all the elements of the inflammatory processes as well as extramedullary hematopoiesis manifested by complex diseases such as myelofibrosis. Due to limited sampling, an extensive examination of the bone marrow immunophenotype and inflammatory cytokine composition was not performed.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the corresponding author, Simrit Parmar.

Materials availability

TregCXCR4 can be accessed through material transfer agreement.

Data and code availability

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

• This paper does not report original code.

Acknowledgments

The authors thank Dr. Robert Negrin, Stanford University, for careful review of our manuscript.

Author contributions

M.H. was responsible for conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing. Z.K., M.-A.L., and L.M. were responsible for data curation, formal analysis, investigation, and writing – review and editing. T.S. was responsible for resources, materials, formal analysis, and writing – review and editing. C.R.F. was responsible for funding acquisition, project administration, resources, supervision, validation, and writing – review and editing. S.P. was responsible conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing.

Declaration of interests

S.P. has an equity interest in, holds patents for, receives royalties and research funding from, and is a member of the board of directors/advisory committee for Cellenkos Inc. C.R.F. received research funding from 4D, AbbVie, Acerta, Adaptimmune, Allogene, Amgen, Bayer, Celgene, Cellectis, Cellenkos, EMD, Gilead, Genentech/Roche, Guardant, Iovance, Janssen Pharmaceutical, Kite, MorphoSys, Nektar, Novartis, Pfizer, Pharmacyclics, Sanofi, Takeda, TG Therapeutics, Xencor, Ziopharm, Burroughs Wellcome Fund, Eastern Cooperative Oncology Group, National Cancer Institute, and Cancer Prevention and Research Institute of Texas: CPRIT Scholar in Cancer Research and consulting fees from AbbVie, Bayer, BeiGene, CelgeneDenovo Biopharma, Epizyme, Genentech/Roche, Genmab, Gilead, Karyopharm, Pharmacyclics/Janssen, Seagen, and Spectrum. T.S. is an employee of Cellenkos Inc. L.M. is a consultant/advisor/speaker: Cogent, GlaxoSmithKline, MorphoSys, and PharmaEssentia. The authors declare that this study received funding from Cellenkos Inc.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Alexa Floyur532-conjugated anti-human CD3	Invitrogen	Cat# 58-0038-42; RRID: AB_11218675	
BV650-conjugated anti-human CD4	BD Biosciences	Cat# 563875; RRID: AB_2744425	
BV786-conjugated anti-human CD8	BD Biosciences	Cat# 563823; RRID: AB_2687487	
BV421-conjugated anti-human CD25	BD Biosciences	Cat# 562442; RRID: AB_11154578	
PE-conjugated anti-human CD25	BD Biosciences	Cat# 567489; RRID: AB_2916621	
BUV496-conjugated anti-human CD25	BD Biosciences	Cat# 741201; RRID: AB_741201	
PE-Cyaine 7-conjugated anti-human CD127	Invitrogen	Cat# 25-1278-42; RRID: AB_1659672	
APC-conjugated anti-human CXCR4	Invitrogen	Cat# 17-9999-42; RRID: AB_1724113	
PE-conjugated anti-human CCR7	BD Biosciences	Cat# 566742; RRID: AB_2864275	
eFluor450-conjugated anti-human CD62L	Invitrogen	Cat#48-0621-82; RRID: AB_1963590	
BV605-conjugated anti-human CD45RA	BD Biosciences	Cat#562886; RRID: AB_2737865	
BV711-conjugated anti-human CD45RO	Biolegend	Cat#304236; RRID: AB_2562107	
BUV395-conjugated anti-human CCR6	BD Biosciences	Cat# 743356; RRID: AB_2741448	
PerCP eFluor710-conjugated anti-human CCR4	Invitrogen	Cat#46-1949-42; RRID: AB_2573725	
PerCP Cy5.5-conjugated anti-human CXCR3	Biolegend	Cat# 353714; RRID: AB_10962908	
Alexa fLuor700-conjugated anti-human CXCR5	Biolegend	Cat#356916; RRID: AB_2562290	
Alexa Fluor PE594-conjugated anti-human CD39	Biolegend	Cat# 328232; RRID: AB_2686985	
APC/FIre750-conjugated anti-human CD73	Biolegend	Cat#344036: RRID: AB_2876649	
PE-Cyaine 5-conjugated anti-human CD95	Invitrogen	Cat# 15-0959-42; RRID: AB_11042290	
Alexa Fluor 647-conjugated anti-human HLA-DR	Biolegend	Cat#327012; RRID: AB_893570	
FITC-conjugated anti-human HLA-ABC	Biolegend	Cat#311404; RRID: AB_314873	
BUV737-conjugated anti-human PD-1	BD Biosciences	Cat#741881; RRID: AB_2871205	
PE-CF594-conjugated anti-human BCL-2	BD Biosciences	Cat#563601; RRID: AB_2738307	
PerCp-Cy5.5-conjugated anti-human FOXP3	BD Biosciences	Cat# 45-5773-82; RRID: AB_914351	
Alexa Fluor 647-conjugated anti-human Helios	BD Biosciences	Cat# 563951; RRID: AB_2738506	
FITC-conjugated anti-human IL10	Invitrogen	Cat#RHCIL1001; RRID: AB_10375444	
BV421-conjugated anti-human STAT5	BD Biosciences	Cat#562984; RRID: AB_2737931	
BV785-conjugated anti-human T-bet	Biolegend	Cat#644835; RRID: AB_2721566	
APC-R7-conjugated anti-human IFNGR	BD Biosciences	Cat# 561024; RRID: AB_2033976	
BUV737-conjugated rat anti-human IL4	BD Biosciences	Cat#612835; RRID: AB_2870157	
BV605-conjugated anti-human IL17	Biolegend	Cat#512326; RRID: AB_2563887	
BUV395-conjugated anti-human GITR	BD Biosciences	Cat# 747660; RRID: AB_2744222	
BV786-conjugated rat anti-mouse CD45	BD Biosciences	Cat# 564225; RRID: AB_2716861	

Experimental model and study participant details

In vivo xenogeneic homing mouse model

Female non-obese diabetic (NOD)-immunodeficient (SCID) IL-2Rγ-/- (NSG) mice at 5 weeks of age were acquired from Jackson Laboratory (Farmington, CT). All animal experiments were carried out in accordance with the MDACC Institutional Animal Care and Use Committees. Expanding human Tregcontrol and TregCXCR4 cells were obtained by the Cellenkos® Inc (Houston, TX, USA) and incubated with Cell Trace Violet (CTV) cell proliferation kit (Life Technologies Corp., Oregon, USA) as per manufacturers’ instruction. 1x107 cells of CTV labeled Tregcontrol or TregCXCR4 cells were injected into tailvein (t.v.) of NSG mice. At 12 hours and 24 hours post infusion, mouse peripheral blood mononuclear cells (PBMCs), hepatocytes, splenocytes and BM cells were harvested to detect CTV-labeled cells using BD Fortessa X-20 and Cytek Aurora. To determine the homing index, the ratio of frequency of CTV+ cells in the positive group (TregCXCR4) to the number of CTV+ cells in the control group (Tregcontrol) was calculated.53

To prepare a single-cell suspensions, mouse peripheral blood was drawn into ethylenediaminetetraacetic acid (EDTA)-coated tubes, and for tissue samples, fresh liver and spleen were processed by mashing and filtering through a 40-μm cell strainer. BM cells were harvested by cutting both ends of the long bones (femur) of the hind limbs and flushing them with FACS buffer (PBS, 2%FCS, 2 mM EDTA; Lonza). Red blood cells were removed using 1x RBC lysis buffer (Miltenyi Biotec, Gladbach, Germany). All cells were resuspended in FACS buffer (Lonza, Walkersville, MD, USA) for tallying and immunolabeling.

Bone marrow inflammation xenograft model

Five-week-old female NOD-SCID IL-2Rγ-/- (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were subjected to 1.5 Gy irradiation followed by t.v. injection of 10×106 human PBMCs. In the treatment arm 10×106 TregCXCR4 cells injected by t.v. on days 1, 8, 15, 22, and 29. On day 30, harvested bone marrow samples from mouse femur were fixed with 10% buffered formalin and embedded in paraffin for processing into 5-μm tissue sections. Deparaffinized and rehydrated tissue sections were stained using anti-CD8, anti-TNF-α, anti-IFN-γ, and DAPI, and imaged on the Leica DMi8. Immunofluorescent images were exported using LAS X (Leica Microsystems, Wetzlar, Germany).

Institutional approval for animal studies

Animal procedures were performed according to an approved protocol by MD.

Anderson Cancer Center’s Institutional Animal Care and Use Committe.

Method details

UCB Treg cells, and TregCXCR4 cells isolation and in vitro expansion

UCB units were obtained from The University of Texas MD Anderson Cancer Center Cord Blood Bank after Institutional Review Board (IRB) approval. Using magnetic enrichment UCB Tregs (defined as CD4+CD25hiCD127lo) were isolated based on CD25hi cell surface expression.1,8 At day 3 or 4 of culture, Tregs were harvested and labeled with CD184 (CXCR4)-APC and Anti-APC Microbeads kit (CD184 MicroBead Kit, Miltenyi Biotec, Gladbach, Germany) and were positively enriched by CXCR4-postive magnetic cell sorting and cultured for a total of 14 days.

Flow cytometry

Flow cytometry was performed to determine the phenotype of three distinct cell populations, i) pre-selection Tregcontrol, ii) post-enrichment (TregCXCR4), and iii) negative flow through (TregCXCR4neg) populations using a Cytek Aurora flow cytometer system. Three different marker panels were created with 20 colors, 15 colors, and 14 colors (Table S1). Cells from the first and second panels were stained with cell surface markers, while cells from the third and fourth panels were fixed and permeabilized for intracellular staining, using the Foxp3/Transcription Factor Fixation/Permeabilization Staining Buffer Set (ThermoFisher Scientific, Waltham, MA). Dead cells were excluded using a fixable viability dye (Invitrogen, Waltham, MA), and the FCS files were analyzed using FlowJo (Ashland, OR) and Cytobank software (Santa Clara, CA).

viSNE analysis

The FCS files generated from the flow cytometer were analyzed using software programs such as FlowJo (Ashland, OR) and Cytobank 9.0 (Santa Clara, CA) to create viSNE maps. The viSNE maps were constructed by running the algorithm for 1000-7500 iterations using arbitrary seed and tSNE parameters (perplexity=30). This enabled the projection of the high-dimensional data into two dimensions, tSNE1 and tSNE2, which facilitated the visualization of the phenotypic heterogeneity of Tregcontrol and TregCXCR4 subsets.

DNA methylation analysis

FoxP3 Treg-specific demethylation region (TSDR) methylation analysis of TregCXCR4 or Tregcontrol cells was performed using deep amplicon bisulfite sequencing.54,55,56

IL-10 secretion assay

Human IL-10 levels were quantified in the cell culture supernatants using IL10 human ELISA kit (BMS215-2, ThermoFisher Scientific, Waltham, MA) as per manufacturer’s instructions. Samples, standards, and controls were added to wells pre-coated with anti-human IL-10 antibody, incubated with a biotinylated antibody at 37°C for 2 hours, followed by washes and an additional incubation with Streptavidin-HRP. Tetramethylbenzidine substrate was then used to develop color, indicative of IL-10 concentration. The reaction was halted with an acidic stop solution, and the optical density was measured at 450 nm using a BioTek Epoch plate reader. Data analysis was conducted using appropriate software, and quality control was maintained through replicates and standard controls.

Cell suppression assay

Functional ability of TregCXCR4 or Tregcontrol cells to suppress proliferating CD4+25- conventional T cells (Tcon) was quantified using the method of cell suppression assay, where different ratios of Treg:Tcon were examined.1,8,57

Migration assay

The speed of egress of the TregCXCR4 vs. Tregcontrol towards the chemoattractant CXCL12/SDF1α[8] was analyzed using Transwell™ migration assay [9], where chemotaxis was performed using a 24-well Boyden chamber with a 5.0 μm pore size polyvinyl pyrrolidone–free polycarbonate membrane (Corning, NY, USA). 100 ul volume of TregCXCR4 or Tregcontrol cells, were added at a concentration of 0.5 x 106 cells/mL, to the upper chamber. Simultaneously, CXCL12/SDF1α at a concentration of 300 ng/mL was introduced to the lower chamber in serum-free medium. Migration was allowed over various time points in humidified chambers at 37°C, both with and without the CXCR4 antagonist AMD3100 at a concentration of 6 μg/mL. Non-migrating Tregs located on the top of the insert were removed using a cotton swab. Subsequently, the Tregs on the inserts were fixed with 70% ethanol for 10 minutes, air-dried for an additional 10 minutes, and stained with 0.1% crystal violet. The Transwell inserts were then mounted onto slides for imaging using a microscope. The total number of cells in each image was counted for analysis.

In vivo phenotype analysis by flow cytometry

Single cell suspension from PBMCs, hepatocytes, splenocytes and BM cells were stained using CTV proliferation kit, and fluorescent antibodies as shown in Table S1. Dead cells were excluded using fixable viability dye (Invitrogen, Waltham, MA). Anti-CXCL12 antibody used for in vivo staining showed cross species reactivity for human and mouse. Multicolor flow cytometry data acquisition was done with BD LSR Fortessa and Cytek Aurora and the FCS files were analyzed using FlowJo (Ashland, OR) and Cytobank software (Santa Clara, CA). Of CTV+ human Tregcontrol and TregCXCR4 cells, CD4+ and CD25+ co-expression was identified as Tregs. Among the human Treg cells (hTreg), CD45RA+ and CCR7+ were commonly used markers to distinguish resting Treg cells (rTreg), memory Treg cells (mTreg), and activated Treg cells (aTreg) in BM. In addition, CD45RO and CD62L markers could distinguish between resting Treg cells (rTreg), memory Treg cells (mTreg), and activated Treg cells (aTreg) in the spleen.

Cytokine analysis

Plasma samples, collected from the EDTA-treated murine PB, were analyzed for systemic inflammation using a human cytokine 48-plex Discovery assay kit and TGFβ 3-Plex Discovery assay kit (Eve Technologies, Calgary, Alberta, Canada).

Bone marrow immunostaining

Bone marrow samples collected from mouse femur were stained using anti-CD8 (BD Biosciences, Franklin Lakes, NJ), anti-TNF-α (Cell Signaling Technology, Danvers, MA), anti-IFN-γ (Bethyl laboratories, Montgomery, TX), and DAPI (4′,6-Diamidino-2-Phenylindole) (Thermo Scientific, Waltham, MA), and imaged on the Leica DMi8 (Wetzlar, Germany). The immunofluorescence analysis was conducted in a blinded fashion, with the assistance of Image J software.

Interferon-γ ELISA

EDTA-treated plasma samples were analysed for inflammation using a human IFN-γ ELISA kit (ThermoFisher Scientific, Waltham, MA) as per manufacturer’s instructions. Samples, standards, and controls were added to wells pre-coated with anti-human IFN-γ antibody, incubated with a biotinylated antibody at 37°C for 1.5 hours, followed by washes and an additional incubation with Streptavidin-HRP. TMB substrate was then used to develop color, indicative of IFN-γ concentration. The reaction was halted with an acidic stop solution, and the optical density was measured at 450 nm using a BIoTek Epoch plate reader. Data analysis was conducted using appropriate software, and quality control was maintained through replicates and standard controls.

Quantification and statistical analysis

Statistical analysis was performed using Prism Version 9 software (GraphPad Software, La Jolla, CA). Statistical significance was calculated by P value using unpaired t-test, one-way ANOVA and two-way ANOVA. P value<0.05 was considered statistically significant.

Supplemental information

Document S1. Figures S1 and S2 and Tables S1–S5

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110830.
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