Donor Inoculum Research Articles

Fecal microbiota colonization dynamics in dairy heifers associated with early-life rumen microbiota modulation and gut health.

Early-life modulation of rumen microbiota holds promise for enhancing calf growth, health, and long-term production in ruminants. However, limited attention has been given to the impact of rumen microbiota modulation on the establishment of hindgut microbiota. In this study, fecal microbiota development was examined in identical twin calves for 12 months. The treatment group (T-group) received adult cow fresh rumen liquid inoculum during the pre-weaning period, while the control group did not (C-group). The effects of inoculum were assessed on calf gut health and as microbial seeding route into the hindgut. The early rumen modulation had no effect on age-related fecal microbiota development. The fecal bacterial community evolved gradually following dietary changes and categorized into pre-weaning and post-weaning communities. Bacterial richness increased with age and stabilized at month 9, while between-sample variation reduced in post-weaning samples. Archaeal load in fecal samples increased after month 4, while archaeal richness increased and stabilized in both groups by month 9. Between-sample similarity was higher during the pre-weaning period, with increased dissimilarity from month 4 onward. Anaerobic fungi were detected in feces at month 4, with richness peaking at month 7. Before month 6, fungal community composition distinctly differed from mature communities. When colostrum, calf rumen, and donor inoculum were evaluated as seeding sources for hindgut colonization, the calf's own rumen was identified as the primary seeding source for fecal bacteria and fungi. Colostrum was a source for several bacteria detected in feces, but these were of temporary importance until weaning. The donor inoculum had limited impact on gut health as diarrhea rates were similar between the T-group and C-group. In conclusion, early-life microbiota modulation shows potential in ruminant development. However, a more targeted approach with bacteria adapted to the hindgut environment may be necessary to modulate hindgut effectively. This research contributes to our understanding of the complex relationship between gut microbiota and calf health and growth.

Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Acute graft-versus-host disease (GVHD) causes apoptosis of intestinal stem cells (ISCs) that are key targets in the gastrointestinal (GI) tract (Zeiser and Blazar, NEJM 2917; 377:2167). The receptor interactive protein kinase 1 (RIP1) is an important mediator of apoptosis and we investigated the role of the (RIP1) pathway in GI tract and ISC damage in both human and mouse GVHD models. Immunohistochemical staining of human GI biopsies from GVHD patients showed strong phospho-RIP1 staining in biopsies with Lerner grades 2/3 compared to grades 0/1 (12/17 v 0/8, p < 0.01). We then established in vitro models of ISC apoptosis (at least 50% loss) by adding 10 ng/ml of both IFNg and TNFa to organoid cultures from both ileum and colon (organoids cultures with either IFNg or TNFa alone showed no change). Addition to cultures of 0.1 mcg/ml GNE684, a well-characterized small molecule RIP inhibitor (Patel et al, Cell Death Diff 2020; 27:161), dramatically reversed ISC loss in both human and mouse ileal organoids as measured by cross sectional area (Fig 1). WE observed an identical pattern in colon cultures where GNE684 reversed loss of human ISCs (70.0 + 7.7 % v 29.3 + 2.5 %, p < 0.001) and mouse ISCs (100 + 6.6 % v 41 + 6.6 % p < 0.001). In vivo, 14 daily ip injections of GNE684 prevented mortality in a GVHD mouse model (C3H.SW → B6) in a dose dependent fashion (Fig 2). Systemic GNE administration also improved survival in a second model (Balb/c → B6: day 30 survival 7/10 v 0/10, p < 0.001). In the C3H.SW → B6 model there were dramatic reductions in CD4+ lamina propria effector lymphocytes secreting either IFNg (56 + 11 v 9 + 2, p < 0.001) or IL-17 ( 22 + 10 v 7 + 4, p < 0.01) and in CD8+ effector cells secreting IFNg (39 + 11 v 8 + 3, p < 0.001) or IL-17 ( 19 + 8 v 7 + 2, p < 0.001) on day 10 after HCT. Importantly, GVHD was dramatically reduced when administration of GNE684 was used as treatment for GVHD beginning on day +7 after GVHD was established (day 40 survival: 8/12 v 2/12, p = 0.02). Additionally, survival dramatically improved in RIP1 kinase-dead recipients compared to wild-type B6 controls in both the C3H.SW → B6 model (13/16 v 4/16, p < 0.001) and in the Balb/c →B6 model (13/20 v 1/20, p < 0.001). IFNg secreting CD4+ and CD8+ effector lymphocytes that infiltrated the lamina propria were significantly reduced in RIP1 kinase-dead recipients compared to wild-type controls (58 + 13 v 11 + 7 and 39 + 11 v 14 + 4, respectively [p < 0.001 for both]). Infiltrating lymphocytes secreting IL-17 were also reduced four fold. Importantly, administration of GNE684 preserved beneficial GVL effects when lethal doses of myeloid tumor C1498 cells that caused 100% mortality by day 28 in syngeneic BMT recipients were added to the donor inoculum (day 42 survival: 7/10 v 4/13, p = 0.03). We conclude that RIP1 is a key mediator of GI damage during GVHD. In vitro RIP1 inhibition prevents ISC apoptosis in both mouse and human models of GI GVHD. RIP1 inhibition, either through genetic alteration of the RIP1 kinase domain in transplant recipients or through systemic administration of the RIP1 inhibitor GNE684, protects the GI tract from acute GVHD damage as measured both clinically and histologically in preclinical models while preserving beneficial GVL effects. These data suggest that GNE684 a clinical trial of GNE684 is warranted in patients with gastrointestinal GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nrf2 Is a Critical Mediator of CD4 T Cell-Induced Acute Graft-Versus-Host Disease

Recent studies have shown that, in contrast to typical proliferating lymphocytes, alloreactive T (allo-T) cells can be specifically killed by apoptosis through compounds that lead to generation of intracellular reactive oxygen species. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is an ubiquitously expressed transcription factor well-known for its role in regulating the cellular redox pathway, but little is known about its involvement in T-cell biology. In this study, we examined the contribution of Nrf2 to allo-T cells in the context of an allogeneic hematopoietic cell transplant (allo-HCT).To study the role of Nrf2 in T cell function, we first assessed the expression pattern of Nrf2 in activated T cells. We found that total cellular and nuclear Nrf2 expression in T cells were significantly increased 24 hours after in vitro α-CD3/CD28 stimulation (p<0.001), as well as in vivo in donor allo-T cells on day 14 in a well-established MHC-disparate murine allo-HSC model (C57BL/6→BALB/c) (p<0.05) (Fig 1). We next examined the effects of Nrf2 deficiency on T cell alloreactivity by transplanting WT or Nrf2-/- donor T cells in MHC-disparate (C57BL/6→BALB/c), as well as MHC-matched, minor antigen mismatched (C57BL/6→LP) murine models. In both models, allo-HCT recipients of Nrf2-/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality (p<0.01), morbidity (p<0.001), and intestinal pathology (p<0.05) (Fig 2a). To test if Nrf2 differentially regulates CD4+ versus CD8+ T cell function, we mixed and matched WT versus Nrf2-/- CD4+ and CD8+ T cells for transplant. Compared to allo-HCT recipients of donor WT CD4+: WT CD8+ T cells, we observed similar GVHD mortality in allo-HCT recipients of donor WT CD4+: Nrf2-/- CD8+ T cells, while we saw reduced GVHD mortality in allo-HCT recipients of donor Nrf2-/- CD4+: WT CD8+ T cells (p<0.01) (Fig 2b). These data support the notion that Nrf2 predominately regulates donor CD4+ and is dispensable for CD8+ T cells during allo-activation and the development of GVHD.We assessed if Nrf2 could affect polarization of donor CD4 T cells after allo-HCT, and found a two-fold increase in the proportion and absolute numbers of CD25+Foxp3+ regulatory T cells (Tregs) in the spleens of donor Nrf2-/- T cell allo-HCT recipients on day 7 post transplant (p<0.001) (Fig 3a). We next examined if Nrf2-deficiency results in greater induction of Tregs (iTregs) or stabilization of natural Tregs (nTregs). While we found an equal fraction of WT and Nrf2-/- naïve CD4+ T cells induced towards Tregs in vitro, we saw a significantly greater percentage of donor Nrf2-/- Tregs expressing Helios, a marker for thymus-derived nTregs, compared to donor WT Tregs in allo-HCT recipients on day 7 (p<0.01) (Fig 3b). Recent studies have suggested that Helios is required to stabilize the expression of FoxP3, and thus, the abundance and inhibitory activity of Tregs in inflammatory settings. In keeping with this notion, we discovered an increase in the cellular FoxP3 protein expression within donor Nrf2-/- Tregs in allo-HCT recipients (p<0.01) (Fig 3c). To further elucidate the biological significance of Nrf2 function on nTregs, we removed CD4+CD25+ putative Tregs from the donor inoculum and transplanted only conventional T cells (Fig 3d). We found that the survival advantage with donor Nrf2-/- T cells was abolished when Tregs were depleted from the donor graft, suggesting that Nrf2 expression in donor T cells negatively regulates nTregs during allo-activation, most likely through downregulation of Helios and FoxP3 expression.Finally, to determine if Nrf2 was critical for T-cell mediated GVT activities, we challenged allo-HCT recipients with A20 (B-cell lymphoma) cells. Using bioluminescent imaging, we found that Nrf2-/- allo-T cells were equally efficient in clearing tumor burdens, and along with their mitigated GVHD activities, resulting in a significantly improved survival outcome.Taken together, we identified a novel role of Nrf2 in CD4+ T cells in the context of allo-HCT. Nrf2 negatively regulated CD4+ nTreg through Helios downregulation during GVHD, but appeared to be dispensable in CD8+ mediated antitumor responses, further highlighting the possibility of separating GVT from GVHD activities. Our findings suggest that targeted manipulation of Nrf2 activities in allo-T cells may offer a useful clinical strategy to mitigate GVHD while preserving GVT responses. [Display omitted] DisclosuresJenq:Seres: Research Funding. van den Brink:PureTech Health: Consultancy; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; Therakos Institute: Other: Speaking engagement.

A Novel Role for AMP-Activated Protein Kinase (AMPK) in Controlling T Cell Sensitivity to IL-6

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and many non-malignant hematologic disorders. However, the use of alloHSCT as a therapy remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that alloreactive T cells increase phosphorylation of the intracellular energy sensor AMP-activated protein kinase (AMPK) as early as day 3 post-transplant (levels 8-fold higher in alloreactive T cells compared to naive or syngeneic controls (p=0.0003)) and donor T cells lacking AMPK (AMPK KO) cause significantly less GVHD in both major- and minor-histocompatibility mismatch models of GVHD. At the same time, AMPK KO T cells equivalently clear leukemia cells and extend recipient survival in leukemia models with high tumor burden. Transplantation of cells lacking AMPK lowers the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPKKO respectively) but interestingly, increases both the percentage and total number of regulatory T cells (Treg) (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. KO, p=0.004).To understand these findings mechanistically, we sought to study the role of AMPK in donor Treg expansion and stabilization post-alloHSCT. Canonically, interleukin-2 (IL-2) signaling, through phosphorylation of Stat5, promotes Treg generation and stability, while inflammatory signals (e.g. IL-6 signaling through Stat3) are known to decrease Treg stability post-transplant. Studies testing IL-2 sensitivity in KO cells, or the degree of Stat5 phosphorylation post-transplant, were unrevealing. However, AMPK KO Tregs significantly decrease their sensitivity to IL-6, with a 50% reduction in phosphorylated-Stat3 levels following intermediate levels of cytokine stimulation (Figure 1A). To demonstrate the functional consequences of this difference in cytokine sensitivity, we exposed in vitro generated Treg to increasing concentrations of IL-6. As expected, the percentage of wt FoxP3+ cells decreased sharply with increasing IL-6 concentrations (decreasing from 50% to 14%), while AMPK KO Tregs were minimally affected over this dose range (47% versus 42%; Figure 1B, p<0.02).To determine if increased Treg stability was the predominant reason for improvements in GVHD seen with AMPK KO cells, CD25+ cells were removed from the donor inoculum of AMPK KO or wt mice by magnetic selection (to eliminate >90% of Treg), and then either CD25-replete or CD25-depleted sets of cells were transplanted into B6xDBA2 F1 recipients in a major MHC mismatch model of GVHD. Despite a decrease in AMPK KO Treg numbers on day 7 to levels commiserate with wt donors (Figure 2A), elimination of AMPK KO Treg had only a modest effect on GVHD severity (Figure 2B), implicating a substantial role for AMPK in the non-Treg compartment.To explore the role of AMPK in non-Tregs further, we investigated the sensitivity of unmanipulated wt and AMPK KO T cells to IL-6 stimulation. Similar to FoxP3+ cells, both naïve CD4 and CD8 T cells lacking AMPK experienced lower levels of Stat3 phosphorylation in response to graded concentrations of IL-6 (Figure 3A). Furthermore, if IL-6 was added to a 4-day culture with anti-CD3 stimulation, the number of wt T cells recovered after 96 hours increased >2.2-fold. In contrast, AMPK KO cells experienced minimal changes in T cell number over a wide range of IL-6 concentrations (Figure 3B).In conclusion, we define in these studies a novel role for AMPK in controlling donor T cell sensitivity to IL-6, both at the level of Stat3 phosphorylation, and in terms of functional consequences. Not only does this striking result elegantly explain our previous observation of increased Treg percentages, and decreased donor T cell numbers, following transplantation of AMPK KO cells, it continues to make the case that inhibition of AMPK represents an attractive clinical target for the amelioration of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recent developments in prophylaxis and treatment of graft-versus-host disease

Acute graft-versus-host disease (GVHD) is a systemic immune reaction in which mature T cells in the donor inoculum are abnormally activated by the host and donor antigen-presenting cells. These activated cells then attack normal host tissue after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical GVHD develops when the damage exceeds the threshold for tissue tolerance. Furthermore, functional damage of hematopoietic and non-hematopoietic organs due to this acute immune response triggers the subsequent development of chronic GVHD. The endpoint of HSCT is shifting from just surviving the acute phase to having an enriched life until later years; therefore, seamless and detailed immune management is required from the acute to chronic phase after HSCT. Acute GVHD prophylaxis consisted of calcineurin inhibitors and short-term methotrexate for a long time. However, various novel immune-modulating strategies have been developed against the background of recent diversification in donor sources and changes in treatment goals. In this review, we discuss recent clinical developments in basic and clinical research regarding acute or chronic GVHD.

Development of a Concomitant Treg Expansion and Stem Cell Mobilization Protocol Which Enables Peripheral Blood Transplant Amelioration of Gvhd Following Pre-Clinical aHSCT

Graft-versus-host disease (GVHD) remains the major immunological complication preventing more widespread application of allogeneic hematopoietic stem cell transplants (aHSCTs). Mobilized hematopoietic stem / progenitor cells (HSPC) is the predominant choice for adult HSCT (CIBMTR, 2017), but unfortunately still frequently results in the development of GVHD. Our lab and others have been exploring the potential application of CD4+FoxP3+ T cells (Tregs) to ameliorate GVHD. A useful advance in the field would be the ability to concomitantly mobilize stem cell donors and efficiently expand the peripheral Treg compartment. We have utilized a two-pathway Treg expansion strategy previously reported from our lab (Wolf, 2017; Copsel 2018) to amplify suppressor activity in the donor inoculum to diminish GVHD. The present studies developed a protocol to concurrently induce mobilization of mouse progenitor cells and expansion of Tregs for use in pre-clinical aHSCT. Donor mice were treated with recombinant human granulocyte colony stimulating factor (G-CSF) for 4 days (2ug/mouse) followed by plerixafor (5mg/kg) on D-1 and D0 (day of transplant). Peripheral blood was then assessed and significant elevations in granulocytes ( Figure 1 A) as well as multiple progenitor populations including c-Kit+ stem cells, CLP's and Lin-Sca-1ckit+ cells was observed ( Figure 1 B). We then employed a strategy beginning on D-6 through D-1 of transplant administering a fusion protein (TL1A-Ig) targeting TNFRSF25 and low dose IL-2 (two-pathway expansion). Peripheral blood was then assessed for Treg levels in donors on the day of transplant. We identified a marked increase in the frequency of CD4+FoxP3+ Treg cells ranging from (26-31%) within the CD4 compartment during the mobilization process ( Fig. 2 A). Peripheral blood was pooled from donors from both mobilized and Treg unexpanded (“TrUM”) or mobilized and Treg expanded (“TrEM”) donors and transplanted into lethally irradiated, complete MHC-mismatched BALB/c recipients. Recipients of TrUM lost weight and developed clinical signs of GVHD ( Fig. 2 B). In contrast, recipients of TrEM donors exhibited significantly reduced weight loss and clinical GVHD scores ( Figure 2 B). Transplants were also performed utilizing donors similarly prepared and transplanted into MHC-matched mice. The recipients of TrEM peripheral blood exhibited reduced GVHD compared to recipients of TrUM. Notably, mobilized donor HSPC were found to be functional as donor derived progeny (ex. CD19) were identified >1 month post-HSCT. In total, these findings support the notion that during the donor stem / progenitor cell mobilization process, manipulation of Treg cells using our two-pathway strategy can be successfully accomplished in peripheral blood resulting in an effective and translational approach to ameliorate GVHD following allo-HSCT.

In vitro assessment of the factors that determine the activity of the rumen microbiota for further applications as inoculum.

The rumen microbiota has been used as inoculum for in vitro studies and as a probiotic to improve productivity in young animals. However, great variability across studies has been noted depending on the inoculum considered. The present study aims to assess the relevance of different factors (microbial fraction, collection time, donor animal diet, fermentation substrate and inoculum preservation method) to maximize the rumen inoculum activity and set the standards for further in vitro and in vivo applications. Rumen inoculum sampled at 3 h after feeding led to greater microbial growth and activity [+12% volatile fatty acid (VFA), +17% ammonia] compared to before feeding. Similar results were noted when rumen liquid or rumen content were used as inocula. Rumen inoculum adapted to concentrate diets increased microbial activity (+19% VFA) independently of the substrate used in vitro. Freezing-thawing the inoculum, in comparison to fresh inoculum, decreased microbial activity (-14% VFA, -96% ammonia), anaerobic fungi and protozoa, with holotrichs protozoa being particularly vulnerable. Inoculum lyophilization had a stronger negative effect on microbial activity (-51% VFA) and delayed re-activation of the microbes, leading to lower levels of methanogens and anaerobic fungi, as well as almost complete wipe out of rumen protozoa. Fresh rumen fluid sampled at 3 h after feeding from donor animals that were fed concentrate diets should be chosen when the aim is to provide the most diverse and active rumen microbial inoculum. © 2018 Society of Chemical Industry.

Preclinical Canine Model of Graft-versus-Host Disease after In Utero Hematopoietic Cell Transplantation

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allogeneic engraftment and associated donor-specific tolerance without the need for toxic conditioning, as we have previously demonstrated in the murine and canine models. This strategy holds great promise in the treatment of many hematopoietic disorders, including the hemoglobinopathies. Graft-versus-host disease (GVHD) represents the greatest theoretical risk of IUHCT and has never been characterized in the context of IUHCT. We recently described a preclinical canine model of IUHCT, allowing further study of the technique and its complications. We aimed to establish a threshold T cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype. Using a range of T cell concentrations within the donor inoculum, we were able to characterize the phenotype of IUHCT-induced GVHD and establish a clear threshold for its induction between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1% to 3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.

Transient changes in milk production efficiency and bacterial community composition resulting from near-total exchange of ruminal contents between high- and low-efficiency Holstein cows

The objectives of this study were to determine if milk production efficiency (MPE) is altered by near-total exchange of ruminal contents between high- (HE) and low-MPE (LE) cows and to characterize ruminal bacterial community composition (BCC) before exchange and over time postexchange. Three pairs of ruminally cannulated, third-lactation cows were selected whose MPE (energy-corrected milk per unit of dry matter intake) differed over their first 2 lactations. Approximately 95% of ruminal contents were exchanged between cows within each pair. Ruminal pH and volatile fatty acid (VFA) profiles, along with BCC (characterized by sequencing of the variable 4 region of 16S rRNA genes), were assessed just before feeding on d -8, -7, -5, -4, -1, 1, 2, 3, 7, 10, 14, 21, 28, 35, 42, and 56, relative to the exchange date. High-MPE cows had higher total ruminal VFA concentrations, higher molar percentages of propionate and valerate, and lower molar percentages of acetate and butyrate than did LE cows, and re-established these differences 1 d after contents exchange. Across all LE cows, MPE increased during 7 d postexchange but declined thereafter. Two of the 3 HE cows displayed decreases in MPE following introduction of the ruminal contents from the corresponding LE cow, but MPE increased in the third HE cow, which was determined to be an outlier. For all 6 cows, both liquid- and solids-associated BCC differed between individuals within a pair before contents exchange. Upon exchange, BCC of both phases in all 3 pairs was more similar to that of the donor inoculum than to preexchange host BCC. For 5 of 6 cows, the solids-associated community returned within 10 d to more resemble the preexchange community of that host than that of the donor community. Individual variability before the exchange was greater in liquids than in solids, as was the variability in response of bacterial communities to the exchange. Individual cows varied in their response, but generally moved toward re-establishment of their preexchange communities by 10 d after contents exchange. By contrast, ruminal pH and VFA profiles returned to preexchange levels within 1 d. Despite the small number of cows studied, the data suggest an apparent role for the ruminal bacterial community as a determinant of MPE.

Reprogramming and Recapitulating T Cells in the Immature Immune Environment of the Neonate to Induce Transplant Tolerance

Introduction: Semi-allogeneic bone marrow and spleen cells (BMC/SC) induce transplant tolerance in neonatal mice whereas allogeneic BMC/SC cause lethal graft-versus-host disease (GVHD). To prevent aGVHD and induce transplant tolerance with allo-SC/BMC we reprogrammed donor/host CD4 T cells by co-stimulation blockade to preserve regulatory CD4 T cells while recapitulating depleted donor/host CD8 T cells. Methods: C3H (H-2k) neonatal mice were injected iv with total or depleted B6 (H-2b) GFP+ SC/BMC. CD8 T and CD49b NK cells were depleted from donor inocula using StemCell Technologies kits. In vivo neonatal host CD8 T cells were depleted (Mab 53–6.7) and donor/host CD4 T cells reprogrammed (CD154 Mab MR1). Trafficking and interactions of injected cells were monitored by microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Neonatal mice injected with GFP+ allo-SC/BMC developed aGVHD, with diarrhea, reduced growth and early death. GFP+ cells proliferated and spread throughout injected mice indicating systemic inflammation. Depletion of donor/host CD8 T cells together with co-stimulation blockade of donor/host CD4 T cells (CD154) resulted in GFP signal being reduced and restricted to lymphoid organs (day 6). High resolution microscopy showed donor T and B cells positioned respectively in PALS and follicular regions of host spleen; lack of Ki67 immunostaining indicated many donor cells were not proliferating. Donor DC were detected in host thymus (day 6) suggesting a possible role in central tolerance. Donor-type B6 hearts transplanted into neonatally-treated adult C3H mice (n=5) (CD8 depleted/CD4 co-stimulation blockade) all continued to beat at 100 days post-transplant, 3 grafts with maximal strength and 2 grafts with diminished strength. In untreated control mice (n=3) donor-type hearts stopped beating by day 10. H&E staining of a strongly beating B6 heart graft at 100 days showed undamaged cardiomyocytes with little if any cellular infiltrate and absence of macrophages and T cells by immunostaining. Conclusion: Reprograming CD4 T cells while depleting/recapitulating CD8 T cells in allo-SC/BMC treated neonates leads to 100% cardiac allograft survival at 100days; low beat scores in 2/5 grafts suggests further optimization is required. These findings provide insight into robust tolerance induction in neonates. This project was supported with funding from the Canadian Institutes for Health Research and the Gordon English family.

Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.

Kiwifruit fermentation drives positive gut microbial and metabolic changes irrespective of initial microbiota composition

It is well established that individuals vary greatly in the composition of their core microbiota. Despite differing ecology, we show here that metabolic capacity converges under the pressure of kiwifruit substrates in a model gut system. The impact of pre-digested green and gold kiwifruit on the human colonic microbiota and their metabolic products was assessed using in vitro, pH-controlled, anaerobic batch culture fermenters. Phylogenetic analyses revealed that bacterial composition changed over time, irrespective of whether a substrate was added or not, indicating a natural adjustment period to the gut model environment. Adding kiwifruit substrate caused additional changes in terms of growth of specific bacterial groups, bacterial diversity and metabolite profiles. Relative abundance of Bacteroides spp. increased with both green and gold kiwifruit substrate while Bifidobacterium spp. increased only with green kiwifruit. NMR spectroscopy and GC demonstrated an increase in organic acids (primarily acetate, butyrate, and propionate) and a concomitant decrease in several amino acids and oligosaccharides following addition of green and gold kiwifruit substrate. The experiments demonstrated that despite markedly different baseline profiles in individual donor inoculum, kiwifruit components can induce substantive change in microbial ecology and metabolism which could have consequences for human health.

Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: A practical approach for a critical review

Plasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events.

Transplantation: moving to the next level.

This issue of Immunological Reviews bridges two traditionally separate fields. While the average immunologist may think of “transplantation” as a single entity, it has, for largely practical reasons, been split into two separate fields, namely hematopoietic cell transplantation (HCT) and organ transplantation. In allogeneic HCT, which was originally used as a rescue therapy for the marrow toxicity of high-dose chemotherapy and/or irradiation employed to treat a hematologic malignancy, the recipient’s immune system is severely compromised and the greatest concern has been the attack of immune cells in the donor inoculum on the severely immunocompromised recipient. This attack, which caused the sometimes fatal syndrome known as graft-vs-host disease (GVHD), was so severe when HLA barriers were transgressed that until recently the practice of HCT has been largely confined to the HLA-identical or near-identical donor situation, severely limiting the availability of this curative therapy. Even in the present day, as approaches to performing HLA-mismatched HCT have permitted more widespread transplantation in this setting, the incidence of significant GVHD is in the 30–40% range in the least immunogenic combination involving HLA-identical sibling donors, despite the use of pharmacologic immunosuppression as GVHD prophylaxis(1). Thus, GVHD remains a major cause of morbidity and mortality following HCT.

A Preclinical Canine Model of in Utero Hematopoietic Cell Transplantation–Induced Graft Vs. Host Disease.

AbstractAbstract 3002 Introduction:In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allo-engraftment and associated donor-specific tolerance (DST) without toxic conditioning. To evaluate IUHCT in a clinically relevant large animal model, we have developed the haploidentical canine model of IUHCT. Recent optimization of the model using a CD3 depleted graft has achieved consistent and stable levels of mixed hematopoietic chimerism (average > 10%) without graft versus host disease (GVHD). However, since GVHD represents the greatest theoretical risk of clinical IUHCT, it is important to identify the phenotype and requirements for IUHCT-induced GVHD in the canine model prior to clinical application. In the current study, we attempted to establish a threshold dose of CD3+ cells required for IUHCT-induced GVHD in the haploidentical canine model by increasing the absolute percentage of CD3+ cells in the donor inoculum and we characterized the GVHD phenotype observed. Methods:43 fetuses of 9 pregnant bitches underwent ultrasound-guided intracardiac injection of maternal BM cells at 37.5–43 days gestational age (GA). In our large animal model, bone marrow yields, cellularity and litter size differ between dams. Due to these factors and because the targeted gestational age range coincides with an exponential growth phase for the dog fetus, standardization of cell dose per weight was not possible. Following MACS CD3 depletion, graft composition was standardized by adding back the CD3+ fraction to achieve donor CD3 content of 1%, 3%, 7.5%, and 16% (whole bone marrow [WBM] positive control) (Table 1). Live born puppies were monitored daily for signs of GVHD, including skin lesions, diarrhea, hematochezia, jaundice, weight loss and lethargy. Chimerism was measured by a PCR-based VNTR assay using commercially available primers. Results:Though prenatal survival of 75% was not significantly different than baseline (76%) and pups were not runted at birth, acute GVHD was observed postnatally in all ten live born recipients of either 7.5% CD3 cells or WBM, with onset ranging from 1–14 days of age. Recipients of grafts containing 1% and 3% CD3 cells showed no clinical evidence of GVHD.Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD in our model. Various affected animals manifested additional symptoms in all GVHD target organs, including increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin lesions. The phenotype was severe and all ten ultimately required euthanasia between 2 and 21 days of age.Necropsies performed during acute GVHD revealed significant erythematous and/or ulcerated skin change, hepatosplenomegaly, and punctate hemorrhage throughout the gastrointestinal tract and lung. Histology (H&E) demonstrated lymphocytic infiltrates and focal necrosis in target tissues as well as effacement of the dermal-epidermal junction and villous surfaces. Canalicular bile stasis was the most uniform and characteristic microscopic finding irrespective of hepatic functional status.Chimerism levels in pups who survived to birth were compared based upon CD3 content of the donor graft (Table 1). Increasing CD3 percentage trended toward an increase in chimerism, though only the WBM group reached statistical significance (p=.043). Conclusions:In the preclinical haploidentical canine model of IUHCT the threshold for induction of GVHD appears to be between 3% and 7.5% graft CD3+cell content. Interestingly, the absolute dose of CD3+ cells/kg fetal weight does not entirely correlate with the occurrence of GVHD, suggesting that the composition of the graft rather than the absolute T-cell content may determine the likelihood of GVHD. Given the complete absence of GVHD at a CD3 dose of 1% and the excellent engraftment achieved, it appears that there is a safe therapeutic index for achievement of chimerism without GVHD. When IUHCT-induced GVHD occurs, it is severe and clinically similar to postnatal GVHD. Notably, clinical GVHD only occurred after birth, suggesting suppressive mechanisms in utero. Additional studies are needed to further optimize the graft for support of engraftment without GVHD.Table 1DosenGA at IUHCTCD3 cells/kgCD34 cells/kgMean ChimerismGVHD1%2039–41.37–2.72 × 10 (9)5.8–58.2 × 10 (8)12.1%N3%4381.26 × 10 (9)9.8 × 10 (8)7.3%N7.5%341.52.26 × 10 (9)7.0 × 10 (8)17.7%Y16% (WBM)7432.0 × 10 (9)5.1 × 10 (8)20.3%Y Disclosures:No relevant conflicts of interest to declare.

Hepatitis C Virus Envelope Glycoprotein Fitness Defines Virus Population Composition following Transmission to a New Host

Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission.

Ecological Succession of Bacterial Communities during Conventionalization of Germ-Free Mice

Little is known about the dynamics of early ecological succession during experimental conventionalization of the gastrointestinal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jejunum), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacterial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum and analyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunum had lower diversity and were distinct from the input community (dominated by either Escherichia or Bacteroides). However, by days 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of the donor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to 21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity compared to each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is more dynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do not simply reassemble at mucosal sites during conventionalization (they first transform into a "pioneering" community and over time take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal environment plays a role in shaping the community.

Post-Transplant Cyclophosphamide Treatment Ameliorates Experimental Gvhd While Permitting Lymphopenic Expansion of Non-Host Reactive Donor T Cells.

AbstractAbstract 3751Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Vβ Spectratype-guided Separation of Graft-Versus-Host Disease and Graft-Versus-Leukemia Effects in a MHC-Matched miHA-Mismatched Mouse Model of Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation has been proven to be one of few curative treatment options for patients with hematological malignancies. One drawback to this procedure is the development of graft-versus-host disease (GVHD), which can lead to high rates of morbidity and mortality. It is well established that donor T cells are primarily responsible for anti-host activity. Removal or delayed-administration of donor T cells reduces the incidence or severity of GVHD, but is often associated with an increased risk of tumor relapse due to lack of donor T cells that mediate graft-versus-leukemia (GVL) effects. Utilizing CDR3-size spectratype analysis we have focused on identifying host- and tumor-reactive T cells. In this study, a MHC-matched, miHA-mismatched murine transplantation model (B10.BR→CBA) was used to characterize CD8+ T cell repertoire anti-host responses as well as anti-tumor responses to a host-derived myeloid leukemia cell line (MMC6). This model is particularly relevant in that it mimics matched-sibling donor transplants for AML. Spectratype analysis has shown that 14 of 20 Vβ families exhibit biased CDR3-size usage in the B10.BR anti-CBA CD8+ T cell repertoire. In response to MMC6, 7 of 20 Vβ families exhibit biased CDR3-size usage, with Vβ4 and Vβ13 uniquely skewed in the anti-tumor response. We sought to test the ability of spectratype-identified Vβ families, uniquely skewed in the B10.BR CD8+ T cell anti-MMC6 response, to mediate GVL effects in the absence of GVHD in tumor challenged transplant recipients. To this end lethally irradiated (11Gy;split dose) CBA recipients were transplanted with B10.BR ATBM alone, ATBM + B10.BR CD8+ T cells, or ATBM + either B10.BR CD8+ Vβ4+ or Vβ13+ T cells. Duplicate groups of mice received the same transplant conditions and were challenged with MMC6 on day +1 post-transplant. All mice were monitored for symptoms of GVHD and tumor growth. Mice receiving MMC6 in the absence of T cells succumbed to tumor within 30 days post-transplant. Tumor challenged mice receiving CD8+ T cells displayed a statistically significant GVL effect (p<0.0001), however, all eventually succumbed to GVHD. Mice receiving either Vβ4+ or Vβ13+ T cells along with MMC6 displayed minimal GVL effects, with all mice succumbing to tumor challenge. As expected based on the spectratype analysis, mice receiving either Vβ4+ or Vβ13+ T cells in the absence of tumor challenge did not develop any signs of GVHD and survived until termination of the experiment. In order to test the possibility of enhancing the GVL potential, duplicate groups of mice received the same transplant conditions and MMC6 tumor challenge, as above with an additional group that was co-transplanted with CD8+Vβ4+ and Vβ13+ T cells. This combined transplantation of CD8+Vβ4+ and Vβ13+ T cells did not mediate severe lethal GVHD (80% survival). However, together, CD8+Vβ4+ and Vβ13+ T cells significantly prolonged survival (p=0.001) of tumor challenged recipients. Taken together, the results of this study provide proof of principle to support the hypothesis that Vβ spectratype analysis can be used to identify the donor GVH- and GVL-reactive T cells. Furthermore, manipulation of the donor inoculum to enrich for the GVL-reactive and/or deplete the GVH-reactive Vβ families can provide an effective way to separate GVHD from GVL effects.

TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection

AbstractCytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) are synthetic ODNs with unmethylated DNA sequences that mimic viral and bacterial DNA and protect against infectious agents and tumor challenge. We show that CpG ODNs markedly accelerated graft-versus-host disease (GVHD) lethality by Toll-like receptor 9 (TLR9) ligation of host antigen-presenting cells (APCs), dependent upon host IFNγ but independent of host IL-12, IL-6, or natural killer (NK) cells. Imaging studies showed significantly more green fluorescent protein–positive (GFP+) effector T cells in lymphoid and nonlymphoid organs. In engraftment studies, CpG ODNs promoted allogeneic donor bone marrow (BM) rejection independent of host IFNγ, IL-12, or IL-6. During the course of these studies, we uncovered a previously unknown and critical role of donor BM APCs in modulating the rejection response. CpG ODNs promoted BM rejection by ligation of donor BM, but not host, TLR9. CpG ODNs did not impair engraftment of TLR9−/− BM unless wild-type myeloid (CD11b+) but not B-lineage (CD19+) BM cells were added to the donor inoculum. The importance of donor BM APCs in modulating the strength of the host antidonor rejection response was underscored by the finding that B7-1/B7-2−/− BM was less likely than wild-type BM to be rejected. Collectively, these data offer new insight into the mechanism of alloresponses regulating GVHD and BM rejection.