Molecular immune monitoring in kidney transplant rejection: a state-of-the-art review

Although current regimens of immunosuppressive drugs are effective in renal transplant recipients, long-term renal allograft outcomes remain suboptimal. For many years, the diagnosis of renal allograft rejection and of several causes of renal allograft dysfunction, such as chronic subclinical inflammation and infection, was mostly based on renal allograft biopsy, which is not only invasive but also possibly performed too late for proper management. In addition, certain allograft dysfunctions are difficult to differentiate from renal histology due to their similar pathogenesis and immune responses. As such, non-invasive assays and biomarkers may be more beneficial than conventional renal biopsy for enhancing graft survival and optimizing immunosuppressive drug regimens during long-term care. This paper discusses recent biomarker candidates, including donor-derived cell-free DNA, transcriptomics, microRNAs, exosomes (or other extracellular vesicles), urine chemokines, and nucleosomes, that show high potential for clinical use in determining the prognosis of long-term outcomes of kidney transplantation, along with their limitations

Dialysate White Blood Cell Change after Initial Antibiotic Treatment Represented the Patterns of Response in Peritoneal Dialysis-Related Peritonitis

Background. Patients with peritoneal dialysis-related peritonitis usually have different responses to initial antibiotic treatment. This study aimed to explore the patterns of response by using the changes of dialysate white blood cell count on the first five days of the initial antibiotic treatment. Materials and Methods. A retrospective cohort study was conducted. All peritoneal dialysis-related peritonitis episodes from January 2014 to December 2015 were reviewed. We categorized the patterns of antibiotic response into 3 groups: early response, delayed response, and failure group. The changes of dialysate white blood cell count for each pattern were determined by multilevel regression analysis. Results. There were 644 episodes in 455 patients: 378 (58.7%) of early response, 122 (18.9%) of delayed response, and 144 (22.3%) of failure episodes. The patterns of early, delayed, and failure groups were represented by the average rate reduction per day of dialysate WBC of 68.4%, 34.0%, and 14.2%, respectively (p value < 0.001 for all comparisons). Conclusion. Three patterns, which were categorized by types of responses, have variable rates of WBC declining. Clinicians should focus on the delayed response and failure patterns in order to make a decision whether to continue medical therapies or to aggressively remove the peritoneal catheter

SRL effectively inhibited B-cell proliferation.

<p>Purified CD19⁺ B cells were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 (BCR method) in the absence (control; CTRL) or presence of TAC (6ng/ml) or SRL (2ng/ml or 6ng/ml) and flow cytometric analyses were performed after 6 days in culture. <b>(A)</b> A representative experiment: cells were gated on viable lymphocytes and analyzed for CFSE diluting proliferating cells. This scheme of analysis was used in all subsequent experiment, unless indicated otherwise. <b>(B)</b> The percentage of proliferating CD19⁺ B cells as obtained in A from 7 different experiments. <b>(C)</b> Absolute number of proliferating CD19⁺ B cells was calculated in each experiment by multiplying the recovered cell counts with the percentage of proliferating cells as in A (n = 7). Statistically significant (*p < 0.05) inhibition of B cell proliferation was observed with SRL at both subtherapeutic (2ng/ml) and therapeutic (6ng/ml) doses.</p

SRL, but not TAC effectively inhibited the differentiation of B cells into plasma cells.

<p>Purified CD19⁺ B cells were cultured as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129658#pone.0129658.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129658#pone.0129658.g002" target="_blank">2</a>. <b>(A)</b> A representative experiment showing flow cytometric profile indicative of putative plasma cells (CD19^low) in proliferated B cells (gated on viable lymphocytes; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129658#pone.0129658.g001" target="_blank">Fig 1A</a>). <b>(B)</b> The mean ± SD percentage of such CD19^low B cells from 4 different independent experiments. <b>(C)</b> Mean ± SD (n = 4) percentage of CD19^lowCD38⁺⁺ plasmablasts, CD19^lowCD138⁺ plasma cells, Blimp1⁺PAX5^- cells and CD138⁺Blimp1⁺ cells in the proliferating CD19^low cells. *p < 0.05, **p < 0.01. CTRL, control; TAC, 6 ng/ml TAC; SRL, 6 ng/ml SRL.</p

SRL-treated B cells enhances the proliferation and differentiation of CD4⁺ T cells.

<p>Purified CD19⁺ B cells were pre-stimulated for 6 days with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of 6ng/ml TAC or SRL. These cultured B cells were used as stimulators in 6-day MLRs of allogeneic CFSE-labelled CD4⁺CD25⁻ T cell responders. (<b>A</b>) Level of proliferation differentially induced by pre-cultured B cells as detected by CFSE dilution in the allogeneic CD4 responder cells (representative experiment on the left, and compiled data from 8 independent experiments on the right). (<b>B</b>) Percentage of responding T cells positive for memory marker (CD45RO) and activation markers (CD62L, CD25, CD69, CD95) after co-culture with pre-stimulated B cells (n = 8); (<b>C</b>) Mean ± SD (n = 4) percentage of responding proliferating T cells expressing intracellular cytokines (top row) and transcription factors (bottom row). Taken together the data indicated that B cells that proliferated in presence of SRL on a per cell basis were capable of inducing alloreactive T cell proliferation towards a Th1 phenotype. *p < 0.05. ** p < 0.01.</p

SRL was more effective in inhibiting IL-6 and IL-10 production in B cells.

<p>Cytokine levels were measured in the culture supernatants of stimulated B cells in the absence or presence of 6ng/ml SRL or TAC on day 6. SRL markedly inhibited production of IL-10 and IL-6 by B cells compared to control and TAC. No significant difference was found for IL-1beta, IL-4, IL-7, IL-8, TNF-alpha and GM-CSF. *p < 0.05, **p < 0.01.</p

B-cell stimulation in the presence of SRL resulted in a population shift toward an activated phenotype.

<p>Purified CD19⁺ B cells were stimulated with anti-IgM, anti-CD40 mAb and IL-21 and multi-color flow cytometric analyses were performed on day 6 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129658#pone.0129658.g001" target="_blank">Fig 1</a>. The figures <b>A</b>, <b>B</b> and <b>C</b> show the expression of various surface markers on stimulated B cells (Percentage of positive cells/total proliferating CD19⁺ cells) p < 0.05, **p < 0.01. CTRL, control; TAC6, 6 ng/ml TAC; SRL2, 2 ng/ml SRL; SRL6, 6 ng/ml SRL.</p

SRL but not TAC inhibited the proliferation of CD19⁺CD27⁻ naïve and CD19⁺CD27⁺ memory B cells.

<p>B cells were purified by depleting non-B cells resulting in >95% CD19⁺ cells which subsequently sorted into CD27⁻ (naïve) and CD27⁺ (memory) B-cell fractions. These subsets were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of TAC or SRL at 6ng/ml and were analyzed by multicolor flow cytometry after 6 days in culture. <b>(A)</b> A representative experiment: histogram plots show dilution of CFSE in the proliferating CD19⁺CD27⁻ (upper panel) or CD19⁺CD27⁺ (lower panel) cells. <b>(B)</b> Data are from four different independent experiments are shown as mean ± SD percent proliferating naïve CD19⁺CD27⁻ and memory CD19⁺CD27⁺ B cells. <b>(C)</b> B cell subsets showing indicated surface markers were analyzed and plotted as mean ± SD (n = 4) percent of proliferating cells in the cultures of naïve CD19⁺CD27⁻ (upper panel) and memory CD19⁺CD27⁺ B cells (lower panel). The residual cells that proliferated in presence of SRL demonstrated an activated phenotype. *p < 0.05, **p < 0.01.</p

Mechanistic analyses in kidney transplant recipients prospectively randomized to two steroid free regimen-Low dose Tacrolimus with Everolimus versus standard dose Tacrolimus with Mycophenolate Mofetil.

Calcineurin inhibitors (CNI), the cornerstone of immunosuppression after transplantation are implicated in nephrotoxicity and allograft dysfunction. We hypothesized that combined low doses of CNI and Everolimus (EVR) may result in better graft outcomes and greater tolerogenic milieu. Forty adult renal transplant recipients were prospectively randomized to (steroid free) low dose Tacrolimus (TAC) and EVR or standard dose TAC and Mycophenolate (MMF) after Alemtuzumab induction. Baseline characteristics were statistically similar. EVR levels were maintained at 3-8 ng/ml. TAC levels were 4.5±1.9 and 6.4±1.5 ng/ml in the TAC+EVR and TAC+MMF group respectively. Follow up was 14±4 and 17±5 months respectively and included protocol kidney biopsies at 3 and 12 months post-transplantation. Rejection-rate was lower in the TAC+EVR group. However patient and overall graft survival, eGFR and incidence of adverse events were similar. TAC+EVR induced expansion of CD4+CD25hiFoxp3+ regulatory T cells as early as 3 months and expansion of IFN-γ+CD4+CD25hiFoxp3+ regulatory T cells at 12 months post-transplant. Gene expression profile showed a trend toward decreased inflammation, angiogenesis and connective tissue growth in the TAC+EVR Group. Thus, greater tolerogenic mechanisms were found to be operating in patients with low dose TAC+EVR and this might be responsible for the lower rejection-rate than in patients on standard dose TAC+MMF. However, further studies with longer follow up and evaluating impact on T regulatory cells are warranted

Cellular and molecular immune profiles in renal transplant recipients after conversion from Tacrolimus to Sirolimus

Tacrolimus and Sirolimus are commonly used maintenance immunesuppressants in kidney transplantation. Since their effects on immune cells and allograft molecular profiles have not been elucidated, we characterized the effects of Tacrolimus to Sirolimus conversion on frequency and function of T cells, and on graft molecular profiles. Samples from renal transplant patients in a randomized trial of 18 patients with late Sirolimus conversion and 12 on Tacrolimus maintenance were utilized. Peripheral blood was collected at 0, 6, 12 and 24-months post-randomization with T cell subpopulations analyzed by flow cytometry and T cell alloreactivity tested by IFN-γ ELISPOT. Graft biopsy samples obtained 24-months post-randomization were used for gene expression analysis. Sirolimus conversion led to an increase in CD4+25+++Foxp3+ regulatory T cells. While Tacrolimus-maintained patients showed a decrease in indirect alloreactivity over time post-transplant, Sirolimus conversion increased indirect alloreactive T cell frequencies compared to Tacrolimus-maintained patients. No histological differences were found in graft biopsies, but molecular profiles showed activation of the antigen presentation, IL-12 signaling, oxidative stress, macrophage-derived production pathways, and increased inflammatory and immune response in Sirolimus-converted patients. Thus, chronic immune alterations are induced after Sirolimus conversion. Despite the molecular profile being favorable to calcineurin inhibitor-based regimen, there was no impact in renal function over 30 months of follow-up