Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome

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Science Immunology  07 Oct 2016:
Vol. 1, Issue 4, eaah3732
DOI: 10.1126/sciimmunol.aah3732

Transplantation stalemate

Successful organ transplantation depends on both preventing rejection of the graft by host cells [host versus graft (HvG)] and blocking graft cells that attack the host (GvH). Zuber et al. demonstrated that a balance in this two-way alloreactivity affected clinical outcomes in organ transplant recipients. They examined gut-resident T cell turnover kinetics in human intestinal allografts and found that HvG-reactive cells persisted long-term in the graft, acquiring a tissue-resident phenotype. However, GvH-reactive cells expanded within the graft in the absence of rejection. The GvH-reactive cells may balance out the HvG-reactive cells, preventing rejection.


One paradigm in transplantation is that graft-infiltrating T cells are largely nonalloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time have not been investigated in detail in animals or humans. We used polychromatic flow cytometry and high-throughput T cell receptor sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intragraft host-versus-graft (HvG) and graft-versus-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted in the long term in the graft. Early expansion of GvH clones in the graft correlated with the rapid replacement of donor antigen-presenting cells by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ αβ T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady-state tissue-resident phenotype but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in nonrejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.


Small-bowel transplantation is complicated by high rates of rejection (1), infection, and, in 5 to 9% of cases, graft-versus-host (GvH) disease (GVHD) (2), resulting in about 50% 5-year patient survival rates. Large numbers of donor lymphohematopoietic cells are transferred within these grafts (2). This large load of passenger donor-derived T cells combined with reductions of recipient T cell mass may promote GVHD (3). Conversely, donor-specific antibodies (DSAs) and associated intractable rejections have emerged as leading causes of intestinal allograft failure (1), and pretransplant DSAs are associated with accelerated clearance of graft-derived circulating donor cells (4) and lower rates of GVHD (3) after intestinal transplantation. We hypothesized that clinical outcomes might largely reflect the balance between host-versus-graft (HvG) and GvH reactivities.

Long-lived tissue-resident memory T cells (TRM) have been implicated in murine and human tissue-specific inflammatory disease (5). TRM display hallmark phenotypic markers (CD103 and CD69) (6), demonstrate tissue-specific T cell repertoires (5, 7), and persist in the long term in mucosal tissues (8, 9). However, little is known about how HvG and GvH responses influence the turnover, phenotype, and repertoire of tissue-resident T cell populations after transplantation. Studies in mice (10) and humans (11) have indicated that recipient-derived T cells repopulate the gut-associated lymphoid tissues of intestinal allografts early after transplantation. However, the turnover rate of mucosal T cells after intestinal transplantation has been investigated in only a few patients (12) in cross-sectional studies (11), without phenotype or repertoire analysis (11, 12). We took advantage of serial protocol biopsies in intestinal transplant recipients to address the hypothesis that recipient cells would replace the donor leukocytes rapidly in the presence of rejection and that homeostatic leukocyte turnover could be measured in the absence of rejection. Moreover, we used a novel method of identifying and tracking the alloreactive T cell repertoire to determine the role of GvH and HvG clones in this turnover. Our studies revealed that donor T cells often persist much longer than previously thought in nonrejecting intestinal allografts. The replacement rate of donor T cells varied greatly between patients, depending on the presence or absence of rejection. Recipient T cells repopulating the graft ultimately acquire a tissue-resident phenotype. Contrary to the current paradigm (13), our alloreactive T cell receptor (TCR) tracking method allowed us to demonstrate that most of the graft-infiltrating recipient T cell clones during pure cellular rejection are donor antigen–specific and that the balance of in situ GvH and HvG responses correlates with the kinetics of graft leukocyte turnover. Graft-resident GvH clones preexisted in donor lymphoid organs as circulating memory cells with an intestinal mucosa counterpart.


Greatly variable graft lymphocyte turnover rates

Using recipient- and/or donor-specific monoclonal antibodies (table S1) in combination with a pan–HLA (human lymphocyte antigen) class I monoclonal antibody, we investigated the phenotypes and origins of intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) populations with multicolor flow cytometry. Single-cell suspensions were obtained from 183 fresh ileum graft biopsies from 14 intestinal transplant patients (Fig. 1, A and B, figs. S1 and S2, and table S2), including 9 patients followed from transplantation to the last follow-up (Fig. 1B and fig. S2, bottom). CD45 nonhematopoietic cells, found mainly in IELs and assumed to be epithelial cells, remained of donor origin as expected (Fig. 1, A and B, and fig. S2). In contrast, recipient T cell replacement occurred over time (Fig. 1B) but with highly variable kinetics between patients (Fig. 1, B and C, and fig. S2). Overall, recipient replacement rates were less uniform and slower for CD45+ CD3+ T cells than for CD56+ CD3 natural killer (NK)/innate lymphoid cells (Fig. 1C), and donor graft lymphocytes persisted much longer than previously reported (Fig. 1, B and C, and fig. S2) (11, 12).

Fig. 1 Relationship between rejection and recipient chimerism in intestinal allografts.

(A) Representative plots (from patient 14) depicting recipient chimerism in cell subsets isolated from graft biopsies (n = 183). Chimerism was detected using recipient HLA allele–specific and pan-HLA class I antibodies. (B) Replacement rate of donor CD3+, CD3 CD56+, and CD45 cells by recipient cells (recipient chimerism) over time in IELs and LPLs, isolated from serial protocol biopsies. The two selected patients depict different T cell turnover kinetics. Pt, patient. (C) to (E) depict 170 biopsies obtained from nine patients, with a longitudinal follow-up from POD0 up to POD600. (C) Kaplan-Meier curves plot the proportion of transplants for which recipient cells permanently exceed 50% in the indicated cell populations over time. Survival curves were compared using log-rank (Mantel-Cox) test. (D) Graphic representation showing the severity/intensity and period of rejection episodes (left) and DSAs (right) during the first 3 months after transplant. MFI, mean fluorescence intensity. (E) Recipient chimerism in CD4 and CD8 IELs and LPLs within the first 45 days (Mann-Whitney U test; **P < 0.01 and ***P < 0.001). (F) Recipient chimerism over time in CD4+ and CD8+ αβ TCR+ IELs isolated from serial biopsies, according to the occurrence of mixed rejection, TCMR, or no rejection. Dots and triangles represent multivisceral transplant and isolated intestinal transplant recipients, respectively. AUC, area under the curve. Turnover kinetics were assessed by the integration of recipient chimerism over time (AUC) and by the 50% turnover rate (right), plotting the proportion of transplants for which recipient cells permanently exceed 50% in the indicated cell population over time. Survival curves and AUC were compared using log-rank (Mantel-Cox) and Mann-Whitney tests, respectively (*P < 0.05 and **P < 0.01). Tx, transplantation.

Faster replacement rate of gut-resident T cell subsets in rejecting intestinal allografts

We hypothesized that uncontrolled rejection would hasten the turnover of gut-resident T cells. We thus analyzed the chimerism of CD3+ γδ TCR-negative (αβ) CD4 and CD8 T cell populations according to the occurrence of biopsy-proven rejection and DSA (Fig. 1D). Seven patients experienced early rejections, within 90 days after transplantation, including four with high-titer (mean fluorescence intensity > 2000) anti-HLA class I and II DSA and histological features of mixed (cellular and antibody-mediated) rejection (Fig. 1D). Low levels of recipient chimerism among CD4+ and CD8+ αβ TCR+ IELs and LPLs during the first 45 days were significantly associated with the absence of biopsy-proven rejection (Fig. 1E and table S3). Over time, cell replacement rates in the graft were significantly faster for CD4+ and CD8+ αβ TCR+ LPLs and IELs over the first 3 and 6 months after transplant in patients with mixed rejection compared with those without (Fig. 1F and fig. S3). Recipient chimerism that peaked at the time of early pure T cell–mediated rejection (TCMR), especially in CD4+ IELs, then returned to baseline after rejection resolution (Fig. 1F). In contrast, recipient chimerism peaked at a greater level in mixed rejection compared with TCMR and remained much greater than previous rejection even after treatment (Fig. 1F). Differences in treatment-induced tissue lymphodepletion did not account for these differences, because absolute CD3+ IEL counts on immunostained biopsy sections were similar in both groups after antithymoglobulin induction and at 3 months (fig. S4). Together, our findings demonstrate that strong antidonor immune responses accelerated recipient replacement of donor αβ T lymphocytes in human intestinal allografts.

Recipient T cells involved in rejection initially exhibit a different phenotype from those arising from physiological turnover

Human mucosal–resident T cells show reduced expression of CD28, especially on CD8 cells, and high expression of CD103 and CD69 compared with peripheral blood cells (6, 14). We analyzed IEL and LPL phenotypes from 171 ileal graft biopsies from nine intestinal transplant recipients (Fig. 2) and from 10 ileum specimens obtained from deceased organ donors (fig. S5A and table S2). Donor-derived IELs and LPLs exhibited roughly the same phenotypic features as those from organ donors (Fig. 2A and fig. S5A). However, the phenotype of recipient-derived IEL and LPL cells isolated from early biopsies differed significantly from that of donor cells (Fig. 2, A to C), displaying an intermediate phenotype between those of blood-borne and gut-resident T cells (Fig. 2A). Recipient T cells eventually acquired tissue-resident phenotypic markers (Fig. 2, B and C).

Fig. 2 Relationship between recipient intestinal T cell phenotype and rejection.

(A and B) Comparison of recipient and donor IEL and LPL T cell subset phenotypes with that of blood T cells. Representative IELs and LPLs at early (A) and late (B) time points are shown. (C) Evolution of recipient and donor T cell phenotype in IELs (left) and LPLs (right) over time. CD28, CD103, and CD69 phenotypic information was available for 127, 83, and 120 biopsies, respectively. (D) Representative contour plots (from patient 6) showing CD28 up-regulation on recipient but not donor CD8+ IELs during rejection. (E) Summary of CD28 expression by recipient CD8 IEL T cells in relationship to rejection (R) episodes. Frequencies of CD28+ cells were compared using Mann-Whitney test (****P < 0.0001). HC, healthy control.

Whereas CD28 expression among recipient IEL T cells was initially higher than that of resident donor T cells even in the absence of rejection (Fig. 2, C and E), a significantly greater proportion of recipient CD8+ IEL T cells expressed CD28 within a week of rejection episodes compared with biopsies more temporally removed from rejections (Fig. 2, D and E). CD28+ CD8+ IELs from late rejections expressed high levels of CD103 and CD69, like their CD28 counterparts (Fig. 2D). In the context of gut inflammation, up-regulation of natural killer (NK) group 2 member D (NKG2D) by CD8+ IELs, with increased expression of its ligands, has been correlated with enhanced cytotoxic potential (15, 16). Whereas most recipient CD8+ IELs expressed less NKG2D than peripheral blood CD8 T cells, similar to donor CD8+ IELs and those from organ donors (fig. S5B), CD8+ CD28+ recipient IELs exhibited high NKG2D expression in rejecting biopsies (fig. S6A). In parallel, the epithelium of rejecting biopsies expressed much higher levels of the NKG2D ligand MICA than nonrejecting grafts or normal duodenum (fig. S6B). Together, these data suggest that recipient-derived CD28+ NKG2DHi CD8+ IELs play a role in graft rejection, whose late forms may have a TRM component.

Origin and turnover of the ileal T cell repertoire after transplantation

High-throughput TCRβ CDR3 analysis was performed on 18 ileal biopsies, including 6 with overt rejection, from seven intestinal transplant recipients (Fig. 3A). As expected, complete replacement of donor graft–resident T cells by the recipient was accompanied by extensive repertoire turnover (patient 9; patient 10, T1 to T2; patient 14, T1 to T3), whereas the TCR repertoire was more conserved over time when the graft-resident T cells were mostly of the same origin (donor versus recipient) across different biopsies (Fig. 3, B and C, and fig. S7). We examined repertoire overlap between early and late biopsies among the clones in the biopsies that could be mapped to pretransplant donor or recipient lymphoid samples (table S4). Considering that different sites were biopsied at different time points, donor-derived T cells exhibited a remarkably stable repertoire as long as they were maintained in the graft, as reflected by the high repertoire overlap over a period of 55 to 200 days in patients whose grafts showed little T cell replacement by the recipient in this period (patients 7, 13, and 15; Fig. 3, B to D). In contrast, recipient cells from the same biopsies displayed much greater turnover but still with significant overlap between early and late time points (Fig. 3, D to F). In patient 14, for instance, 19.5% of the 773 clones identified as derived from the recipient [postoperative day (POD) 16 biopsy] were shared with those detected at POD156 (Fig. 3E). In terms of cumulative frequency, 21.3 and 67.6% of the recipient clones found in the POD156 biopsy (T3′) overlapped with recipient clones from POD16 (T2′) and POD226 (T4′) biopsies, respectively (Fig. 3F). Similarly for all patients, recipient cell repertoire became more stable over time, after complete replacement of donor lymphocytes by the recipient (patients 4, 10, and 14; Fig. 3F). Together with the finding that recipient cells eventually expressed high levels of CD69 (Fig. 3G), these data suggest that the first T cells entering the graft were mainly CD69 effector cells with a short life span, a minor subset of which gave rise to recipient-derived long-lasting CD69+ TRM.

Fig. 3 Clonal analysis of recipient T cells in intestinal allografts.

(A) Timeline for biopsies showing rejection (red) or nonrejection (black) analyzed by multicolor flow cytometry (T1 to T4) or repertoire analysis (T1′ to T4′) in seven patients. (B) Frequency of recipient and donor cells in isolated LPLs for seven patients. IEL cell origin, which was almost superimposable with LPLs, is shown in fig. S7. (C) Cumulative frequency of T cell clones (defined by TCRβ CDR3 sequences) overlapping in sequential biopsies. (D) Venn diagram representing two biopsies (T1′ and T2′) from patient 7, showing the number of clones detected in each biopsy alone or in both, according to their donor (D) or recipient (R) origin. (E) Venn diagram representing the number of recipient (R) clones overlapping across three biopsies taken from patient 14 at POD16 (T2′), POD156 (T3′), and POD238 (T4′). (F) Cumulative frequency of the clones identified in a given biopsy (indicated within the brackets) overlapping in other biopsies taken earlier (purple) (patients 4 and 9) or both earlier (purple) and later (green) (patients 10 and 14). (G) Histograms representing three biopsies taken from patient 14. PBMCs, peripheral blood mononuclear cells.

HvG-reactive clones accumulate in rejecting biopsies

We hypothesized that rejection-associated increases in recipient T cell repopulation of the graft reflected infiltration of HvG clones. To test this hypothesis, we used pretransplant samples to identify and then tracked the donor-reactive TCR repertoire in posttransplant biopsies using the high-throughput sequencing approach we recently described (table S4) (17).

We investigated five biopsies with early rejection, from days 12 to 24 (Figs. 3A and 4A). HvG-reactive clones were highly enriched relative to non–HvG-reactive recipient-mappable clones in the biopsies with rejection compared with pretransplant peripheral lymphoid samples. HvG clones subsequently declined yet persisted at a lower frequency, after the resolution of rejection (Fig. 4A). The greatest enrichment in HvG clones (up to 80% of recipient-mappable clones), in both CD4 and CD8 T cells, was observed in biopsies with pure TCMR. Patient 15, who remained free of rejection within the first 3 months after transplant, showed enrichment of HvG clones in two early biopsies (POD27 and POD55) compared with pretransplant lymphoid tissues, but to a lesser degree than those in patients with early rejections (Fig. 4A). The increased frequency of HvG clones was less compelling in late rejection and did not change significantly after successful treatment (Fig. 4A). In addition, blood TCR repertoire was analyzed at the time of rejection before any treatment in one patient and showed markedly less enrichment for HvG-reactive clones than what was seen in the rejecting graft (Fig. 4B). Thus, we concluded that HvG clones accumulated within the graft at the time of early rejection and then declined but persisted long after rejection resolution.

Fig. 4 Evolution of HvG-reactive recipient T cell clones in intestinal allografts.

(A) Frequency of HvG clones in rejecting biopsies (red) compared with pretransplant lymph node (patients 4, 9, 13, and 14) or spleen (patients 7, 10, and 15) samples (black) and nonrejecting biopsies (gray). The denominator includes all the clones that were identified in pretransplant recipient samples (HvG plus all other clones). HvG clone frequencies were compared using Fisher’s exact test (***P < 10−25, **P < 10−10, and *P < 10−3). (B) Frequency of HvG clones in the blood (purple) and in the graft (red) at the time of rejection (POD20) compared with pretransplant spleen sample in patient 7(Fisher’s exact test; ***P < 10−25 and **P < 10−10).

HvG-reactive clones take up long-term residency after rejection resolution

The expression of CD69 and CD103 by CD28+ NKG2DHi T cells isolated from late-rejection biopsies along with the persistence of HvG-reactive clones after the resolution of rejection suggested that HvG-reactive clones took up long-term residency within the graft. Remarkably, HvG-reactive clones seeded the entire intestinal tract, including the native colon, with roughly the same frequency of HvG-reactive clones in the transplanted and nontransplanted colon segments (fig. S8A) (18). This observation, combined with the high level of overlap of HvG clones from ileal biopsies taken from different locations at different times, suggests that HvG-reactive clones replenished the total gut-resident TRM compartment, possibly after activation in lymphoid tissues. Apparently, this seeding was independent of the local presence of alloantigens (18, 19). Nevertheless, it seemed possible that repetitive allogeneic stimulation in the graft would result in the likelihood that the same HvG-reactive clones will persist over time and across different graft sites compared with non-HvG clones. We thus compared the clonal overlap between HvG and non-HvG clones identified from two late biopsies taken concurrently from the native and transplanted colon with T cell clones detected in the graft at the time of rejection. HvG clones isolated from the transplanted, in contrast to the native, colon segment displayed a greater overlap with those in the ileum and grafted colon at an earlier time point compared with non–HvG-reactive clones (fig. S8B). These data suggest that the continual donor antigenic stimulation in both lymphoid tissues and the graft may account for greater expansion, recruitment, and persistence of HvG-reactive than non–HvG-reactive recipient clones in the graft.

Intragraft expansion of GvH clones associated with low donor cell replacement in the graft

Passenger lymphocytes from intestinal transplants can cause GVHD (3). However, the origin within the graft and site of activation of these GvH-reactive T cells is unknown. GvH clones were identified as previously described (17) and tracked in posttransplant biopsies (Fig. 5, A and B, and table S4). In all early biopsies in six of six patients (POD9 to POD27), GvH-reactive clones were markedly enriched, compared with pretransplant donor spleen, among the clones identifiable as donor-derived (Fig. 5B). The greatest GvH clonal enrichment in CD4+ cells was found in three patients (patients 7, 13, and 15) in whom the replacement rate of graft donor T cells by recipient cells was the slowest (Figs. 1F and 5B and fig. S3). The decline in HvG clones after rejection resolution in patients 7 and 13 (Fig. 4A) was associated with a reversal of the HvG-to-GvH clonal ratio (Fig. 5, C and D). In contrast, the progressive disappearance of donor cells in other patients (patients 9, 10, and 14) was associated with increased HvG-to-GvH clonal ratios (Fig. 5C).

Fig. 5 Expansion of GvH clones that may counteract the replacement of donor cells by recipient cells.

(A) Venn diagram depicting the number of T cell clones overlapping between donor spleen total clones, GvH-reactive clones, and clones identified as donor-derived in the POD24 biopsy from patient 7. Bx, biopsy. (B) Frequency of GvH clones in early biopsies (red) compared with pretransplant donor spleen samples (black). The denominator includes all clones that could be identified in pretransplant donor samples (Fisher’s exact test; ***P < 10−25, **P < 10−10, and *P < 10−3). (C) Evolution of the HvG-to-GvH clonal ratio after rejection resolution or in the absence of rejection in all patients for whom both HvG and GvH clones were identified in biopsies. (D) Proportion of HvG- and GvH-reactive clones in rejecting (R) and nonrejecting (No R.) biopsies, at POD12 and POD142, respectively, using the clones identified in pretransplant recipient or donor samples in patient 13 as the denominator. (E) From the same patient (patient 13), whose graft exhibited a slow donor T cell replacement rate, proportion of GvH and non-GvH clones from early biopsies (POD12) that were detected in subsequent biopsies (POD142). OR, odds ratio; CI, confidence interval. To permit comparison in the analysis in (F), only GvH clones that were also detectable in the unstimulated populations were used for this analysis. However, analysis that included all GvH clones provided similar results (P < 0.05) yet with a lower OR (~1.5). (F) Comparison of individual clonal frequencies of the GvH and non-GvH clones identified in the early biopsy and pretransplant samples in patient 13. Pretransplant and posttransplant frequencies were compared using Fisher’s exact test (****P < 10−4). Horizontal bars represent medians. ns, not significant.

In patients 7, 13, and 15, in whom donor cells persisted long-term in the graft, GvH-reactive clones mapped in the first biopsy were more likely to persist over time than non-GvH donor-mappable clones (Fig. 5E and fig. S9A). Additionally, the median frequency of individual GvH clones was significantly greater than that of non-GvH clones in early biopsies (Fig. 5F and fig. S9B), in contrast to pretransplant donor spleens. These results suggest that GvH clones accumulated in the graft in an alloantigen-driven manner.

Graft-resident GvH-reactive clones expand from preexisting TRM

TRM clones preexisting in the graft might be expected to have a circulating memory counterpart in draining and remote lymphoid tissues (18). In contrast, GvH clones derived from naïve T cells that are primed in the graft after transplant would preexist only in gut-associated lymphoid tissues such as mesenteric lymph nodes (MLNs) (18). We therefore identified and compared clones present in both donor MLN and spleen with those detected in the MLN or spleen alone in two donors (patients 13 and 10) from whom both tissues were available (Fig. 6 and fig. S10). A far greater proportion of clones originally detected in both donor spleen and MLN were detectable in posttransplant biopsy specimens compared with clones detected in only one of these tissues before transplant (Fig. 6B and fig. S10B). The clonality was low and comparable for CD4 and CD8 TCRs detected in the MLN alone, as is characteristic of naïve cells, whereas the clonality of CD8 cells was higher than that of CD4 cells for clones detected in both spleen and MLN (Fig. 6C and fig. S10C), as is typical for memory cells. Although clones also detected in the spleen accounted for a minority of the MLN clones, they made up roughly 40 to 80% of the CD8+ donor clones detected in the biopsies (Fig. 6D and fig. S10D) and were thus likely tissue-resident memory clones with a recirculating counterpart at the time of transplantation.

Fig. 6 Origin of donor-mappable clones detected in early biopsies from patient 13.

(A) Scatterplots showing donor clones identified in the MLN alone (green) and in the spleen alone (red) and those found in both the MLNs and spleen (purple) in patient 13. (B) The bar graphs depict the proportion of the three donor-derived cell subsets (green, red, and purple) also detected in the posttransplant intestinal biopsies with a sizeable donor population. (C) Clonality of CD4 and CD8 clones from the three subsets. (D) Proportion of blue, green, and red clones among the total donor clones in the MLN, spleen, and posttransplant biopsies.

Rapid graft infiltration by recipient-derived CD14+ myeloid cells

The expansion of GvH-reactive T cell clones in the graft raised the question of the source of recipient antigen in the donor allograft. Whenever sufficient cell yields were obtained, we analyzed the origin of lamina propria antigen-presenting cell (APC) populations, most of which were CD14+ CD11c+ myeloid cells that also expressed CD33, CD11b, and HLA-DR (Fig. 7A). These were very rapidly replaced by recipient-derived cells, which accounted for up to 75% of this subset as early as 10 days after transplant (Fig. 7, B to D). This early replacement of myeloid APCs is uniform between patients, regardless of the rate of recipient T cell replacement. Paired comparison of recipient chimerism between T cell and CD14+ myeloid cell populations showed significantly greater recipient chimerism in myeloid cells (Fig. 7C), demonstrating the rapid turnover of CD14+ myeloid populations within the graft.

Fig. 7 Recipient APCs replace graft APCs early after transplant.

(A) CD14+ CD11c+ CD45+ lamina propria cells express CD33, CD11b, and HLA-DR. (B) Representative contour plots (from patient 14), showing recipient chimerism in CD14+ CD11c+ myeloid cells and in T cells. (C) Comparison of recipient chimerism between myeloid cells and T cells in individual biopsy specimens (paired t test, ***P < 0.0001). (D) Recipient chimerism over time in CD14+ CD11c+ myeloid cells and CD3+ T cells.


The small-bowel mucosa of a healthy human adult is estimated to contain more than 30 billion TRM (20). Rodent experiments have shown that intestinal IELs do not equilibrate with circulating T cells and have low turnover rates (9), but turnover studies of mucosal lymphocytes in allogeneic settings are largely lacking in humans. Our study examines the dynamics of this turnover in detail and provides evidence that two-way alloreactivity has a marked impact on the rate of recipient T cell replacement in the graft mucosa after intestinal transplantation in humans. TCR tracking data demonstrate remarkable stability of the ileal αβ T cell repertoire over time and space as long as gut-resident CD69+ T cells remain of the same origin (donor or recipient). Mixed rejection, which includes an antibody-mediated component, was associated with accelerated replacement of donor αβ TCR+ T cells in the graft. These data show that vigorous antibody- and cell-mediated HvG responses lead to the clearance and early replacement of the donor lymphocytes in the graft. Whereas human skin TRM are spared from circulating cytotoxic antibodies because of the lack of cellular mediators of antibody-dependent cellular cytotoxicity (ADCC) (21), human gut contains CD16-expressing NK cells, which may be cytotoxic under inflammatory conditions (22). Thus, the accelerated clearance of donor graft–resident T cells through ADCC mechanisms might take place in the graft itself. Alternatively, but not exclusively, donor cells bound by DSA might be eliminated from the MLN, where the priming of the HvG T cell response takes place (10).

So far, studies of graft-infiltrating T cells have been limited by the lack of reliable tools to distinguish donor-specific T cells from those recruited in a bystander inflammatory response. Studies in a rodent sponge allograft model have led to the suggestion that donor-specific T cells represent only a small proportion of graft-infiltrating cells (13). In contrast, our study, which specifically tracks the presence of preidentified donor-reactive TCR, shows that donor-specific clones account for up to 80% of the T cells identifiable as recipient-derived in rejecting intestinal allografts, overturning the long-standing dogma that most graft-infiltrating lymphocytes are “bystanders.” At the times of rejection, we detected a large population of CD28+ NKG2DHi CD8+ cells among recipient-derived but not donor-derived IELs, associated with increased expression by epithelial cells of the well-characterized NKG2D ligand MICA, which senses cellular stress. Besides celiac disease, in which the NKG2D/MICA axis has emerged as a major pathogenic pathway (15, 16), NKG2D has been implicated in alloimmune-driven intestinal epithelium damage in rodent models (23). Our data now implicate it in human allograft rejection.

Recipient T cells in the graft ultimately acquired CD69 expression and displayed a stable repertoire over time, which are two hallmarks of TRM (6, 14). A significant proportion of recipient-derived TRM repopulating the graft had HvG-reactive TCR. The limited number of cells available from small forceps biopsies did not allow us to assess the graft-resident lymphocytes in functional assays. It is thus possible that immunosuppression or a “physiological” increase in TCR activation threshold (24) prevented graft-residing HvG-reactive T cell activation at steady state. Consistent with this hypothesis, long-term graft-resident recipient CD8 cells down-regulated CD28 and NKG2D, and this down-regulation may contribute to the increased activation threshold observed for IELs. However, establishment of a local HvG-reactive T cell repertoire poses a constant threat to the allograft that could reactivate in the context of inflammatory triggers. After the loss of CD28 and NKG2D and acquisition of CD103 and CD69 by recipient CD8 T cells that adopted the tissue-resident phenotype, the observed reacquisition of CD28 and high levels of NKG2D with retention of CD69 and CD103 by recipient CD8+ IELs during late rejections suggest that tissue-resident CD8 cells can dynamically up-regulate CD28 and NKG2D during rejection.

Colonization of the native colon by HvG-reactive clones is consistent with the concept from rodent studies that, after activation in MLNs and spleen, a subset of TRM expresses gut-homing molecules and seeds the entire intestinal tract, independent of cognate antigen (19). Broad tissue localization by resident TRM was also recently demonstrated in mouse and human skin (18). In addition to recruiting memory cells from the circulation (25), there is also some evidence that TRM can expand locally through proliferation in response to cognate antigen stimulation (26). However, the extent of this proliferation remains unclear (27). In three patients with long-persisting donor T cells in the graft, GvH clones, which were significantly enriched compared with pretransplant lymphoid tissues, were far more likely to overlap than non-GvH clones across two graft samples. This observation suggests that intragraft stimulation led to intragraft expansion of GvH clones, accounting for their greater persistence across time points and graft biopsy sites.

Studies in mice have shown early emigration and recipient replacement of graft dendritic cells (28, 29). Consistent with a similar mechanism, donor-derived CD14+ myeloid cells were rapidly replaced by their recipient counterparts in intestinal transplant recipients, to a much greater extent than lymphoid populations at the same early time points. The early entrance of recipient-derived APCs seems to induce local activation and expansion of donor GvH-reactive clones. Because we did not systematically include HLA-DR in the myeloid panel, we cannot exclude the possibility that some of the CD14+ myeloid cells were HLA-DRlow, a hallmark of human myeloid-derived suppressor cells (MDSCs) that could restrain the two-way immune response. MDSCs have indeed emerged as an important immunoregulatory population, including in inflammatory bowel disease (30). Long-term persistence of donor cells in the graft was associated with decreased HvG-to-GvH clone ratios, raising the possibility that GvH-reactive clones may selectively attack recipient T cells, restraining the HvG response.

We took advantage of the availability of spleen and MLN in two donors to investigate the origin of donor-mappable clones found in posttransplant biopsies. Donor clones identified in both spleen and MLN were more likely to be found in posttransplant biopsies than those found in spleen or MLN alone. The lower clonality of CD4 versus CD8 T cell populations, which has been shown to be characteristic of memory but not naïve human T cells (31), suggested that the clones detected in both lymphoid tissues were enriched for memory cells, whereas those detected in MLN alone may be enriched for naïve T cells. TRM clones have been shown to have circulating counterparts in draining and remote lymphoid tissues in mice (18). Similarly, our study showed that a large proportion of donor-derived intestinal clones shared the same TCR as recirculating memory clones detected in both donor spleen and MLN. Thus, we conclude that many graft-resident GvH-reactive clones preexisted as circulating memory clones before transplantation. Moreover, GvH-reactive clones were very stable over time, another characteristic of TRM.

Our study was limited by the relatively low number of patients, an issue inherent to intestinal transplantation. This caveat precluded assessment of the utility of HvG-reactive clone tracking as a biomarker or predictor of rejection. In addition, our method did not permit assessment of the role of HvG-reactive clones generated de novo after transplantation in graft infiltration. Thus, our strategy may underestimate the posttransplant HvG response, especially in children with high thymic output.

In conclusion, our study provides insights into the role of two-way alloreactivity in driving human intestinal allograft repopulation by recipient cells. We demonstrated that HvG-reactive clones accumulated in intestinal allografts at the time of rejection and persisted long after resolution, despite clonal contraction. These HvG-reactive TRM may be reactivated later to cause rejection. In the absence of overwhelming cellular and antibody-mediated HvG reactivity, preexisting donor TRM with GvH reactivity may expand in the graft and prevent the replacement of donor cells by recipient T cells. Our study suggests that resident TRM can mount an immune response that counteracts rejection. Therapeutic approaches to preventing the entry of HvG-reactive T cells and hence their establishment as TRM could potentially have a major impact on outcomes of transplants with large mucosal TRM compartments, such as lungs and intestines.


Study design

Twelve consecutive small intestinal transplant (either isolated or as part of a multivisceral allograft) recipients, transplanted between November 2011 and November 2015 at our institution, were prospectively enrolled in a noninterventional cohort study. The study primarily aimed at correlating intragraft recipient chimerism and local alloreactive immune responses with clinical outcomes. Nine of the patients (patients 4, 5, 6, 7, 9, 10, 13, 14, and 15) were enrolled at the time of the transplant and were monitored until the last follow-up (data cutoff in May 2016). Three additional patients (patients 8, 11, and 12), who had received a transplant in other centers, were included late after the transplant. Patient 12 was excluded from the study because of the lack of a suitable anti-HLA allele monoclonal antibody to distinguish recipient cells from donor cells.

Approval was obtained from the Columbia University Institutional Review Board (IRB #AAAJ5056 and IRB #AAAF2395). All patients or legal guardians provided their written informed consent. When intestinal transplant recipients underwent protocol or “for cause” biopsies, excess fresh biopsy specimens were either immediately processed (into single-cell suspension) or frozen and stored.

HLA-specific staining and cellular staining

Monoclonal HLA-specific antibodies that readily distinguished donor cells from pretransplant recipient peripheral blood or spleen mononuclear cells were included in lineage-specific panels of antibodies (fig. S11 and table S1), as previously reported (4). More information about the multicolor T cell panel and flow data analysis is provided in the Supplementary Materials.

Carboxyfluorescein diacetate succinimidyl ester–mixed lymphocyte reactions and cell sorting

These were performed as previously described (17). More details are provided in the Supplementary Materials. DNA was frozen down to −20°C and shipped on dry ice to Adaptive Biotechnologies (Seattle, WA) for high-throughput TCR sequencing. The TCR sequencing data were retrieved from Adaptive Biotechnologies’ immunoSEQ software.

Identification and tracking of HvG- and GvH-reactive clones

The initial steps of the analysis are described in the Supplementary Materials. Alloreactive clones were defined by fivefold or greater expansion in stimulated compared with unstimulated pretransplant cells and by a minimum frequency of 0.001% in CFSElo (carboxyfluorescein diacetate succinimidyl ester), which serves to ensure 85% repeatability, as determined by power analysis (17). This ensures that a clone appearing only in CFSElo without a match in unstimulated samples, considered infinitely expanded by the fivefold expansion criterion, and therefore alloreactive, is sufficiently high frequency to be recaptured in a repeated mixed lymphocyte reaction (MLR). We experimentally observe stabilization of captured alloreactive clone counts and cumulative frequency at this frequency threshold, especially for CFSElo clones that are relatively low frequency but still have a nonzero match in unstimulated samples from which they are expanded fivefold, many of which are discounted by a more stringent minimum frequency.

Diversity measures are calculated for total clones and alloreactive clones in all samples, including entropy [H ≡ Σpi log 2(pi)], where pi is the frequency of clone i; the related clonality (S ≡ 1 − Hobs/Hmax); and Simpson’s index (D ≡ Σpi2), which is more sensitive to changes in frequency of dominant clones. Clonality, which ranges from 0 to 1, is primarily used such that higher clonality indicates less diversity and a less polyclonal distribution. Pairwise Jensen-Shannon Divergence is calculated as Embedded Image to indicate overlap between biopsies for all patients, where a JSD of 0 indicates complete overlap and a JSD of 1 indicates complete divergence (32).

Contingency tables of clone counts are created to compare biopsies to pretransplant cells and to each other, with the total clone count N mappable to pretransplant MLR in unstimulated samples, stimulated samples, or both, and to a subset A of N clones that are alloreactive. These are used in Fisher’s exact tests of (N1-A1,A1:N2-A2,A2), and odds ratios with 95% confidence interval are calculated for alloreactive clone fraction between the two samples being compared, along with P value for the comparison. Cumulative frequencies f(N) and f(A) are also reported for these clonal populations, without associated P values, because these cannot be straightforwardly derived without repeated MLR.

Correction:Authors have revised the figure titles for Fig. 6 and fig. S10 to more accurately reflect the data shown. The data and conclusions in the paper have not changed.


Materials and Methods

Fig. S1. Representative gating strategy for graft-resident lymphoid populations.

Fig. S2. Intragraft mixed chimerism long after intestinal transplantation.

Fig. S3. Recipient chimerism in LPL according to clinical outcome.

Fig. S4. Absolute CD3+ IEL counts at day 10 and month 3 after transplant.

Fig. S5. Phenotypic features of IEL and LPL populations obtained from deceased organ donors.

Fig. S6. Role of NKG2D/MICA interaction in rejection.

Fig. S7. Frequency of recipient and donor cells in isolated IELs for seven patients.

Fig. S8. Frequency and overlap of HvG and non–HvG-reactive clones in native and transplanted intestines.

Fig. S9. Trajectory of GvH and non-GvH clones over time.

Fig. S10. Origin of GvH clones detected in early biopsies from patient 10.

Fig. S11. Representative contour plots demonstrating recipient specificity of anti–HLA-B8 antibody in patient 10.

Table S1. HLA class I typing and anti-HLA allele antibodies used to distinguish donor from recipient cells in intestinal transplant recipients.

Table S2. Epidemiological and clinical characteristics of patients and deceased donors.

Table S3. Source data for Fig. 1E.

Table S4. Number of unique clones detected in each population/specimen, and proportion of biopsy-detected clones identifiable as recipient, donor, or neither.


Acknowledgments: We thank J. Colozzi for assistance with the submission of the manuscript. We are indebted to N. Cerf Bensussan for helpful review of the manuscript. The authors are also grateful to M. Velasco and T. Gonda for their care of intestinal/multivisceral transplant recipients. Funding: J.Z. was supported by Fulbright, Monahan Foundation, Société Francophone de Transplantation, and Schaefer research scholarships. J.T. was supported by NIH F31AG047003. S.D.W. was supported by a Kidney Research Student Scholar Grant from the American Society of Nephrology. The study was funded by the Schaefer research scholar program and National Institute of Allergy and Infectious Diseases grant P01 AI106697. The LSR II FACS analyzer used in this study was purchased with NIH award 1S10RR027050-01A1. This work was made possible in part by samples made available through the support of V. Segal and S. Segal for the Columbia Center for Translational Immunology Biobank. Author contributions: J.Z., M.M., T.K., and M.S. designed the study. J.Z., B.S., S.-P.L., J.F., M.L., S.C., and J.W. performed the experiments. J.Z., B.S., S.-P.L., J.F., S.Y., J.T., A.G., and M.M. collected the data. J.Z., B.S., S.-P.L., A.O., M.L., S.D.W., D.L.F., Y.S., S.C.-Z., G.B., A.G., M.M., T.K., and M.S. performed data analysis. Y.S. (senior biostatistician) and A.O. wrote the codes to identify and track the alloreactive clones and performed the statistical analyses. J.Z., A.O., and M.S. wrote the final report. All authors contributed to the editing of the final report. All authors agreed to all of the content of the submitted manuscript. Competing interests: The authors declare that they have no competing interests. Data and material availability: Raw TCR sequences data are freely accessible through (username: projectreview{at}; password: ProjectReview). The codes used to analyze TCR sequences are available and can be uploaded from a public GitHub repository (
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