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Serial EBV load monitoring of clinically asymptomatic pediatric thoracic organ transplant patients has identified three groups of children who exhibit undetectable (16,000 copies/ml) EBV loads in peripheral blood. Chronic high EBV load patients have a 45% rate of progression to late-onset posttransplant lymphoproliferative disorders. In this article, we report that asymptomatic patients carrying EBV loads (low and high) expressed increased frequencies of EBV-specific CD8+ T cells, as compared with patients with undetectable EBV loads. Although patients with low viral load displayed EBV-specific CD8+ T cells with moderate signs of activation (CD38+/−/CD127+/−), programmed death 1 upregulation and effective IFN-γ secretion, high EBV load carriers showed significant CD38+ upregulation, features of cellular exhaustion (programmed death 1+/CD127−) accompanied by a decline in IFN-γ release. Immunopolarization of EBV-specific CD8+ T cells was skewed from the expected type 1 (IFN-γ) toward type 0 (IFN-γ/IL-5) in patients, and Tr1 (IL-10) in high load carriers. These results indicate the importance of chronic EBV load and of the levels of antigenic pressure in shaping EBV-specific memory CD8+ T cells. Concomitant phenotypic and functional EBV monitoring is critical for identifying the complex “functional” versus “exhausted” signature of EBV-specific CD8+ T cells, with implications for immunologic monitoring in the clinic.

Experimental models describe immune exhaustion in liver transplant tolerance. It is not known if immune exhaustion is also occurs in clinical operational tolerance. We tested this in a cross-sectional cohort of pediatric liver transplant recipients with stable allograft function in the absence of immunosuppression (Operationally Tolerant, OT) in comparison to those Weaned off Immunosuppression (WI), continued on Maintenance Immunosuppression (MI) and Healthy Controls (HC). Results: In OT recipients, B cell frequencies (p

Monocytes are an important source of antigen-presenting cells and macrophages in tissues affected by infection or inflammation and in organ transplantation. Monocytes have been grouped into classical (CD14+CD16-), non-classical (CD14dimCD16+), and intermediate (CD14+CD16+) subsets. The biologic significance of these subsets is not completely understood. We phenotyped monocytes in peripheral blood from 25 healthy subjects and performed functional studies on monocyte subsets sorted from leukapheresis products to better characterize these subsets in humans. Monocyte subset frequencies were found to be tightly controlled over time and across healthy individuals: 83.95% classical, 95% CI [81.12, 86.78], 6.65% intermediate [4.32, 8.99], and 9.28% non-classical [7.12, 11.44]. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to Toll-like receptor agonists, and cytokine secretion by classical and intermediate monocytes was suppressed when all three subsets were recombined in physiologic ratios. After incubation with IL-4 and GM-CSF, classical monocytes acquired dendritic cell markers and morphology; intermediate and non-classical monocytes did not. From these studies we conclude that classical monocytes appear to be the principal source of antigen-presenting dendritic cells, and non-classical monocytes suppress secretion of inflammatory cytokines by the other subsets in mixed cultures, perhaps indicating a regulatory role.

Rapamycin (RAPA) is a tolerance- and Treg-sparing immunosuppressant. Prolonged exposure of myeloid dendritic cells (mDC) to RAPA confers resistance to phenotypic and functional maturation following exposure to inflammatory stimuli. Infusion of RAPA-conditioned mDC (RAPA-DC) promotes transplant survival and inhibits GVHD. However, RAPA is also reported to have pro-inflammatory properties, mediated by increased DC IL-12. Our aim was to define how DC cytokine production and CD4+ T helper (Th) cell polarization is modified by prolonged RAPA exposure. Methods: Bone-marrow derived murine mDC were generated in 7 day (d) cultures, with RAPA added d2-d7. Following purification, RAPA-DC or control mDC (CTR-DC) remained unstimulated or were exposed to LPS. Co-stimulatory molecule expression and intracellular IL-12p40 was determined by flow cytometry. Th polarization was assessed using these DC to stimulate alloreactive CD4+ T cells, followed by analysis of T cell proliferation, apoptosis, and intracellular cytokines. Western blot was used to assess total and phosphorylated signaling molecules. Results: RAPA-DC displayed reduced costimulatory molecules and allostimulatory capacity before and after exposure to LPS. IL-12p40 and p70 were reduced in unstimulated RAPA-DC compared to CTR-DC. LPS induced a significant increase in IL-12p70 production by RAPA-DC, due to failed inhibition of glycogen synthase kinase-3. However, RAPA-DC stimulated with LPS remained poor allostimulators that induced apoptosis in alloreactive CD4+ T cells and failed to promote Th1 responses. Conclusions: Prolonged exposure of mDC to RAPA, although promoting LPS-induced IL-12p70 production, inhibits T cell stimulatory capacity and Th1 polarization.