Cell Therapy in Kidney Transplantation: Focus on Regulatory T Cells.

Renal transplantation is the renal replacement modality of choice for suitable candidates with advanced CKD or ESRD. Prevention of rejection, however, requires treatment with nonspecific pharmacologic immunosuppressants that carry both systemic and nephrologic toxicities. Use of a patient's own suppressive regulatory T cells (Tregs) is an attractive biologic approach to reduce this burden. Here, we review the immunologic underpinnings of Treg therapy and technical challenges to developing successful cell therapy. These issues include the selection of appropriate Treg subsets, ex vivo Treg expansion approaches, how many Tregs to administer and when, and how to care for patients after Treg administration.

Homeostatic expansion as a barrier to lymphocyte depletion strategies.

PURPOSE OF REVIEW: Following lymphodepletion, lymphocytes repopulate the immune space both through enhanced thymopoiesis and proliferation of residual nondepleted peripheral lymphocytes. The term homeostatic proliferation (alternatively homeostatic expansion or lymphopenia-induced proliferation) refers to the latter process. Homeostatic proliferation is especially relevant to reconstitution of the lymphocyte compartment following immunodepletion therapy in transplantation. Repopulating lymphocytes can skew toward an effector memory type capable of inducing graft rejection, autoimmunity, or, in the case of allogeneic bone marrow transplantation, graft versus host disease. Here we review recent studies exploring the biologic mechanisms underlying homeostatic proliferation and explore implications for therapy in transplantation. RECENT FINDINGS: Two immune-depleting agents, alemtuzumab and rabbit antithymocyte globulin, have been well characterized in their abilities to induce an effector-memory phenotype in repopulating lymphocytes. Additionally, we have gained new understandings of the mechanisms by which the cytokines interleukin-7 and interleukin-15 regulate this process. Recent studies have also explored the functions of noncytokine and signaling molecules in lymphopenia-induced proliferation. Finally, we have seen the promise and limitations of several therapeutic approaches, including recombinant interleukin-7 therapy, CD8-targeted antibodies, and peri-transplant cyclophosphamide, to treat posttransplant lymphopenia and reduce the risks of immune dysregulation following homeostatic proliferation. SUMMARY: Immune dysfunction following homeostatic proliferation is a special challenge in transplantation. A deeper understanding of the underlying biology has led to a number of promising new therapies to overcome this problem.

A case of levamisole-induced systemic vasculitis and cocaine-induced midline destructive lesion: a case report.

We describe a case of antineutrophil cytoplasmic antibody-positive vasculitis from levamisole-tainted cocaine with concomitant cocaine-induced midline destructive lesions of the palate and nasal septum. The diagnosis was confirmed after extensive clinical, laboratory, pathologic, and radiographic testing. Timely recognition of this clinical entity is critical to avoid misdiagnosis and unnecessary treatment with potentially harmful cytotoxic agents. Given the high rate of levamisole contamination within the nation's cocaine supply, clinicians should be alerted to this emerging health threat.

A case of atypical hemolytic uremic syndrome in a second renal transplant.

Atypical hemolytic uremic syndrome (aHUS) has gained increased visibility over several years as an important cause of renal failure. Unfortunately, diagnosis is often difficult because individual courses can be highly variable depending the causative genetic mutations. Here we present the case of a patient with a failed renal allograft and acute failure of a second allograft who was ultimately diagnosed with aHUS. Interestingly, he developed early de novo donor specific antibodies (DSA) after the second renal transplant in context of likely recurrent aHUS. Terminal complement inhibition with eculizumab resulted in prompt improvement of renal allograft function.

An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry.

BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets. RESULTS: TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38-), and late memory (CD24-CD38-) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation. CONCLUSIONS: We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.

Transplantation immunology in 2013: New approaches to diagnosis of rejection.

In 2013, a key theme of research in renal transplantation was the diagnosis of rejection. Data from key studies published in the past year highlight aspects of rejection that warrant further investigation and should prompt the consideration of adjunctive tests to complement traditional histological assessment of allograft biopsy samples.

Identification of phosphorylation-dependent binding partners of aquaporin-2 using protein mass spectrometry.

Vasopressin-mediated control of water permeability in the renal collecting duct occurs in part through regulation of the distribution of aquaporin-2 (AQP2) between the apical plasma membrane and intracellular membrane compartments. Phosphorylation of Ser-256 at AQP2's cytoplasmic COOH-terminus is well-accepted as a critical step for translocation. The aim of this study was to identify binding partners to phosphorylated versus nonphosphorylated forms of the AQP2 COOH-terminus via a targeted comparative proteomic approach. Cytosol from inner medullary collecting ducts isolated from rat kidneys was incubated with "bait" peptides, representing the COOH-terminal AQP2 tail in its nonphosphorylated and phosphorylated forms, to capture differentially bound proteins prior to LC-MS/MS analysis. Mass spectrometric results were confirmed by immunoblotting. Immunoprecipitation was performed using an AQP2 COOH-terminal antibody combined with immunblotting against the proposed binding partners to demonstrate interactions with native AQP2. Our studies confirmed previously identified interactions between AQP2 and hsc70, hsp70-1 and -2, as well as annexin II. These proteins were found to bind less to the Ser-256-phosphorylated AQP2 than to the nonphosphorylated form. In contrast, another heat shock protein, hsp70-5 (BiP/grp78), bound to phosphorylated AQP2 more avidly than to nonphosphorylated AQP2. Immunogold EM studies demonstrated that BiP is present not only in the ER but also in the cytoplasm and apical plasma membrane of rat collecting duct cells. Furthermore, confocal immunofluorescence studies showed partial colocalization of BiP with AQP2 in non-ER compartments. These results suggest that phosphorylation of AQP2 at Ser-256 may regulate AQP2 trafficking in part by mediating differential binding of hsp70 family proteins to the COOH-terminal tail.