Lorea Beloki

Isolation of highly suppressive CD25+FoxP3+ T regulatory cells from G-CSF-mobilized donors with retention of cytotoxic anti-viral CTLs: application for multi-functional immunotherapy post stem cell transplantation.

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.

Manufacturing of highly functional and specific T cells for adoptive immunotherapy against virus from granulocyte colony-stimulating factor–mobilized donors

BACKGROUND AIMS Cytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF-mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs. METHODS CMV-T were isolated from G-CSF-mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation. RESULTS We successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF-mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer. CONCLUSIONS We confirm that the use of an aliquot from G-CSF-mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors.

Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8(+) T cells by flow cytometry.

Multimer technology is widely used to screen antigen‐specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo‐HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus‐specific T‐cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen‐specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice.

Impact of T cell selection methods in the success of clinical adoptive immunotherapy.

Chemotherapy and/or radiotherapy regular regimens used for conditioning of recipients of hematopoietic stem cell transplantation (SCT) induce a period of transient profound immunosuppression. The onset of a competent immunological response, such as the appearance of viral-specific T cells, is associated with a lower incidence of viral infections after haematopoietic transplantation. The rapid development of immunodominant peptide virus screening together with advances in the design of genetic and non-genetic viral- and tumoural-specific cellular selection strategies have opened new strategies for cellular immunotherapy in oncologic recipients who are highly sensitive to viral infections. However, the rapid development of cellular immunotherapy in SCT has disclosed the role of the T cell selection method in the modulation of functional cell activity and of in vivo secondary effects triggered following immunotherapy.

The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by Multimer Technology in Adoptive Immunotherapy

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood

BackgroundAdoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional.MethodsMHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers.ResultsCMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products.ConclusionsThe translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.

Generation of memory T cells for adoptive transfer using clinical-grade anti-CD62L magnetic beads.

Pre-clinical studies of allogeneic stem cell transplantation suggest that depletion of naive T cells from donor lymphocytes will reduce the risk of GvHD but preserve immunity to infectious pathogens. In this study, we have established a clinical-grade protocol under good manufacturing practice conditions for purging CD62L+ naive T cells from steady-state leukapheresis products using the CliniMACS system. The efficacy of immunomagnetic CD62L depletion was assessed by analysis of cell composition and functional immune responses. A median 2.9 log CD62L depletion was achieved with no evidence of CD62L shedding during the procedure and a mean T-cell yield of 47%. CD62L− cells comprised an equal mix of CD4+ and CD8+ T cells, with elimination of B cells but maintenance of regulatory T cells and natural killer cell populations. CD62L-depleted T cells were predominantly CD45RA− and CD45RA+ effector memory (>90%) and contained the bulk of pentamer-staining antivirus-specific T cells. Functional assessment of CD62L− cells revealed the maintenance of antiviral T-cell reactivity and a reduction in the alloreactive immune response compared with unmanipulated cells. Clinical-grade depletion of naive T cells using immunomagnetic CD62L beads from steady-state leukapheresis products is highly efficient and generates cells suitable for adoptive transfer in the context of clinical trials.

Functional specific-T-cell expansion after first cytomegalovirus reactivation predicts viremia control in allogeneic hematopoietic stem cell transplant recipients

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo‐HSCT) recipients might result in over‐treatment, inducing drug‐related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow‐up, in combination with the use of streptamers (STs) and cytokine‐intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV‐specific CD8+ T‐cell (CMV‐CTL) populations, we monitored 25 patients who have undergone allo‐HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV‐specific T cells, determined by early interferon γ production or by significant T‐cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV‐CTLs does correlate with CMV recurrence. These results support that behavior of CMV‐specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers

BackgroundCytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product.MethodsIn the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples.ResultsAfter the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved.ConclusionsG-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.