Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer’s patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.

CD45CD34cells have been reported in adult human small intestine () and liver (), but detailed analysis of those in the intestine was not reported. We hypothesized that graft-resident hematopoietic stem and progenitor cells (HSPCs) contribute to long-term multilineage blood chimerism after human ITx. Using the current differentiation scheme for human hematopoiesis (), we identified HSCs and multiple types of progenitors, including multipotent progenitors (MPPs), lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), myeloid progenitors (MPs), and unclassified progenitors co-expressing CD56 () in human ileum mucosa, Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and liver. Colony-forming-cell (CFC) assays, long-term-culture-initiating-cell (LTC-IC) assays, and humanized mouse models demonstrated the differentiation potential of gut HSPCs. We further investigated the dynamics of HSPC replacement by the recipient within the intestinal allograft and demonstrate replenishment from a circulating pool.

Having it all? Stem cells, haematopoiesis and lymphopoiesis in adult human liver.

Having it all? Stem cells, haematopoiesis and lymphopoiesis in adult human liver.

Blood chimerism denotes coexistence of hematopoietic cells from both the donor and recipient after a transplant. Blood chimerism, either durable or transient, can be achieved without GVHD and, even when transient, can promote donor-specific tolerance in animals and humans (). We recently reported () that a high level of donor T cell macrochimerism (denoting ≥4% donor CD3T cells) often appears in the blood following ITx, usually without GVHD. This occurs more often in MVTx than in iITx recipients. Macrochimerism correlates with less graft rejection () and slower T cell replacement by the recipient within the allograft (). Long persisting donor blood chimerism can be observed in T, B, and natural killer (NK) cells, especially for MVTx patients. Myeloid chimerism is more transient ().

Preclinical and clinical studies on the induction of renal allograft tolerance through transient mixed chimerism.

Intestinal transplantation (ITx) provides the only long-term option for patients with end-stage intestinal failure (). ITx may be performed in isolation (iITx) or as part of a multivisceral transplant (MVTx) (). ITx is associated with high rates of graft failure, ∼50% at 5 years (), and these rates are reduced in MVTx compared to iITx (). The large lymphoid load in intestinal allografts results in graft-versus-host disease (GVHD) in 5%–9% of patients (). Thus, ITx involves a delicate balance between graft rejection, GVHD, and opportunistic infections due to over-immunosuppression.

While all patients with persistent T cell chimerism (>0.2%) had persistent intestinal HSC chimerism (pts 2, 16, and 19), the duration of intestinal HSC chimerism was greater than that of T cell chimerism ( Figure 7 G). One patient (pt 16) who had early transient donor blood T cell chimerism that became low or undetectable (0%–0.33%) during POD 23–463 showed resurgent donor T cell chimerism to >4% by POD 786, when there were still >50% donor HSCs persisting in the allograft ( Figure 7 G). Overall, persistent donor chimerism (POD >100) was significantly more common in the allograft than in the peripheral blood ( Figure 7 H, p = 0.0062). Together, our data indicate that graft-derived HSPCs likely contribute to the long-term persistence of donor blood chimerism, and these donor cells are gradually replaced by recipient HSPCs in the circulation that home to the intestine.

We tracked the origin of HSPCs in intestinal allograft biopsy specimens (jejunum, ileum, or colon) collected from 10 ITx recipients whenever sufficient samples were available from stoma revision or closure or small bowel removal (POD 28–1,606). Representative FCM gating to distinguish donor- versus recipient-derived HSPCs is shown in Figure 7 A. A specimen taken from the native colon of pt 14 on POD 532 served as a control containing recipient-derived HSPCs ( Figures 7 B–7D and 7G). Variable percentages of CD34cells were detected among DAPICD45Lincells in the intestinal mucosa post-Tx ( Figure 7 B). Remarkably, as early as 1 month post-Tx, recipient HSPCs already started to populate some grafts (pt 23 POD 28 ileum and pt 19 POD 32 ileum, Figure 7 C). Early recipient contributions included mainly LMPP, CLP, and MP cells in pt 16 (POD 47), pt 19 (POD 32), and pt 23 (POD 37) ( Figure 7 C). HSCs tended to be more enriched (>80%) among donor than recipient LinCD45CD34cells regardless of patients’ ages or early clinical outcomes ( Table S1 ), and this trend was maintained long-term (POD >100) post-Tx in at least 6 patients (pt 20, POD 104; pt 19, POD 105; pt 17, POD 243; pt 16, POD 786; pt 15, POD 347, 1,041; pt 2, POD 1,544), even beyond 4 years post-Tx ( Figures 7 C and 7G). In other patients, the replacement of donor HSCs by the recipient progressed over time (e.g., pt 17, Figures 7 C and 7G) and in others complete or nearly complete replacement of donor HSPCs by the recipient was observed long-term (e.g., pt 4 on POD 1,606 and pt 13 on POD 1,032; Figures 7 C and 7G). Overall, donor gut HSCs persisted long-term (POD 100–1,600) in the allograft in 6 out of 8 patients ( Figure 7 G, right). In general, LinCD45CD34donor cells in graft LPL included a high percentage of HSCs and MPPs, while the recipient compartment mainly consisted of MPs ( Figure 7 D). For all specimens from POD >100, donor LinCD45CD34cells in the graft LPL (n = 9 specimens) included significantly greater proportions of HSCs than those of the recipient (n = 13 specimens) ( Figure 7 E), while the recipient compartment (n = 13) contained significantly greater proportions of MPs than the donor (n = 9) ( Figure 7 F).

(H) Percentage of donor HSCs in the gut and the percentage of donor T cells in the blood at a similar time point (POD >100) for individual specimens from patients (circles for MVTx, squares for LITx, and triangles for iITx) who underwent stoma revision or closure or small bowel removal at various time points post-Tx. Chi-square test was performed with a cut-off for donor chimerism of 0.2% in peripheral blood (for T cells) or intestinal allograft (for HSCs).

(G) Percentages of donor T cells in peripheral blood (left) and donor HSCs in gut (right) up to 2,000 days post-Tx.

(E and F) Percentage of HSCs (E) and MPs (F) among donor or recipient Lin − CD45 +/dim CD34 + LPLs isolated from patients’ intestinal mucosa after 100 days post-Tx shown in box-and-whisker plots (the line inside the box indicates the median). A two-tailed unpaired Student’s t test was used to compare the donor and recipient compartments.

(D) Composition by percentage of HSC, MPP, LMPP, CLP, and MP among donor or recipient DAPI − Lin − CD45 +/dim CD34 + LPLs in different patients at multiple time points post-Tx (refer to A).

(B and C) The percentage of CD34 + cells among DAPI − CD45 +/dim Lin − LPLs (B) and the percentage of each cell type of donor versus recipient origin (C). DAPI − Lin − CD45 +/dim CD34 + cells, HSC, MPP, LMPP, CLP, MP, and CD56 + progenitors in gut LPLs were collected from different patients during stoma revision or closure or small bowel removal at multiple time points post-Tx (POD 28–1,606).

Dynamics of Replacement of Donor HSPCs within the Graft by Recipient Cells

Figure 7 Dynamics of Replacement of Donor HSPCs within the Graft by Recipient Cells

We used a humanized mouse model to determine the ability of human intestinal LinCD45CD34cells to reconstitute hematopoietic lineages in vivo ( Table S4 ). In one animal (#110), gut HSPC-derived cells of different lineages were identified by FCM ( Figures 6 C–6E). When residual T cells carried by the human thymus graft had nearly disappeared in NSG recipient (#110) blood (around 12 weeks post-Tx) ( Figure 6 D), human gut origin cells from D#293 dominated among human CD45cells ( Figure 6 D), demonstrating de novo generation of T, B, NK, and myeloid lineages ( Figure 6 E). At 24 weeks post-Tx, human gut origin cells (HLA-A2A9A3) showed multilineage differentiation in BM harvested from the recipient (#110) ( Figure 6 C). A small population of gut-derived CD34cells remained within the BM ( Figure 6 C), suggesting that HSCs had engrafted. In another animal with a human fetal thymus implant (#558), gut-derived CD45CD34progenitors and CD4CD8T cells accounted for 0.21% and 60% of gut origin thymocytes (HLA-A2A3A9) in the human thymus graft at 24 weeks ( Figure 6 C). In two additional mice (#329 and #382) that did not receive a human thymus implant, a 6.4:1 or 4:1 ratio of ileum versus BM origin HSPCs was injected ( Figure S6 ). Human intestine-derived cells from D#238 or D#332 accounted for >90% of hCD45cells in mouse PBMCs ∼2 weeks post-transfer. This proportion declined to 10%–50% by week 8 and to <1.5% at week 10 post-transfer, with temporary recovery from week 17 to 22 ( Figures S6 D and S6F). Of ten humanized mice in our study ( Table S4 ), four were followed only short-term (2–3 weeks). Among six mice with long-term (>24 weeks) follow-up, human gut HSPC-derived cells were detectable in the peripheral blood of all and chimerism persisted >12 weeks in four mice. At termination at week 24 or 28 post-adoptive transfer, human gut HSPC-derived cells were detectable in one or more tissues (BM and/or spleen and/or thymus) of five of the six mice ( Table S4 ). CD4 T cells, NKT cells, B cells, and monocytes were the major lineages derived from intestinal HSPC. Together, our data indicate that human gut HSPCs have multilineage differentiation potential (T, B, NK, and myeloid) and can likely self-renew, although less than BM HSPCs in some experiments.

Both ileum LPL and BM LinCD34-enriched cells formed myeloid and erythroid colonies in CFC assays, although those from ileum were less efficient than BM CD34 cells ( Figures 6 A and S6 A). Long-term LTC-IC assay ( Figures 6 B, S6 B, and S6C) using single-sorted ileum LPL HSC co-culture with MS5-DL1feeder cells () demonstrated their lymphoid (T/B) differentiation potential. BM and ileum LPL HSCs sorted from the same donor showed similar percentages of colony-forming cells in LTC-IC assays ( Figure 6 B).

(E) Percentage of different lineages repopulating in NSG mouse (#110) blood among HLA-A3 − A2 + A9 − compartment originating from D#293 ileum.

(D) Percentage of ileum (red), BM (purple), or thymus-derived (green) cells among hCD45 + mCD45 − populations in NSG mouse (#110) blood at different times post-adoptive transfer.

(C) Sublethally irradiated representative NSG mouse (#110) of 3 mice analyzed with human thymus implant (HLA-A3 + A2 − A9 − ) received FACS sorted CD45 +/dim CD34 + cells (1 × 10 6 ) from D#293 ileum LPLs (53 y/o female, HLA-A3 − A2 + A9 − ) and CD34 + magnetic-activated cell sorting (MACS)-isolated cells (1 × 10 5 ) from D#291 BM (26 y/o female, HLA-A3 − A2 − A9 + ) at a ratio of 10:1. Representative FCM gating shows T, B, NK, and myeloid lineage reconstitution in mouse blood ∼19 weeks post-adoptive transfer (second row) and in mouse BM at termination at week 24 (third row). Human HC PBMCs (HLA-A3 − A2 + A9 − ) serve as positive controls (first row). Human thymus implant (HLA-A3 + A2 − A9 − ) harvested from another NSG mouse (#558) at termination at week 24 (fourth row) contains gut-derived CD4 + CD8 + T cells and CD34 + progenitors. Mouse #558 received CD45 +/dim CD34 + FACS sorted cells (4.5 × 10 5 ) from D#291 ileum LPLs and CD34 + MACS-isolated cells (2.5 × 10 5 ) from D#293 BM at a ratio of 1.8:1.

(B) Representative colonies from long-term LTC-IC assay of single cell sorted HSCs from D#305 (28 y/o female) and D#332 (38 y/o male) BM or ileum LPLs co-cultured with MS5-DL1 ind100 stromal cells. CD3 + T cells (green), CD20 + B cells (red) and CD14 + myeloid cells (purple) were detectable within the colony on day 42 by fluorescence microscopy. HC human PBMCs were positive staining controls. Scale bar, 20 μm. Bar graph shows the percentage of colonies forming from single-cell sorted BM or ileum LPL HSC.

(A) Representative colonies from short-term CFC assay of Lin − CD34-enriched cells from BM or ileum LPL of D#259 (46 y/o male). Scale bar, 100 μm. Bar graph shows normalized colony number per 35 mm dish.

We used CyTOF, including barcodes to distinguish BM and ileum LPL-derived CD45cells from the same organ donor, and analyzed markers of particular hematopoietic lineages (CD3/CD4/CD8/γδ TCR/CD14/CD11c/CD11b/CD56) and progenitors (CD34/CD38/CD10/CD90/CD45RA) and of thymus homing ability (CCR9). LinCD45CD34cells from ileum LPL and BM of the same donor demonstrated similar phenotypic properties (localization) in viSNE plots ( Figure 5 A). Both ileum LPL and BM LinCD45CD34cells contained a subpopulation that expressed progenitor thymus homing marker CCR9 () ( Figure 5 B), and T/NK lineage-polarized progenitors that co-expressed CD45RA and CD7 () ( Figure 5 C). These phenotypes were observed across a wide age range (26–76 years old).

(C) Analysis of HSPCs coexpressing CD45RA and CD7 in BM and ileum LPL of adult deceased organ donors D#280, D#305 and D#337. NA, not applicable.

(B) Expression of HP thymus-homing marker CCR9 on viSNE plot of CD45 +/dim lineage-depleted BM cells and ileum LPLs in each individual organ donor. CD34 + gating was kept as shown in (A). Overlap between CCR9 + cells (red) and these gated populations is shown in the viSNE plot.

(A) viSNE plot and density dot plot of CD45 +/dim CD34 + cells from lineage-depleted BM cells and ileum LPLs in each individual organ donor (D#259, D#280, D#305 and D#337). CD45 +/dim CD34 + cells (gated as shown in dot plots) are indicated in red in viSNE plots.

Consistent with the long-term multilineage chimerism in patients with high peak levels (>4%) of donor T cell chimerism in blood ( Figure S1 ), we detected circulating donor HLAhematopoietic progenitors (LinCD45CD34) ( Figure 4 A) in 7 of 7 patients ( Figure 4 B). We hypothesized that these originated in the graft, as CD45CD34cells have been reported in adult human small intestine () and liver (). Using the current differentiation scheme for human hematopoiesis (), we further assessed the presence in such tissues of HSCs and progenitors, including MPPs, LMPPs, CLPs, MPs, and CD56progenitors (), by flow cytometry (FCM) ( Figures 4 C and S4 ). We analyzed HSPCs in intestinal mucosa, PPs, MLNs, and BM from multiple deceased organ donors ( Figures 4 C, 4D, and S4 Table S3 ). All of these tissues contained HSPCs at variable levels ( Figure 4 D). Ileum LPLs included higher percentages of HSCs than IELs or cells from MLN ( Figure 4 D). Remarkably, the percentages of HSCs and LMPPs were greater among CD45CD34cells of ileum IEL, LPL, and PPs than among adult BM or fetal liver cells ( Figure 4 D). The percentage of LinCD45CD34cells or of HSCs among LinCD45CD34cells in ileum LPLs was not dependent on donor age or sex ( Figure S5 ). In a small number of transplant patients for whom pre-ITx donor liver biopsies (n = 2) and donor organ perfusates (n = 8) were available, we were able to detect variable levels of HSPCs ( Figures 4 E and 4F). Therefore, both liver and intestines are reservoirs for HSPCs that may partially explain the long-lasting donor mixed chimerism that is more frequently seen in MVTx patients than iITx patients.

(E and F) Percentages of CD45 +/dim CD34 + cells among DAPI − Lin − CD45 +/dim cells and percentages of HSC, MPP, LMPP, CLP, MP, and CD56 + progenitors among DAPI − Lin − CD45 +/dim CD34 + cells in liver biopsies (E) and organ perfusates (F) collected from donors of pts 15, 16, 17, 18, 20, 21, 23, and 24 pre-ITx.

(D) Percentage and composition of HSPCs in human ileum (IEL, LPL), PPs, MLN, and BM among multiple deceased organ donors. Student’s t test was used for statistical comparisons between paired tissues, including ileum IEL, LPL, PPs, and MLN ( ∗ p < 0.05, ∗∗ p < 0.01). Means and SDs are shown. Cells from two fetal liver donors are used as reference.

(B) Percentage (left) and concentration (right) of donor cells among DAPI − HLA-ABC + Lin − CD45 +/dim CD34 + HPs in blood from indicated patients and POD.

Having it all? Stem cells, haematopoiesis and lymphopoiesis in adult human liver.

Having it all? Stem cells, haematopoiesis and lymphopoiesis in adult human liver.

These data suggest that tolerance to the recipient was induced among long-term donor T cells in the recipient lymphoid compartment, consistent with de novo development of the naive T cells in the recipient thymus and with the possibility that recipient-specific regulatory T cells (Tregs) promote tolerance of GvH T cells from the intestinal allograft.

TCR sequencing on sorted donor HLACD3CFSEcells from pt 15 POD 83 MLR indicated that the weak GvH response revealed by depletion of CD25cells included a dominant pre-existing donor GvH clone ( Figure 3 C), despite the small sample size and low number of unique clones (124), in which four donor-mappable clones were identified. Additional GvH clones identified in pre-Tx MLR by their CDR3 sequences persisted long-term in both the allograft (POD >140), at high frequency (), and recipient peripheral blood (POD >250), at low frequency ( Figure 3 D).

We performed MLRs on sorted T cells from pt 15 PBMCs collected on POD 83 and POD 214, with 27% and 26% of donor T cell chimerism, respectively. In contrast to pre-Tx donor T cells from spleen, which reacted vigorously to recipient antigens, circulating donor T cells from post-Tx PBMCs did not proliferate to recipient antigens but reacted strongly to third party antigens ( Figure 3 A). However, depletion of CD25cells from the sorted T cell responders revealed a donor CD4 T cell response to recipient stimulators ( Figure 3 A). Similar specific donor T cell hyporesponsiveness to the recipient was observed using PBMCs from pt 7 (POD 253, 1.5% donor T cell chimerism) and POD 786 splenocytes from pt 16, who still showed 3.7% donor T cell chimerism in the spleen. Hyporesponsivenss of donor T cells in all 3 patients was recipient-specific ( Figure 3 B), despite equal or even greater HLA mismatching to the recipient than to the 3party ( Figure 3 E)

(E) HLA typing (HLA-A, -B, -C, DRB1, and DQ) and number of HLA mismatches (red) of donor with recipient and 3 rd party is shown for pt 15. Pt 15’s donor had a greater number of HLA mismatches to the recipient (8/10) than to the 3 rd party (7/10). Pt 7 and pt 16’s donors each had equal numbers of HLA mismatches to the recipient and the 3 rd party, 5/10 and 8/10, respectively (data not shown).

(D) Total frequencies among all TCR sequences for representative GvH clones that were detected in pre-Tx donor spleen or MLN, post-Tx early (POD <70) and late (POD >140) ileum biopsies (Bx), and early (POD <70) and late (POD >250) PBMCs in pts 4, 7, 13, and 15.

(C) Donor HLA + CD3 + CFSE low cells were sorted on day 6 of a MLR in which CD25 − T cells sorted from POD 83 PBMCs were used as responders, and irradiated recipient pre-Tx splenocytes were used as stimulators. TCRβ CDR3 DNA sequencing was performed on sorted donor HLA + CD3 + CFSE low cells, and the cumulative frequency of GvH clones as a percentage of all sequences within donor mappable clones is shown. “Donor mappable clones” refer to clones that were detectable in sequenced pre-Tx spleen, lymph node, and/or MLR CFSE low T cell populations from the donor. Four such clones were identified among a total of 124 unique sequences.

(B) Summary of %CFSE low donor CD4 and CD8 T cells in functional MLRs using pre-Tx donor splenocytes (pts 15, 7, and 16) or post-Tx PBMCs (pt 15 POD 214 and pt 7 POD 253) or splenocytes (pt 16 POD 786) as responders against irradiated stimulators. Student’s t test was used to compare paired data as indicated ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n.s.: no significant difference). Means and SDs are shown in bar graphs.

(A) Functional MLR using pt 15 pre-Tx donor splenocytes or post-Tx total or CD25 − T cells sorted from recipient PBMCs collected on POD 83 and POD 214 as responders, and irradiated recipient pre-Tx splenocytes or 3 rd party PBMCs as stimulators. CFSE proliferation profile of gated donor CD4 and CD8 T cells is shown.

Sorting and sequencing donor T cells in pt 18’s and pt 15’s PBMCs on POD 357 and POD 143, respectively, similarly revealed that a high proportion (96.3% and 98.2%, respectively) of TCRs were undetectable (i.e., “non-mappable”) in the pre-Tx donor lymphoid tissues ( Figures 2 B and 2C). The 48,042 non-mappable clones identified from pt 15’s sorted donor PBMC T cells on POD 143 showed similarly high diversity as pre-Tx donor unstimulated spleen and MLN ( Figure 2 C, D) using a new diversity measure (slope) that captures the majority of clones () and by clonality measurements. In sum, the lack of repertoire overlap with pre-Tx donor lymphocytes combined with the enrichment of RTEs and TRECs among donor circulating T cells suggests that these donor circulating naive T cells developed de novo in the recipient post-Tx.

Naive T cells demonstrate high repertoire diversity, making it difficult to detect clonal overlap among different tissues within the same individual (). Consistently, when we sorted naive and memory donor T cells from pt 7 PBMCs collected from day 101 to 136 post-Tx and performed high-throughput TCRβ CDR3 sequencing, clones overlapping with those in pre-Tx donor lymphoid tissue were detected only among memory (209/579 unique sequences = 36.1%) but not naive donor T cells ( Figure 2 A). Naive donor T cells in PBMC also showed much less overlap with early ileum biopsy T cells (POD 24) compared to their memory counterparts ( Figure 2 A). Indicative of greater diversity, the clonality (0.061) of naive circulating donor T cells at POD 101–136 was lower than that of their memory counterparts (0.150).

(D) Abundance plots of nonmappable clones detected from sorted donor T cells in pt 15 PBMCs POD 143 (green), and pre-Tx D4U (black) and D8U (red) T cells from spleen or total T cells from MLN (blue), showing relative log abundance of TCR sequences (y axis) against their log rank in abundance (x axis: low to high frequency). Power-law slopes of abundance plots, whose absolute values vary directly as diversity (), are shown.

(B and C) Proportional Venn diagram showing TCR clonal overlap among sorted donor T cells in pt 18 PBMCs POD 357 (B) or pt 15 PBMCs POD 143 (C) and pre-Tx donor spleen or MLN. “Non-mappable” percentage is the percent of TCR sequences in donor PBMC that were not detected in pre-Tx donor spleen or MLN.

(A) Venn diagram showing TCR clonal overlap among donor memory and naive T cells sorted from PBMCs POD 101–136, pre-Tx donor spleen (including unstimulated [D4U and D8U], and CFSE low -stimulated T cells [D4L and D8L]) and post-Tx ileum biopsy (POD 24) from pt 7. D4U and D8U represent unstimulated donor CD4 and CD8 T cells, respectively; D4L and D8L represent CFSE low donor CD4 and CD8 T cell responders in MLRs, respectively. Clonality of each population is shown.

Unlike the donor intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) T cells, which displayed a tissue-resident phenotype () and consisted mostly of memory T cells, circulating donor T cells expressed a CD28CD69CD103phenotype consistent with circulating T cells and not tissue-resident T cells (CD28CD69CD103) and included both naive (CD45RA) and memory phenotypes (CD45RO) ( Figures 1 A and 1B ). Circulating donor CD4 T cells were markedly enriched for the recent thymic emigrant (RTE) phenotype ( Figure 1 C) compared with recipient cells in patients ≥5 years old ( Figures 1 D, 1G, S2 A, and S2B). Younger recipients generally had high recipient and donor RTE levels ( Figures 1 D and S2 B), presumably reflecting robust thymic function. A similar trend was observed for circulating donor CD8 T cells ( Figures 1 E–1G, S2 A, and S2B). RTEs were significantly enriched in circulating donor compared to recipient CD4 T cells at early (POD ≤100), intermediate (POD 100–200), and late (POD >200) stages post-Tx ( Figure S2 C). A similar increase was seen for RTEs among donor versus recipient CD8 T cells from POD 100–200. CD31 expression on CD4 T cells is a well-validated RTE marker (), whereas its expression on CD8 T cells may be affected by other factors (). Analysis of T cell receptor excision circles (TRECs) as an indicator of recent thymic origin () confirmed that CD45RACD4 T cells expressed much higher levels of TRECs than CD45ROcells in both donor-derived and recipient T cells in pt 1 and in healthy controls (HCs) ( Figure 1 H). Both donor and recipient CD45RACCR7T cells obtained 167–786 days post-Tx from pts 15, 16, 18, and 19 were markedly enriched for TRECs ( Figure 1 I). Overall, enrichment of RTE phenotypes and TRECs strongly supports the notion of de novo generation of circulating donor T cells post-Tx. Circulating donor-derived δ1T cells and CD19B cells in patients with long-term multilineage chimerism were also enriched for naive populations ( Figure S3 ).

(I) Number of TRECs detected in naive (CD45RA + CCR7 + ) or non-naive CD3 + T cells from one HC’s PBMCs and pts 15, 16, 18, and 19’s PBMCs collected on POD 255, 786, 314, and 167, respectively.

(H) Number of TRECs detected in CD45RA + or CD45RO + CD4 T cells from two HCs’ PBMCs and pt 1’s PBMCs POD 246/365.

(G) Percentage of RTEs among CD4 and CD8 T cells in circulating donor (“D”) or recipient (“R”) T cell populations in the same sample (shown connected with a line) post-Tx of recipients at least 5 years old. Donor and recipient phenotypes in the same sample were compared using a two-tailed paired Student’s t test.

(C–F) FCM gating of CD4 + RTEs (CD45RA + CD45RO − CD31 + ) in pt 1 PBMCs POD 246 (C) and CD8 + RTEs (CD45RA + CCR7 + CD31 + ) in pt 15 PBMCs POD 83 (E). Percentage of RTEs among CD4 (D) or CD8 (F) T cells in healthy control (HC, gray) PBMCs or circulating donor- (red) or recipient-derived (black) CD4 or CD8 T cells post-Tx.

(B) Expression of CD45RA and CD45RO on donor or recipient CD4 T cells from pt 7’s ileum IELs and LPLs on POD 45, and PBMCs on POD 45 and POD 121.

(A) Expression of CD28, CD69, and CD103 on donor CD4 and CD8 T cells from pt 7’s PBMCs or ileum IELs and LPLs isolated on POD 127.

Circulating Donor-Derived T Cells Are Enriched for RTE Phenotypes and TRECs Regardless of Donor Age

Figure 1 Circulating Donor-Derived T Cells Are Enriched for RTE Phenotypes and TRECs Regardless of Donor Age

CD31 (PECAM-1) is a marker of recent thymic emigrants among CD4+ T-cells, but not CD8+ T-cells or gammadelta T-cells, in HIV patients responding to ART.

We extended our previous longitudinal prospective study of peripheral blood chimerism (patients [pts] 1–7, 9, and 10) () by recruiting 12 more patients (pts 13–24) to include a total of 10 MVTx, 1 LITx, and 10 iITx recipients ( Table S1 ) followed up to 5 years post-ITx. By combining donor- and/or recipient-specific HLA markers with a pan-HLA-ABC monoclonal antibody (mAb), we could reliably distinguish donor from recipient cells as described () ( Figure S1 A; Table S2 ). High level (>10%) and long-lasting (postoperative day [POD] >90) donor lymphoid (T, B, NK) and myeloid chimerism was more frequently seen in MVTx recipients than iITx recipients ( Figures S1 B–S1D and S1F–S1H). A lower level of peak donor T cell chimerism (<4%) in blood was significantly associated with the development of early (POD <90) moderate to severe graft rejection ( Figure S1 J), strengthening our previous observations (). Myeloid chimerism was generally at a low level and transient even in MVTx patients (). However, we observed recurrent donor chimerism among CD14monocytes (>10%) late post-Tx in MVTx recipients pt 15 (POD 255) and pt 18 (POD 314) and relatively persistent (>2 months) donor chimerism among CD14monocytes in MVTx recipient pt 19 ( Figures S1 E and S1I).

Discussion

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Darche S.

Nussenzweig M.C.

Kourilsky P.

Vassalli P. Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. Guy-Grand et al., 2003 Guy-Grand D.

Azogui O.

Celli S.

Darche S.

Nussenzweig M.C.

Kourilsky P.

Vassalli P. Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. While the phenotype of circulating donor-derived T cells strongly suggests a recipient thymic origin, other sites of potential T cell development, such as the intestine itself, must be considered. Linc-kitHPs have been identified in mouse gut cryptopatches and found to give rise to TCR αβ and γδ IEL T cells (). However, αβ T cells are a minor population generated in this site, and this pathway is suppressed in the presence of a normal thymus (). Extrathymic T lymphopoiesis in MLNs and intestinal mucosa was enhanced in lymphopenic conditions (), and ATG-induced lymphopenia might also promote de novo local generation of T cells from intestinal HSPCs in patients after ITx. This would only be likely to occur early post-Tx, because ATG induction is generally given within the first week in our center.

Howie et al., 1998 Howie D.

Spencer J.

DeLord D.

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MacDonald T.T. Extrathymic T cell differentiation in the human intestine early in life. Lynch et al., 2006 Lynch L.

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Golden-Mason L. Detection and characterization of hemopoietic stem cells in the adult human small intestine. Williams et al., 2004 Williams A.M.

Bland P.W.

Phillips A.C.

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Brooklyn T.

Shaya G.

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Probert C.S. Intestinal alpha beta T cells differentiate and rearrange antigen receptor genes in situ in the human infant. Howie et al., 1998 Howie D.

Spencer J.

DeLord D.

Pitzalis C.

Wathen N.C.

Dogan A.

Akbar A.

MacDonald T.T. Extrathymic T cell differentiation in the human intestine early in life. Williams et al., 2004 Williams A.M.

Bland P.W.

Phillips A.C.

Turner S.

Brooklyn T.

Shaya G.

Spicer R.D.

Probert C.S. Intestinal alpha beta T cells differentiate and rearrange antigen receptor genes in situ in the human infant. −CD45+/dimCD34+ cells obtained from BM versus ileum intestinal LPLs, including subsets expressing the progenitor thymus-homing marker CCR9 ( Haddad et al., 2006 Haddad R.

Guimiot F.

Six E.

Jourquin F.

Setterblad N.

Kahn E.

Yagello M.

Schiffer C.

Andre-Schmutz I.

Cavazzana-Calvo M.

et al. Dynamics of thymus-colonizing cells during human development. Zlotoff and Bhandoola, 2011 Zlotoff D.A.

Bhandoola A. Hematopoietic progenitor migration to the adult thymus. −CD45+/dimCD34+ progenitors in the circulation of patients with long-term chimerism, supporting the hypothesis that donor HSPCs from the graft migrate into the recipient circulation and into the BM and other sites of potential hematopoiesis, where they differentiate and contribute to the circulating leukocyte pool. Evidence for extrathymic T cell development in human intestine is more limited (). Immature T cells and lymphoid progenitors, along with TdT and RAG gene expression, have been reported in fetal and infant intestines, but disappeared by 18 months of age (). Many (12/21 = 57.1%) of our ITx donors were older than 18 months (2–48 years old, Table S1 ), arguing that circulating naive donor T cells did not develop in situ in the graft. In our study, the CD8αα phenotype did not clearly identify cells of intestinal origin ( Figure S7 ), as the vast majority of blood, IEL, and LPL CD8 T cells are CD8αβ and few donor or recipient CD8αα T cells were detected in peripheral blood. Thus, while our studies do not rule out an intraepithelial origin for the RTE-like donor T cells detected in recipient blood after ITx, the phenotype, high TREC content, and high TCR diversity of these cell populations, as well as their tolerance to recipient stimulators in MLR, favor the hypothesis that these cells develop in the recipient thymus from progenitors carried in the donor graft that enter the circulation and either migrate directly to the thymus or differentiate from earlier progenitors after settling in the recipient BM. Indeed, CyTOF analysis revealed similar phenotypes for LinCD45CD34cells obtained from BM versus ileum intestinal LPLs, including subsets expressing the progenitor thymus-homing marker CCR9 (). We detected donor LinCD45CD34progenitors in the circulation of patients with long-term chimerism, supporting the hypothesis that donor HSPCs from the graft migrate into the recipient circulation and into the BM and other sites of potential hematopoiesis, where they differentiate and contribute to the circulating leukocyte pool.

−CD45+/dimCD34+ cells or of HSCs among Lin−CD45+/dimCD34+ cells in the ileum of multiple deceased donors 9–93 years of age. In previous studies, T/NK lineage-polarized CD34+CD45RA+CD7+ HPs corresponding to candidate prethymocytes were detected in fetal BM and cord blood, found to decline around birth, and persisted at a low level in adult BM ( Haddad et al., 2004 Haddad R.

Guardiola P.

Izac B.

Thibault C.

Radich J.

Delezoide A.L.

Baillou C.

Lemoine F.M.

Gluckman J.C.

Pflumio F.

Canque B. Molecular characterization of early human T/NK and B-lymphoid progenitor cells in umbilical cord blood. Haddad et al., 2006 Haddad R.

Guimiot F.

Six E.

Jourquin F.

Setterblad N.

Kahn E.

Yagello M.

Schiffer C.

Andre-Schmutz I.

Cavazzana-Calvo M.

et al. Dynamics of thymus-colonizing cells during human development. +CD7+ cells among Lin−CD45+/dimCD34+ populations in BM of three donors 26–76 years of age. Lin−CD45+/dimCD34+ CD45RA+CD7+ cells were also detected in ileum LPLs of some but not all donors. We did not observe any relationship between age and the percentage of LinCD45CD34cells or of HSCs among LinCD45CD34cells in the ileum of multiple deceased donors 9–93 years of age. In previous studies, T/NK lineage-polarized CD34CD45RACD7HPs corresponding to candidate prethymocytes were detected in fetal BM and cord blood, found to decline around birth, and persisted at a low level in adult BM (). In contrast, we detected high proportions (9%–76%) of CD45RACD7cells among LinCD45CD34populations in BM of three donors 26–76 years of age. LinCD45CD34CD45RACD7cells were also detected in ileum LPLs of some but not all donors.

Granick et al., 2012 Granick J.L.

Simon S.I.

Borjesson D.L. Hematopoietic stem and progenitor cells as effectors in innate immunity. Saenz et al., 2010 Saenz S.A.

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et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. Lefrançais et al., 2017 Lefrançais E.

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et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Extramedullary hematopoiesis has been reported in diverse tissues including the gastrointestinal tract, usually in association with pathologic processes, such as myelofibrosis, inflammation, and infection (). Thus, we cannot rule out the possibility that circulating donor monocytes also emerged from the donor graft, perhaps in response to inflammation, without intermediate residence in the recipient BM. A study in mice demonstrated that lung is a primary site of terminal platelet production (), suggesting that hematopoietic progenitor cells can travel through the bloodstream and differentiate in various organs.

Zuber et al., 2016 Zuber J.

Shonts B.

Lau S.P.

Obradovic A.

Fu J.

Yang S.

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Weiner J.

Thome J.

et al. Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome. Fu et al., 2017 Fu J.

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Xia A.

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Miron M. Differing mechanisms for early versus persistent donor T cell chimerism in peripheral blood of human intestinal transplant recipients. Fu et al., 2017 Fu J.

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Xia A.

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Yang S.

Miron M. Differing mechanisms for early versus persistent donor T cell chimerism in peripheral blood of human intestinal transplant recipients. Zuber et al., 2016 Zuber J.

Shonts B.

Lau S.P.

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Fu J.

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Thome J.

et al. Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome. Sykes et al., 1988a Sykes M.

Sheard M.A.

Sachs D.H. Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance. Sykes et al., 1988b Sykes M.

Sheard M.A.

Sachs D.H. Graft-versus-host-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells. Our recent work has suggested a role for two-way alloreactivity in determining intestinal allograft T cell repopulation by the recipient () and blood T cell macrochimerism (). Enrichment of graft-versus-host (GvH) compared to host-versus-graft (HvG) clones in the graft, the absence of de novo Class I donor-specific antibodies (DSA), and freedom from moderate and/or severe rejection are associated with donor T cell macrochimerism in blood (). We hypothesize that early lymphohematopoietic GvH responses (LGVHR; GvH responses that do not induce clinical GVHD) () attack recipient hematopoietic cells, creating “space” in the recipient BM that permits engraftment of donor cells, including HSPCs carried within the graft.

Zuber et al., 2015 Zuber J.

Rosen S.

Shonts B.

Sprangers B.

Savage T.M.

Richman S.

Yang S.

Lau S.P.

DeWolf S.

Farber D.

et al. Macrochimerism in intestinal transplantation: association with lower rejection rates and multivisceral transplants, without GVHD. Wang et al., 2012 Wang X.Q.

Lo C.M.

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Chen Y.X.

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Zhang W.

et al. Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver. Zuber et al., 2016 Zuber J.

Shonts B.

Lau S.P.

Obradovic A.

Fu J.

Yang S.

Lambert M.

Coley S.

Weiner J.

Thome J.

et al. Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome. Zuber et al., 2015 Zuber J.

Rosen S.

Shonts B.

Sprangers B.

Savage T.M.

Richman S.

Yang S.

Lau S.P.

DeWolf S.

Farber D.

et al. Macrochimerism in intestinal transplantation: association with lower rejection rates and multivisceral transplants, without GVHD. We previously demonstrated only low levels and transient lymphoid or myeloid chimerism in recipients of liver transplants alone (). However, adult human livers () contain pluripotent HSCs with repopulating potential that may contribute to donor chimerism in MVTx recipients. The large load of GvH-reactive clones that expand locally in the intestinal mucosa () may promote the greater and more prolonged chimerism in MVTx compared to LTx recipients ().

Our study reveals the underlying mechanism for the persistence of donor mixed chimerism in recipient blood and provides unique information on the dynamics of human HSPCs. Our data suggest that the human gut serves as a significant site of HSPC residence that contributes to circulating leukocytes. Whether this is true under physiological conditions in addition to ITx needs further investigation. The contribution of graft-derived HSPCs to sustained mixed chimerism in the blood suggests the possibility of promoting allograft tolerance in ITx recipients.