Minor antigen–mismatched skin graft survival was prolonged in antibiotic-pretreated mice. To determine the consequence of reduced bacterial diversity on allograft outcome, 6- to 8-week-old male and female purchased C57BL/6 (B6) mice were left untreated or received a daily gavage of antibiotics (Abx) for 10 days. Skin from male donors was transplanted onto the flanks of female mice 1 day later, and recipients did not receive further Abx treatment. Abx-pretreated recipients of skin grafts from Abx-pretreated donors displayed prolonged skin allograft survival (mean survival time [MST] = 53 ± 23 days) compared with controls (MST = 27 ± 12 days) (Figure 1A). Similar results were obtained when comparing untreated and Abx-pretreated in-house–bred littermates derived from the same maternal lineage to minimize microbiota differences between groups prior to treatment (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI85295DS1). In contrast, Abx pretreatment of donors or recipients alone did not delay rejection (Figure 1A), indicating that Abx-induced changes in both the donor skin and the recipient mice are required for improved graft survival.

Figure 1 Abx pretreatment results in prolonged skin graft survival and reduced bacterial diversity. (A) B6 females untreated or pretreated for 10 days with Abx received a skin graft from B6 males untreated or pretreated for 10 days with Abx. SPF → SPF, n = 12; SPF (Abx) → SPF, n = 5; SPF → SPF (Abx), n = 5; Abx → Abx, n = 19; syngeneic, n = 5. log-rank test. (B) Bacterial load by qPCR in fecal samples of GF and SPF B6 females at the indicated time points after initiation of Abx treatment. n = 3 (1 SPF and 1 GF samples shown for reference). LOD, level of detection. (C–E) Bacterial DNA was isolated from gut and skin of female SPF controls and age-matched mice on day 10 of Abx treatment and was analyzed by high-throughput sequencing. Data are displayed as richness (C), diversity (D), and PCA (E). Each line (C) and dot (B and E) represents an individual mouse. PC1, principal component 1; Exp, experiment. Bars (D) represent the mean ± SEM of 4 mice per group. (C–E) Results are representative of 4 experiments with n = 3–4. *P < 0.05; ***P < 0.001, Student’s t test.

To understand the changes that had occurred in the microbiota prior to transplantation, bacterial DNA was extracted from tail skin and fecal samples of female hosts. This Abx regimen in adult mice did not reduce overall fecal bacterial burden, as determined by qPCR using universal primers for the 16S rRNA gene (Figure 1B), in contrast with the reduced bacterial load that occurs when a similar regimen is started in 2-week-old mice prior to weaning (7). However, 10-day Abx pretreatment resulted in significantly reduced bacterial richness and diversity on the day of transplantation (Figure 1, C and D) in both fecal and skin samples, as determined by 16S rRNA sequencing using the MiSeq platform. Furthermore, principal component analysis (PCA) showed that microbial communities of fecal and skin samples on day 10 of Abx pretreatment clustered separately from those of the control and the pretreatment groups (Figure 1E). In particular, Abx-pretreated mice had a relative expansion of Lactobacillales in their feces and a reduction of Clostridiales in both feces and skin (Supplemental Figure 1, B and C). Thus, Abx pretreatment changed the composition of the microbiota, but not the overall bacterial load.

Minor antigen–mismatched skin allograft survival was prolonged in GF mice. As a second approach to testing the role of the microbiota on graft outcome, we used germ-free (GF) mice, devoid of live bacteria, and transplanted them in a biological safety cabinet in the gnotobiotic facility using sterile techniques (Figure 2A and ref. 8). GF female recipients of GF male skin transplants also displayed prolonged graft survival when compared with specific pathogen–free (SPF) mice transplanted in a sterile manner (Figure 2B). This was not due to a reduced susceptibility of GF skin to rejection, as GF grafts were rejected as promptly as SPF grafts by SPF mice (Figure 2B). To test the causal role of the microbiota, GF mice received a gavage with feces from SPF mice 5 to 7 days prior to transplantation (GF-SPF.F). Although the microbial communities that established in the intestine of GF-SPF.F mice differed from those in the donor fecal samples (Supplemental Figure 2, A and B), restoration of the microbiota was sufficient to accelerate rejection (Figure 2B), indicating that prolonged graft survival in sterile GF mice is due at least in part to their lack of microbiota.

Figure 2 GF mice display prolonged skin graft survival. (A) Schematics of transplantation in the gnotobiotic facility. (B) B6 female SPF, GF, and GF mice gavaged with PBS-diluted fecal material from SPF mice (GF-SPF.F) or Abx-pretreated SPF mice (GF-Abx.F) 5 to 7 days prior, were transplanted with male skin grafts from the indicated donors. SPF → SPF, n = 8; GF → GF, n = 8; GF → SPF, n = 11; GF-SPF.F → GF-SPF.F, n = 10; GF-Abx.F → GF-Abx.F, n = 7. log-rank test. (C) Representative cecum appearance at day 7 after gavage. (D) qPCR of 16S rRNA gene to assess bacterial load in SPF, GF, and GF-Abx.F mice before and after gavage. n = 3–5. One-way ANOVA. (E) Normalized abundance of B. coccoides and Lactobacillus spp. as determined by qPCR in feces of SPF, 10-day Abx-treated SPF mice, and GF-Abx.F mice after fecal gavage. n = 3–5. One-way ANOVA. Data are representative of 2 experiments (D–F) or combined from 2 to 3 experiments (B). (B and E) Data represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

To determine whether all microbial communities accelerated graft rejection, GF mice were gavaged with feces from Abx-pretreated SPF mice (GF-Abx.F) prior to transplantation. In contrast with GF-SPF.F mice, GF-Abx.F mice did not display faster rejection than GF mice (Figure 2B) and their cecal appearance remained enlarged, as is typical of GF mice (ref. 9 and Figure 2C), despite a bacterial load close to that in SPF mice (Figure 2D). The dysbiosis induced by Abx pretreatment in the fecal donors was transferred and remained stably established in GF-Abx.F recipients that were not exposed to other environmental bacteria, as supported by the persistent contraction of Blautia coccoides (formerly classified as Clostridium coccoides; ref. 10) and expansion of Lactobacillus spp. (Figure 2E), although their total bacterial communities were still distinct from those in the donor fecal samples (Supplemental Figure 2, A and B). Interestingly, fecal transfer into GF mice not only resulted in intestinal, but also skin colonization (Supplemental Figure 2, A and B), such that whether it is the gut and/or skin microbiota that influences skin graft outcome remains to be elucidated.

Abx pretreatment resulted in reduced alloimmunity. To investigate whether improved graft survival in Abx-pretreated mice was due to reduced alloimmunity, we analyzed graft-infiltrating leukocytes isolated on day 10 after transplantation. Abx pretreatment was associated with a reduced percentage of CD4+ T cells and fewer IFN-γ–producing cells among them (Figure 3, A–C). To test whether the reduced percentage of intragraft effector CD4+ T cells was due to diminished expansion of alloreactive T cells, H-Y–specific CD45.1+ congenic CD4+ TCR/RAG-KO-Tg (Marilyn) T cells were CFSE-labeled and adoptively transferred into 10-day–Abx–pretreated female recipients 1 day prior to transplantation with skin grafts from Abx-pretreated male donors. Marilyn T cells isolated 4 days later from the skin graft draining LNs (dLNs), the site of initial T cell priming after skin transplantation (11), displayed reduced proliferation in Abx-pretreated mice compared with that in untreated hosts (Figure 3, D and E). Thus, Abx pretreatment triggered reduced priming of graft-reactive T cells, supporting the conclusion that the microbiota that associates with SPF B6 mice in our colony promotes the activation of alloreactive T cells and/or that Abx pretreatment promotes a microbial community that reduces alloreactivity.

Figure 3 Abx pretreatment and GF status result in reduced allogeneic T cell priming. (A–C) Graft-infiltrating cells were isolated from control and Abx-pretreated SPF mice on day 10 after transplantation for flow cytometric analysis to determine the percentage of TCR-β+ (A) or CD4+ (B) cells among CD45+ cells and the percentage of IFN-γ–producing cells (C) among PMA/ionomycin-stimulated CD4+ cells. (D and E) Congenic Marilyn T cells were labeled with CFSE and transferred (106 cells/mouse) into SPF female recipients 1 day prior to transplantation with male skin grafts; donors and recipients were both untreated or both Abx pretreated. Mice were sacrificed 4 days after transplantation, and cells were isolated from the graft dLNs for analysis of CFSE dilution. Representative plots (D) and quantitation (E) of divided Marilyn T cells. (F and G) APCs from skin dLN cells were isolated from control or 10-day Abx-pretreated SPF B6 males or females and cultured with CFSE-labeled T cells from naive Marilyn females. (F) Quantitation of CFSE dilution in Marilyn-gated T cells on day 4 of culture. (G) Percentage of IFN-γ+ cells among Marilyn T cells after a 3-day culture and following restimulation with PMA/ionomycin for 4 hours. (A–G) n = 3–5 mice per group. Experiments were repeated 3 to 4 times. No txp, no transplant. Student’s t test. (H) Marilyn T cell–CFSE dilution after culture as in F with male APCs from different groups. APCs were harvested 7 days after gavage of GF mice. Quantitation of divided Marilyn T cells (I) and IFN-γ production after restimulation (J). n = 2–8. One-way ANOVA. Data represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

In order to modulate T cell priming, microbial signals might directly or indirectly affect T cells, antigen-presenting cells (APCs), or both. To determine whether the Abx pretreatment affected APCs, we isolated T cell– and NK cell–depleted APCs from the skin dLNs of Abx-pretreated or control SPF male mice and used them to stimulate CFSE-labeled Marilyn T cells from naive mice in vitro. APCs from Abx-treated mice stimulated Marilyn T cells less vigorously than control APCs (Figure 3F). Marilyn T cell proliferation was also reduced when stimulated with H-Y peptide–pulsed APCs from Abx-pretreated females (Figure 3F) when compared with peptide-pulsed APCs from untreated females, suggesting that the APC defect is unlikely to be at the antigen-processing level. Marilyn T cells stimulated with APCs from Abx-treated mice also produced less IFN-γ (Figure 3G). Differences in T cell–priming ability were not due to an alteration in the composition of skin dLN APCs after Abx treatment, as the proportion of B cells, CD11b+ cells, and CD11c+ DCs as well as subsets of CD103+, CD8α+, or Siglec-H+ DCs was comparable in control and Abx-treated mice (Supplemental Figure 3, A–D), with the exception of a reduction in CD11b+ DCs. Moreover, expression levels of CD40, PDL1, CD80, CD86, ICOSL, OX40L, and I-Ab were similar on DCs from Abx-pretreated and control mice (Supplemental Figure 3E). Thus, Abx treatment reduced the capacity of APCs to prime alloreactive T cells. In the gnotobiotic setting, skin dLN APCs from male GF and GF-Abx.F mice also induced less proliferation and IFN-γ production by Marilyn T cells than did APCs from SPF or GF-SPF.F mice (Figure 3, H–J). Together, these data argue against a direct effect of oral Abx on immune cells or skin grafts and suggest instead that Abx-sensitive taxa play a critical role in poising skin dLN APCs for alloreactive T cell priming.

Abx treatment downregulates the type I IFN pathway in DCs. Since DCs are the main APC subset that activates alloreactive H-Y–specific T cells (12), skin dLN CD11c+ DCs were sorted from control and 10-day–Abx–treated mice for gene expression profiling. DCs from Abx-treated mice displayed reduced expression of genes associated with the type I IFN pathway, such as IRF3, IRF7, OAS2, OAS1G, OASL1, and OASL2, as well as of genes related to activation of the NF-κB pathway, such as RSAD2, SPHK1, and IRAK2. In addition, genes involved in cytokine regulation and production were also significantly less expressed in DCs from Abx-treated than control mice (Figure 4, A and B). In keeping with this gene profiling analysis, survival of type I Ifnar-deficient male skin grafts was significantly prolonged in type I Ifnar-deficient females when compared with control mice (Figure 4C), supporting a role for type I IFN signaling in the response to H-Y+ skin grafts. IFN-αR expression had to be absent in both donor and recipient mice for graft survival to be prolonged (not shown). Notably, Abx pretreatment of Ifnar-deficient mice did not prolong survival compared with untreated counterparts; they were all rejected within 50 days (not shown), further suggesting that the effects of Abx pretreatment were dependent at least in part on a reduction of the type I IFN pathway.

Figure 4 Abx pretreatment results in downregulation of the type I IFN pathway. (A and B) DCs from skin dLN of control and 10-day Abx-pretreated SPF mice were sorted, and cDNA was analyzed for gene expression profiling. (A) Examples of genes differentially expressed (n = 3 per group). (B) DAVID analysis of enriched pathways in DCs from control relative to Abx-treated groups. (C) Female B6 control and Ifnar–/– SPF mice were transplanted with skin grafts from males of the host genotype. Results are combined from 2 experiments. WT, n = 12; Ifnar–/–, n = 8. log-rank test. **P < 0.01.

Abx pretreatment also prolongs survival of major antigen–mismatched skin and MHC class II–mismatched cardiac allografts. It was conceivable that the effects of the microbiota on graft outcome were limited to the H-Y skin graft mouse model or applicable only to transplantation of colonized organs. To this end, donor and recipient mice were pretreated with Abx prior to transplantation of BALB/c skin or Bm12 heart into B6 animals. Abx pretreatment resulted in a significant delay in graft rejection in both models (Figure 5, A and B), arguing for a more generalizable effect of the microbiota on allograft rejection.