The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas

Persistent CMV infection after allogeneic hematopoietic stem cell transplantation in a CMV-seronegative donor-to-positive recipient constellation: Development of multidrug resistance in the absence of anti-viral cellular immunity

We describe a case of persistent cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with discordant and high-risk (D-/R+) constellation of CMV serostatus. Despite the use of different and innovative antiviral strategies, viral replication could not be suppressed successfully promoting a protracted CMV colitis associated with severe gastrointestinal graft-versus-host disease (GI GVHD). We illustrate that the development of multidrug viral resistance, the failure to mount a CMV-specific cellular immune response, as confirmed by QuantiFERON (R)-CMV (Qiagen) assay, and the refractory GVHD requiring prolonged immunosuppression were the main factors contributing to persistent viral replication and the fulminant unfavorable course. (C) 2015 Elsevier B.V. All rights reserved

Galectin-3 prevents TCR synapse formation in late-stage T cells.

<p>(A) CD7⁻ and CD7⁺ T cells were activated by incubation with anti-CD3 mAb plus anti-human CD28 mAb or as control by an isotype-matched IgG1 (medium). Cells were stained for TCR-alpha/beta (green), CD7 (blue), CD3 (red) and for galectin-3 (yellow). Immunofluorescence was visualized by a LSM. Alternatively cells were stained with mAbs specific to CD7 and subsequently recorded for gal-3 expression by staining with the anti-human gal-3mAb or as control by an isotype-matched IgG and analyzed by flow cytometry. A representative experiment out of five is shown. Galectin-3 specific signals were quantitatively recorded by a LSM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells for each data point was recorded. (B) To monitor location of galectin-3 in lipid raft formation, isolated CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine for 24 hrs and then activated for various time intervals (1 min until 20 min) on coverslips coated with the agonistic anti-CD3 mAb plus anti-CD28 mAb. T cell stimulation was stopped with paraformaldehyde and cells were stained with anti-TCR-alpha/betamAb (blue), anti-CD7 mAb (red) and anti-galectin-3 mAb (yellow) together with CtxB (green) for lipid rafts staining. Immunofluorescence was visualized by a LSM and quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. T cell staining after 5 min stimulation of one representative experiment out of five is exemplarily shown. (C) CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine and activated as described in (B). Frequencies of cells producing IFN-gamma and showing degranulation indicated by CD107a was monitored by flow cytometry. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#pone-0030713-g003" target="_blank">Fig. 3</a> represent the mean ± SEM of five experiments and were compared using a paired t-test. * p<0.05. FU: fluorescence unit; MFI: mean fluorescence intensity.</p

TCR synapse formation is impaired in late-stage T cells.

<p>CD8⁺ CD45RO⁺ T lymphocytes were incubated on coverslips coated with the agonistic anti-CD3 mAb (UCHT-1) plus anti-CD28 mAb (CD28.2) or with the anti-CD2 mAbs (L303.1) and (L304.1) plus the anti-CD28 mAb (CD28.2). Cells were activated for various time intervals (1 min until 20 min), incubation was stopped by addition of paraformaldehyde, and cells were stained with TCR-alpha/betamAb (green) plus CD7 mAb (blue) or alternatively with CD2 mAb (blue) plus CD7 mAb (green) together with cholera toxin B (CtxB) (white) for lipid raft staining. Cells were analyzed on a LSM 510 with 630× microscope magnification. A minimum of 100 cells for each data point was recorded. Representative images after 0 min, 1 min, 5 min, 10 min and 20 min stimulation out of five independent experiments are shown. Synapse formation intensity (CTxB intensity) was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells of each cell population on each coverslip was recorded. Data represent mean scores from five experiments ± standard error of the mean and were compared using a paired t-test. * p<0.05. FU: fluorescence unit.</p

Dynamics of Torque Teno virus viremia could predict risk of complications after allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established treatment option for several hematological diseases. However, the first year post-transplantation is often complicated by infections and graft-versus-host disease (GVHD). Improvements in immunological monitoring could reduce such post-transplant complications. Torque Teno virus (TTV), a chronically persisting DNA virus, is reported to be a marker for immune function in immunocompromised patients. In the present study, the TTV kinetics were analyzed to investigate the potential role of TTV viremia as immune-competence read-out after allo-HSCT. Twenty-three monocentric allo-HSCT recipients were retrospectively tested for TTV-DNA in whole blood at given day post-transplant. Dynamics of TTV viremia was analyzed with respect to episodes of non-TTV viral reactivations (CMV, EBV, and BKPyV), acute GVHD, and recovery of immune cells. Recipients affected by persisting viral infections and/or GVHD during the first 100 days after allo-HSCT showed a significantly higher median TTV load at day +30 than patients with a less complicated clinical course (p = 0.005). This was also associated with a total lymphocyte count <5.5E+08 cells/L in this high-risk group (p = 0.039). These findings suggest that TTV could represent an additional parameter to identify patients at higher risk for complications in the first 100 days following allo-HSCT. Prospective studies, including the monitoring of lymphocyte subsets, are required to define the potential use of TTV in immunological monitoring after allo-HSCT