Eva Distler

Rapid identification and sorting of viable virus-reactive CD4+ and CD8+ T cells based on antigen-triggered CD137 expression

Current methods for the detection and isolation of antigen-specific CD4(+) and CD8(+) T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4(+) and CD8(+) memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137(+) T cells did not produce IFN-gamma, suggesting that CD137 detects a broader repertoire of antigen-specific T cells. Activated CD137(+) T cells could be easily purified by MACS and expanded in vitro thereafter. This CD137-based enrichment method was capable of isolating 2-fold higher numbers of anti-viral CD4(+) and CD8(+) T cells compared to the IFN-gamma secretion assay. In conclusion, antigen-triggered CD137 expression allows the rapid detection and sorting of virus-reactive CD4(+) and CD8(+) T cells. The CD137 assay is most attractive for the simultaneous targeting of anti-viral T helper and effector cells in monitoring studies and adoptive immunotherapy trials.

Alloreactive and leukemia-reactive T cells are preferentially derived from naïve precursors in healthy donors: implications for immunotherapy with memory T cells

Background HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens. Design and Methods We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation. Results We observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RAneg memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells. Conclusions Memory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versus-host disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.

IL-21-treated naive CD45RA+ CD8+ T cells represent a reliable source for producing leukemia-reactive cytotoxic T lymphocytes with high proliferative potential and early differentiation phenotype.

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8+ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA+ CD8+ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8+ CTLs from naive CD45RA+ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8+ CD62L(high)+ T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rγnull mice

OBJECTIVE Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. MATERIALS AND METHODS We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively. RESULTS In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice. CONCLUSION The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.

Recovery of varicella-zoster virus-specific T cell immunity after T cell-depleted allogeneic transplantation requires symptomatic virus reactivation.

Reactivated varicella-zoster virus (VZV) infection causes herpes zoster and commonly occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because VZV-specific T cell immunity is essential to prevent virus reactivation, we developed an interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assay for the sensitive detection of VZV-reactive T cells at the single-cell level ex vivo. We used this assay to monitor the frequency of VZV-reactive T cells in 17 seropositive patients during the first year after T cell-depleted allo-HSCT. The patients did not receive anti-herpesvirus prophylaxis after stem cell engraftment. Independent of the magnitude of transferred donor immunity, VZV-reactive T cell numbers decreased to low levels (median, 2/mL; range, 0 to 35/mL) in peripheral blood early after transplantation. Only patients with subsequent zoster (n = 5) exhibited a dramatic boost in VZV-reactive T cells (median, 366/mL; range, 158 to 756/mL), which was induced by the reactivation event. The postzoster VZV-reactive T cell levels were similar to those seen in healthy virus carriers. In contrast, antiviral T cell levels remained low in patients without VZV disease. Our results demonstrate that VZV-specific T cell immunity recovered efficiently during zoster in T cell-depleted allo-HSCT recipients. It did not reconstitute spontaneously in nonzoster patients, even in the absence of antiviral prophylaxis. Prospective studies should investigate whether VZV vaccination can substitute for natural resensitization by virus disease.

Preservation of dendritic cell function upon labeling with amino functionalized polymeric nanoparticles

Dendritic cells (DCs) are key players in eliciting immunity against antigens, therefore making them the focus of many investigations on immune responses in infections, cancer and autoimmune diseases. Nanosized materials have just recently been investigated for their use as carriers of antigens and as labeling agents for DCs. For this later use nanoparticles should be non-toxic and should most importantly not alter the physiological functions of DCs. Here we demonstrate that by the use of polymeric fluorescent nanoparticles as synthesized by the miniemulsion process immature DCs (iDCs) can be efficiently labeled intracellularly. Amino functionalized nanoparticles are more effective than carboxy functionalized ones. Even after 8 days 95% of DCs have retained nanoparticles with a fluorescence intensity of 67% compared to day 1. Nanoparticle labeling does not influence expression of cell surface molecules on mature DCs (mDCs) like HLA-DR, CD80/83/86, CCR7, CD11c nor does it influence the immunostimulatory capacity of mDCs. This procedure does also not impair the capability of DCs for uptake, processing and presentation of viral antigens as demonstrated by interferon-gamma ELISPOT on T cells stimulated with viral antigens presented by DCs. Therefore polymeric nanoparticles are a promising tool to study migration and homing of DCs in animal studies.

Selective depletion of alloreactive T lymphocytes using patient-derived nonhematopoietic stimulator cells in allograft engineering.

Background. Selective depletion of alloreactive T cells in vitro results in efficient graft-versus-host disease prophylaxis in allogeneic hematopoietic stem-cell transplantation, but it is accompanied by increased recurrence of leukemia. To spare donor T-cell-mediated graft-versus-leukemia immunity against hematopoiesis-restricted minor histocompatibility (minor-H) antigens, we explored the use of patient-derived nonhematopoietic antigen-presenting cells (APC) as allogeneic stimulators for selective allodepletion in leukemia-reactive donor T-cell lines. Methods. Primary keratinocytes, dermal fibroblasts, and bone marrow fibroblasts were generated from skin biopsies and diagnostic bone marrow aspirates of acute myeloid leukemia patients in vitro. Cell cultures were analyzed for expansion, phenotype, and immunostimulatory capacity in comparison with CD40-activated B cells as professional APC. In addition, nonhematopoietic APCs were used for selective allodepletion in vitro. Results. Patient-derived fibroblasts could be reliably expanded to large cell numbers, whereas keratinocytes had limited growth potential. Interferon-&ggr;-pretreated fibroblasts showed increased expression of human leukocyte antigen (HLA)-class I and II molecules, CD40, and CD54. Fibroblasts and CD40-activated B cells comparably stimulated HLA-A*0301-specific CD8+ T cells after transient expression of HLA-A*0301 as a model alloantigen. Finally, fibroblasts could be effectively applied to selectively deplete alloreactivity within leukemia-reactive donor CD8+ T-cell lines by targeting the activation-induced antigen CD137. Conclusions. Primary fibroblasts can be efficiently used as allogeneic nonhematopoietic APC for selective depletion of donor T cells reactive to HLA and ubiquitously expressed minor-H antigen disparities in leukemia-stimulated CD8+ T-cell lines. Therefore, harnessing alloreactivity to hematopoietic minor-H antigens in addition to leukemia-associated antigens might increase graft-versus-leukemia immunity of donor lymphocyte grafts in allogeneic hematopoietic stem-cell transplantation.

In Vitro Stimulation and Expansion of Human Tumour‐Reactive CD8+ Cytotoxic T Lymphocytes by Anti‐CD3/CD28/CD137 Magnetic Beads

Adoptive immunotherapy with tumour‐reactive CD8+ cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen‐independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour‐reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4‐1BB). T cells in PBMCs showed an increased expansion rate of 15‐ to 17‐fold during a 2‐week culture period using antibody‐conjugated beads with interleukin‐2 (IL‐2) added versus IL‐2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour‐reactive CD8+ CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4‐fold ±1.2) compared with CD3/CD28 beads (10.6‐fold ±0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead‐activated cultures expressed consistently higher levels than tumour‐stimulated cultures. CTLs analysed after bead‐induced expansion versus weekly tumour stimulation showed equal IFN‐γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen‐independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti‐CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour‐reactive CD8+ CTLs.

Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation

Background After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. Design and Methods Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4+ and CD8+ memory T cells. Results Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4+ T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4+ and CD8+ T cells. Conclusions Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4+ and CD8+ T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.

Safety and Immunogenicity of a Vero Cell Culture-Derived Whole-Virus H5N1 Influenza Vaccine in Chronically Ill and Immunocompromised Patients

ABSTRACT The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ≥1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ≥1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at ClinicalTrials.gov under registration no. NCT00711295.)