I. J. M. Ten Berge

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.

Activation of resting T cells has been proposed to purge the reservoir of HIV-1-infected resting CD4+ T cells. We therefore treated three HIV-1-infected patients on antiretroviral therapy with OKT3, a CD3 monoclonal antibody, and recombinant human IL-2. Here we report the profound and partially long-lasting host responses induced by the OKT3 and IL-2 treatment. OKT3/IL-2 induced a strong but transient release of plasma cytokines and chemokines. The percentage CD4+ and CD8+ cells in the blood expressing the activation marker CD38 transiently increased to almost 100%, and in lymph nodes we “observed” a 10-fold increase in the number of dividing Ki67+ cells and increased numbers of apoptotic cells. Following OKT3/IL-2 treatment, a long-lasting depletion of CD4+ cells in the peripheral blood and lymph nodes occurred, suggesting the physical deletion of these cells. Increases in CD4+T cell numbers during the two year followup period were due mainly to increased memory cell numbers. CD8+ cells were also depleted in the blood, but less severely in lymph nodes, and returned to baseline levels within several weeks.

Meningococcal Sepsis Complicating Eculizumab Treatment Despite Prior Vaccination

Recently, the monoclonal antibody against complementfactor C5, eculizumab, was successfully applied in the treatment of recurrent atypical hemolytic-uremic syndrome (aHUS) in renal transplant recipients (RTR) (1). Guidelines for its use include meningococcal vaccination prior to treatment. Late complement–pathway– component deficiencies predispose to meningococcal infections by the absence of meningococcal lysis via classical and alternative pathways (2). Consequently, protection against meningococcal disease by antibody-mediated killing becomes essential. However, vaccination of patients using immunosuppressive drugs may be ineffective (3,4). Different immunosuppressive regimens vary in their effects on humoral responses after vaccination. Previously, we demonstrated that RTR treated with prednisolone and everolimus mount adequate humoral vaccination responses, measured by ELISA, against immunocyanin, tetanus-toxoid (TT) and pneumococcal polysaccharide (PPS). In contrast, treatment with mycophenolic mofetil (MMF) and prednisolone completely disturbed vaccination responses against these same antigens (3). Although immune responses after vaccination are generally used as markers of efficacy of vaccination, they are not synonymous with protection. Furthermore, in immunocompromised patients vaccination-induced responses may wane rapidly. Protection provided by vaccines after renal transplantation may be limited in quality and/or duration. This is illustrated by the following case.

Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years

CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.

Donor feces infusion for eradication of Extended Spectrum beta-Lactamase producing Escherichia coli in a patient with end stage renal disease.

In an attempt to decolonise the patient from the ESBL producing E. coli, he underwent donor feces infusion in May 2013. Prior to this intervention, the presence of ESBL producing E. coli in the large intestine was again confirmed by a positive rectal culture. In addition his throat and perineum were also screened for the presence of ESBL producing Enterobacteriaceae, but both were negative. Culture of the urine was not possible because of the anuric condition of the patient. The donor feces infusion was performed according to the protocol as used in the FECAL trial [1]. In summary, donor feces were obtained from a young healthy Caucasian adult, who was periodically screened for various infectious and gastro-intestinal diseases. Feces from the donor were collected and processed within 6 h after production. First, the feces were diluted with sterile saline, and then poured through unfolded gauze in a funnel, in order to obtain a solution which was free of debris and solid particles. This solution was immediately infused in the patient through a nasoduodenal tube. Donor feces infusion was preceded by full colon lavage without prior use of antibiotics. Within the first 2 days after donor feces infusion the patient experienced mild diarrhoea and abdominal cramps, but no other adverse events occurred. Follow-up ESBL swab cultures of the rectum, perineum and throat were taken at week one, two, four, and twelve after the donor feces infusion. During these four follow-up time points the ESBL cultures of the

A single dose of rituximab does not deplete B cells in secondary lymphoid organs but alters phenotype and function

A single dose of the anti‐CD20 monoclonal antibody rituximab induces a nearly complete B cell depletion in peripheral blood, but not in secondary lymphoid organs. Modulation of this remaining B cell population due to rituximab treatment may contribute to the therapeutic effects of rituximab. To assess the in vivo effects of rituximab we used lymph nodes (LNs) collected during renal transplant surgery in patients who had received rituximab 4 weeks earlier in preparation for an ABO‐incompatible transplantation. Rituximab treatment resulted in a lower percentage of naïve (IgD+CD27−) and a higher percentage of switched memory (IgD−CD27+) B cells. Remarkably, transitional (CD24++CD38++) B cells were virtually lacking in the LNs of rituximab‐treated patients. Moreover, LN‐derived B cells from rituximab‐treated patients produced different amounts of various Ig‐subclasses after anti‐CD40/IL‐21 stimulation ex vivo. Finally, after stimulation of allogeneic T cells with LN‐derived B cells from rituximab‐treated patients, the proliferated T cells showed a decreased production of IL‐17. In conclusion, after treatment with rituximab there remains a B cell population with different functional capacities. Consequently, the effect of rituximab on the immune response will not only be determined by the extent of B cell depletion, but also by the functional properties of the remaining B cells.

Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection

BACKGROUND Graft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC. METHODS Expression of perforin, GrB and FasL was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies. Specific inhibitors of the perforin/GrB- and FasL-based pathways were used in 51Cr release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines. RESULTS Cells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-FasL polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC. Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas. CONCLUSIONS The perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.

Quantification of Bax/Bcl-2 ratios in peripheral blood lymphocytes, monocytes and granulocytes and their relation to susceptibility to anti-Fas (anti-CD95)-induced apoptosis

Neutrophils have the shortest half‐life among circulating leucocytes and rapidly undergo apoptosis in vitro. The homologous Bcl‐2 and Bax proteins have opposing effects, with Bcl‐2 extending cellular survival and Bax promoting cell death following an apoptotic stimulus. We determined Bcl‐2 to Bax expression ratios in peripheral blood lymphocytes, monocytes and granulocytes and related them to the susceptibility of these cells to anti‐Fas (anti‐CD95)‐induced apoptosis. Here, we show that Bax/Bcl‐2 ratios are high in granulocytes and relatively low in monocytes and lymphocytes. Furthermore, we show a relation between this ratio in the different leucocyte subsets and their susceptibility to anti‐Fas‐induced apoptosis, with granulocytes showing the highest susceptibility, followed by monocytes and lymphocytes. It is concluded that the balance between Bcl‐2 and Bax forms an apoptotic rheostat, which seems to determine sensitivity to apoptosis.

T-lymphocyte subset distribution in human spleen

Background  When analyzing human cellular immune responses, most focus is placed on the peripheral blood (PB) and, to a lesser extent, the lymph nodes. To date the spleen has not been analyzed with regard to its role in adaptive cellular immunity and more notably not with respect to T‐cell immune responses.

Cytomegalovirus infection in solid organ transplant recipients.

Cytomegalovirus (CMV) is the most frequent infectious complication following solid organ transplantation (SOT). The virus, is responsible for both direct (viral syndrome, hepatitis, pneumonitis, colitis, etc.) and indirect effects (rejection, infections by other microorganisms and graft dysfunction). In this evidence-based guideline we deal with the most important aspects of CMV infection in SOT recipients, including pre- and post-transplant diagnosis assessment and risk factors, with special emphasis on the prevention and treatment of this viral infection. Overall, adequate management of CMV infection is a critical aspect of transplant patient care.

High-affinity cytotoxic T lymphocytes after non-HLA-sharing blood transfusion--the other side of the coin.

Previously, we have shown that pretransplantation blood transfusion modulates the T cell repertoire to a great extent. Patients receiving a BT from a donor sharing one HLA haplotype with the patient (HLA-sharing BT) develop CTL nonresponsiveness against cells of the BT donor and show a selective decrease in the usage of T cell receptor V beta families. The present study has focused on the analysis of the T cell repertoire in patients receiving an HLA mismatched (non-HLA-sharing) BT. CTL precursor frequencies were measured against single class I-mismatched antigens in split-well analysis. In addition, blocking studies of CTL-target cell interaction were performed with anti-CD8 monoclonal antibodies. The results demonstrate that non-HLA-sharing BT immunizes and induces the generation of CD8 independent, high-affinity CTL against immunogenic class I-mismatched antigens. Such HLA class I antigens might become nonacceptable mismatches in subsequent organ transplantation.