Tool For Immune Monitoring Research Articles

Preceeding the rejection: In search for a comprehensive post-transplant immune monitoring platform

The survival of a transplanted organ is dependent on maintenance of continuous immunosuppression. However, even the strictest adherence to the recommended drug levels does not prevent the occurrence of numerous complications associated with immunosuppression. The efficacy of immunosuppression therapy protocols would be enhanced greatly by the availability of biotechnologies capable of identifying and predicting immunological events prior to the manifestation of clinical parameters indicating graft failure. The aim of the study was to evaluate the potential contribution of some modern tools for post-transplantation monitoring, and to propose a method for combining them into a comprehensive mechanism for this purpose. The technologies utilized in this study are among a group of ‘cutting edge’ diagnostic methods at the initial steps of evaluation for their potential contribution for post-transplantation immune monitoring. This study was a pioneering opportunity to combine and utilize these tools jointly. The method of research was based on monitoring 13 adult kidney transplant recipients. The Immuknow assay determined cellular immunity status by quantitative measurement of intracellular ATP level in CD4 + lymphocytes after PHA stimulation. Sera were analyzed for concentration of soluble CD30 reflecting primary allo-stimulation and for donor specific anti-HLA antibodies responsible for accelerated and refractory rejection. The results were correlated with clinical and pathological parameters and appraisal of predictive value was attempted. In Immuknow assay analysis ATP incremental changes indicative of rejection or infection were found in 75% and in 50% incidences, respectively. In stable patients, the ATP deviation from the preoperative baseline, indicative of stable engraftment, was much less pronounced than in other habitual clinical tests. CD30 concentrations were measured greatly above normal values prior to biopsy-proven rejection episodes, both before and after the transplant operation. Anti-HLA antibodies were elevated at a later stage, concurrently with clinical manifestation of graft failure and rejection. Anti-HLA antibody level remained negligible in patients going through a stable post-transplant clinical course. Overall, the utilization of the platform of combined biotechnologies could serve as a valuable tool for immune monitoring in organ transplantation, allowing for therapeutic intervention that can favorably affect the clinical outcome.

Decrease and dysfunction of dendritic cells correlate with impaired hepatitis C virus‐specific CD4+ T‐cell proliferation in patients with hepatitis C virus infection

Through the production of stimulatory and suppressive cytokines, dendritic cells (DCs) regulate virus-specific immune responses that are crucial to virus eradication. To explore a possible role of DCs in the persistence of hepatitis C virus (HCV) infection, in this study we analysed peripheral blood DCs (PBDCs) in patients with chronic hepatitis C (CHC) compared with those in both healthy seronegative (HSN) controls and a group of subjects who had spontaneously resolved infection, defined as healthy HCV-seropositive (HSP), and we evaluated the relationships between PBDCs and HCV-specific CD4(+) T-cell reactivity. The number of PBDCs, their immunophenotype and expression of regulatory cytokines were evaluated by flow cytometry on whole-blood samples. HCV-specific CD4(+) T-cell activation, proliferation and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV peptides. We found that PBDCs from CHC subjects were numerically reduced and showed lower interleukin-12 (IL-12) and higher IL-10 expression than those from HSN controls. PBDCs from HSP subjects were similar to those from HSN controls. HCV-specific CD4(+) T-cell proliferation was less frequent and vigorous in CHC than in HSP patients and was directly related to the number of PBDCs and their IL-12 production but inversely related to their IL-10 production. Taken together, these results seem to suggest that cytokines of DC origin contribute to the regulation of HCV-specific immunity in CHC patients and indicate that PBDCs may represent a novel non-invasive tool for immune monitoring of these patients.

A Novel Flow Assay for the Detection of Cytokine Secreting Alloreactive T Cells: Application to Immune Monitoring

The direct and indirect allorecognition pathways play an important role in graft rejection. We hypothesized that the presence of alloreactive memory T cells in the recipient’s circulation increases the risk of rejection after transplantation. The objective of this study was to develop a noninvasive, immune monitoring tool that simultaneously measures donor-specific responses via both the direct and indirect recognition pathways. Our laboratory developed a whole blood flow cytometric cytokine secretion assay to identify interferon (IFN)-γ secreting memory T cells in whole blood of renal transplant patients. The assay readily detected IFN-γ producing CD3+ T cells in response to recall antigens tetanus toxoid, purified protein derivative, and alloantigens in whole blood from healthy controls. Analysis of sequential posttransplant blood samples from 19 renal allograft recipients showed that alloimmune responses were higher in transplant recipients who had undergone acute rejection than in those without acute rejection episodes. In addition, patients showing increased creatinine levels 3 months after transplantation were more likely to exhibit alloimmune responses than recipients with stable graft function. The flow cytokine secretion assay provides a reliable and simple method for identification of patients at risk of acute rejection and early graft dysfunction.

A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs.Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease.

Monitoring of CD4+ and CD8+ T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis

CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7-53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.

Use of cytokine enzyme-linked immunosorbent spot (ELISPOT) assay as an immune monitoring tool in solid organ transplantation

The development of surrogate markers to predict outcome and guide therapeutic interventions after solid organ transplantation is sorely needed by the transplant community. The cytokine enzyme-linked immunosorbent spot (ELISPOT) assay is one such candidate marker that can accurately detect the frequency and cytokine profile of peripheral alloantigen-reactive T lymphocytes in human allograft recipients at high resolution. Published studies provide strongly suggestive evidence that the frequency of donor-reactive interferon γ ELISPOTs in the the peripheral blood of renal transplant recipients directly correlates with the future risk of acute rejection and chronic graft injury. The assay may additionally have utility for directing minimization of toxic immunosuppressants and identifying patients tolerant to their allografts. It is envisioned that ELISPOT-based immune monitoring, complemented by use of several additional surrogate markers, will ultimately provide clinicians with necessary tools to optimize therapy so as to prolong transplant survival with minimal toxicity.

Flow cytometric detection of alloreactive T cells: tool for immune monitoring

Flow cytometric detection of alloreactive T cells: tool for immune monitoring

Evolution of the Enzyme-Linked Immunosorbent Spot Assay for Post-Transplant Alloreactivity as a Potentially Useful Immune Monitoring Tool

Post-transplant monitoring of cellular immunity has the potential to guide alterations in medical therapy. To this end, our laboratory has developed an enzyme-linked immunosorbent spot (ELISPOT) assay for detection of peripheral blood alloimmunity. Peripheral blood lymphocytes (PBLs) from normal volunteers and from renal allograft recipients were tested against donor stimulator cells for their ability to respond in 'one-way' cytokine ELISPOT assays. T cell depletion of donor spleen or PBLs eliminated donor cell cytokine secretion while preserving the ability of these cells to present allo-antigen to responding T cells. Alloreactive IFN-gamma-producing PBLs derive from the memory T cell pool and are readily detectable in recipients of renal allografts taking immunosuppressant medications. A significant expansion of IFN-gamma-producing donor-reactive memory PBLs was detectable at 4-6 months post-transplant in those who had experienced an acute rejection episode compared with those with a stable post-transplant course. The data demonstrate the feasibility of repeated post-transplant monitoring of allograft recipients, and provide the foundation for improving the care of human transplant recipients through rational clinical decision-making based on measures of immune function.

Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.

The Society for Biological Therapy held a Workshop last fall devoted to immune monitoring for cancer immunotherapy trials. Participants included members of the academic and pharmaceutical communities as well as the National Cancer Institute and the Food and Drug Administration. Discussion focused on the relative merits and appropriate use of various immune monitoring tools. Six breakout groups dealt with assays of T-cell function, serologic and proliferation assays to assess B cell and T helper cell activity, and enzyme-linked immunospot assay, tetramer, cytokine flow cytometry, and reverse transcription polymerase chain reaction assays of T-cell immunity. General conclusions included: (1) future vaccine studies should be designed to determine whether T-cell dysfunction (tumor-specific and nonspecific) correlated with clinical outcome; (2) tetramer-based assays yield quantitative but not functional data (3) enzyme-linked immunospot assays have the lowest limit of detection (4) cytokine flow cytometry have a higher limit of detection than enzyme-linked immunospot assay, but offer the advantages of speed and the ability to identify subsets of reactive cells; (5) antibody tests are simple and accurate and should be incorporated to a greater extent in monitoring plans; (6) proliferation assays are imprecise and should not be emphasized in future studies; (7) the reverse transcription polymerase chain reaction assay is a promising research approach that is not ready for widespread application; and (8)there is a critical need to validate these assays as surrogates for vaccine potency and clinical effect. Current data and opinion support the use of a functional assay like the enzyme-linked immunospot assay or cytokine flow cytometry in combination with a quantitative assay like tetramers for immune monitoring. At present, assays appear to be most useful as measures of vaccine potency. Careful immune monitoring in association with larger scale clinical trials ultimately may enable the correlation of monitoring results with clinical benefit.