Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study.

Identification of biomarkers that assess posttransplant risk is needed to improve long-term outcomes following heart transplantation. The Clinical Trials in Organ Transplantation (CTOT)-05 protocol was an observational, multicenter, cohort study of 200 heart transplant recipients followed for the first posttransplant year. The primary endpoint was a composite of death, graft loss/retransplantation, biopsy-proven acute rejection (BPAR), and cardiac allograft vasculopathy (CAV) as defined by intravascular ultrasound (IVUS). We serially measured anti-HLA- and auto-antibodies, angiogenic proteins, peripheral blood allo-reactivity, and peripheral blood gene expression patterns. We correlated assay results and clinical characteristics with the composite endpoint and its components. The composite endpoint was associated with older donor allografts (p < 0.03) and with recipient anti-HLA antibody (p < 0.04). Recipient CMV-negativity (regardless of donor status) was associated with BPAR (p < 0.001), and increases in plasma vascular endothelial growth factor-C (OR 20; 95%CI:1.9-218) combined with decreases in endothelin-1 (OR 0.14; 95%CI:0.02-0.97) associated with CAV. The remaining biomarkers showed no relationships with the study endpoints. While suboptimal endpoint definitions and lower than anticipated event rates were identified as potential study limitations, the results of this multicenter study do not yet support routine use of the selected assays as noninvasive approaches to detect BPAR and/or CAV following heart transplantation.

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype.

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+ Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+ CD25+ CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+ CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+ CD25+ CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

Isolated CD39 expression on CD4+ T cells denotes both regulatory and memory populations.

Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.

Increased soluble 4-1BB ligand (4-1BBL) levels in peripheral blood of patients with multiple sclerosis.

4-1BB ligand (4-1BBL; CD137L) is a member of the tumour necrosis factor superfamily expressed primarily on antigen presenting cells such as B cells, macrophages and dendritic cells. Its engagement with the receptor 4-1BB (CD137) has been shown to promote T-cell activation and regulate proliferation and survival of T cells. The role of the costimulatory molecule in multiple sclerosis (MS) remains unclear. In this study, the expression of 4-1BBL and soluble 4-1BBL (s4-1BBL) protein levels were analysed in peripheral blood of MS patients. Compared with healthy controls, MS patients had an increase in both plasma s4-1BBL protein levels and expression of 4-1BBL in CD14(+) monocytes. In contrast, myelin basic protein-reactive T-cell proliferation was not found to be inhibited by the use of an anti-4-1BBL antibody. The elevated s4-1BBL protein levels in the MS patients may function as a self-regulatory mechanism of 4-1BB/4-1BBL interaction and costimulation.

Multiple sclerosis: a study of chemokine receptors and regulatory T cells in relation to MRI variables.

Magnetic resonance imaging (MRI) remains the most valuable tool for monitoring disease activity and progression in patients with multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS) with presumably autoimmune etiology. Chemokine receptors have been implicated in MS as key molecules directing inflammatory cells into the CNS. Regulatory (CD4+CD25+) T cells (Tr cells) are important in suppressing autoimmunity, and their absolute or functional deficit could be expected in MS. In the present study, venous blood was obtained from MS patients concurrent with MRI examination of the brain, and expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3 and CXCR4 by CD4 T cells and monocytes, proportions of Tr cells, as well as expression of CD45RO, CD95, CTLA-4, HLA-DR and interleukin (IL)-10 by Tr cells and non-Tr (CD25-) CD4 T cells was analyzed by flow cytometry. Surface expression of CXCR3 by CD4 T cells was downregulated in the group of patients with high lesion load (LL) on T2-weighted images and gadolinium (Gd)-enhancing lesions on T1-weighted images, compared to the group with high LL and no Gd-enhancing lesions, and to the group with low LL, suggesting internalization of CXCR3 due to the release of its chemokine ligand (IP-10/CXCL10) from active MS lesions. Proportions of Tr cells amongst all CD4 T cells, and expression of IL-10 by Tr cells were increased in the patients with high LL and Gd-enhancing lesions. These results suggest that there is correlation between MRI parameters, chemokine receptor expression and the status of circulating Tr cells in MS, but further studies need to discriminate between pathogenetically relevant and bystander phenomena.