In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC-defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade-/ sirolimus-mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan-based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient-rather than donor-derived. Surprisingly, even in MHC-matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen-experienced T cells, and transplant rejection was associated with the acquisition of donor-directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the postimmunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen-experienced phenotype, and ultimately, to transplant rejection.

No abstract

Men who have sex with men (MSM) have differences in immune activation and gut microbiome composition compared with men who have sex with women (MSW), even in the absence of HIV infection. Gut microbiome differences associated with HIV itself when controlling for MSM, as assessed by 16S rRNA sequencing, are relatively subtle. Understanding whether gut microbiome composition impacts immune activation in HIV-negative and HIV-positive MSM has important implications since immune activation has been associated with HIV acquisition risk and disease progression. To investigate the effects of MSM and HIV-associated gut microbiota on immune activation, we transplanted feces from HIV-negative MSW, HIV-negative MSM, and HIV-positive untreated MSM to gnotobiotic mice. Following transplant, 16S rRNA gene sequencing determined that the microbiomes of MSM and MSW maintained distinct compositions in mice and that specific microbial differences between MSM and MSW were replicated. Immunologically, HIV-negative MSM donors had higher frequencies of blood CD38+ HLADR+ and CD103+ T cells and their fecal recipients had higher frequencies of gut CD69+ and CD103+ T cells, compared with HIV-negative MSW donors and recipients, respectively. Significant microbiome differences were not detected between HIV-negative and HIV-positive MSM in this small donor cohort, and immune differences between their recipients were trending but not statistically significant. A larger donor cohort may therefore be needed to detect immune-modulating microbes associated with HIV. To investigate whether our findings in mice could have implications for HIV replication, we infected primary human lamina propria cells stimulated with isolated fecal microbiota, and found that microbiota from MSM stimulated higher frequencies of HIV-infected cells than microbiota from MSW. Finally, we identified several microbes that correlated with immune readouts in both fecal recipients and donors, and with in vitro HIV infection, which suggests a role for gut microbiota in immune activation and potentially HIV acquisition in MSM.

In murine models, T-cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti-CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, non-depleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well-established primate bone marrow chimerism-induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T-cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and non-depletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4 ؉ / CD25 high /CD127 low /FoxP3 ؉ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8 ؉ T cells were mobilized to a greater extent than CD4 ؉ T cells, with accumulation of 3.7 ؎ 0.4-fold more total CD8؉ T cells and 6.2 ؎ 0.4-fold more CD8 ؉ effector memory T cells in the leukapheresis product compared with G-CSF alone.Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation. (Blood. 2011;118(25): 6580-6590) IntroductionThe widespread use of cytokine-mediated mobilization has had a major impact on hematopoietic stem cell transplantation (HSCT). For auto-HSCT, peripheral blood-derived stem cell (PBSC) transplantation is associated with more rapid hematopoietic reconstitution and better outcomes compared with bone marrow transplantation. [1][2][3][4][5] For allo-HSCT, the choice is more complex. A metaanalysis showed that PBSC transplants in adults resulted in more rapid hematopoietic reconstitution, decreased relapse, and increased disease-free survival compared with bone marrow transplantation 6 but did not lead to an overall survival advantage compared with bone marrow, except in patients with late-stage disease. 6 This was probably because of the higher T-cell content of PBSC grafts (10-to 50-fold more than bone marrow-derived allografts), [7][8][9] leading to a significantly greater risk of GVHD. 6 In pediatrics, this increased risk of GVHD and transplant-related mortality shifted the risk/benefit balance, favoring bone marrow over PBSCs. 10 These dichotomous results between pediatric and adult patients suggest that a narrow therapeutic window exists for infused lymphocytes.With the FDA approval of AMD3100 (Plerixafor or Mozobil), 11 mobilization can now occur by multiple regimens, including G-CSF alone, AMD3100 alone, or G-CSF plus AMD3100. Therefore, the risks and benefits of each of these mobilization strategies must be understood and compared with those associated with bone marrow transplantation. AMD3100 is US Food and Drug Administration (FDA)-approved for auto-HSCT, and the combination of G-CSF and AMD3100 was shown to be superior to G-CSF for stem cell mobilization. 12-14 Furthermore, there was accelerated lymphocyte recov...