• Cell therapy
• FAIR data
• Metadata sharing
• Minimum information model
• Open data sharing
• Reporting guidelines
• Standardised data reporting
• Tolerogenic dendritic cell

• autoimmune disease
• disease endotypes
• immune intervention
• immune monitoring
• immunotherapy
• mechanism of action
• precision medicine
• type 1 diabetes

• Cellular immunity
• SARS-CoV-2
• T-cells
• qPCR

• P30 CA196521 NCI NIH HHS
• T32 CA078207 NCI NIH HHS

• Cancer
• Lymphocytes
• Prognosis
• Tumor-infiltrating cells

• Breast Neoplasms
• Colorectal Neoplasms
• Female
• Humans
• Lymphocytes, Tumor-Infiltrating
• Prognosis
• Tumor Microenvironment/genetics

• R01 CA168628 NCI NIH HHS
• R01 CA256068 NCI NIH HHS
• U01 DE029759 NIDCR NIH HHS

• Gastric cancer
• Peripheral lymphocyte count
• Prognosis
• Recurrence

• Aged
• Gastrectomy/adverse effects
• Humans
• Lymphocyte Count
• Neoplasm Recurrence, Local
• Prognosis
• Retrospective Studies
• Stomach Neoplasms/surgery

• Clinical trials
• EBV
• HCMV
• Immune monitoring
• Recall antigens
• T cells

• Cancer
• Immunology
• Immunomonitoring
• MHC class I
• MUC1
• Tumor antigen

The purpose of this study was to evaluate if an early exposure to human papillomavirus (HPV) during the prenatal period or infancy could result in HPV16-specific T helper (Th) responses resembling those of adults with HPV-induced lesions. We tested HPV16-specific cell-mediated immunity (CMI) in children born with HPV-positive umbilical cord blood and/or placenta or having persistent oral HPV infection and in constantly oral HPV-negative controls. Peripheral blood mononuclear cells from 33 children from the Finnish HPV Family Study cohort (mean age 14.7 years) were stimulated with peptide pools covering the amino acid sequence of the HPV16 E2, E6, and E7 proteins. Lymphocyte proliferation, secretion of cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, IL-17A), and the frequency of Foxp3+ regulatory T-cells were determined in relation to the HPV DNA status during a 14-year follow-up. 73.6% of cases and 85.7% of controls responded against HPV16 E2, while reactivity against E6 was found in 10.5 and 35.7%, respectively. The proliferative response against E6 and E7 was more frequent in controls than in cases (p = 0.047). No HPV16-specific CMI response or antibodies were detected in two children with persistent oral HPV16. The profiles of induced cytokines indicated higher levels of IL-5, IL-10, and IL-17A in children with HPV DNA in placenta and/or cord blood than in other children. HPV16-specific CMI is common in HPV DNA-negative children. The cytokine profile in children infected with HPV16 during early life suggests that the viral dose and/or specific environment created by the placenta may have significant impact on the type of HPV-specific immunity.

• Adolescent
• Antigens, Viral/immunology
• Cell Proliferation
• Child
• Child, Preschool
• Cytokines/metabolism
• Female
• Fetal Blood/virology
• Finland
• Follow-Up Studies
• Human papillomavirus 16/immunology
• Human papillomavirus 16/isolation & purification
• Humans
• Infant
• Infant, Newborn
• Longitudinal Studies
• Male
• Maternal-Fetal Exchange
• Mouth/virology
• Placenta/virology
• Pregnancy
• Th2 Cells/immunology

• Suomen Hammaslääkäriseura Apollonia
• Lounais-Suomen Syöpäyhdistys
• Suomen Akatemia
• Syöpäsäätiö
• Sohlberg Foundation
• the Government Special Foundation (EVO)
• Ida Montin Foundation
• Finnish-Norwegian Medicine Foundation
• Finnish National Graduate School

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question.

Although numerous candidate biomarkers have been described, there are currently three FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated to identify patients who are more likely to benefit from a single-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurate prediction of clinical benefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, we discuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.

• Assay
• Biomarker
• Cancer
• Immunotherapy
• Standardization
• Validation

Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

• Antigen-presenting cells
• Autoimmune disease
• Cell therapy
• Minimum information model
• Regulatory macrophages
• Reporting guidelines
• Tolerogenic dendritic cells
• Transplantation

The FluoroSpot assay, an advancement of the ELISpot assay, enables simultaneous measurement of different analytes secreted at a single-cell level. This allows parallel detection of several cytokines secreted by immune cells upon antigen recognition. Easier standardization, higher sensitivity and reduced labour intensity render FluoroSpot assays an interesting alternative to flow-cytometry based assays for analysis of clinical samples. While the use of immunoassays to study immunological primary and secondary endpoints becomes increasingly attractive, assays used require pre-trial validation. Here we describe the assay validation (precision, specificity and linearity) of a FluoroSpot immunological endpoint assay detecting Interferon γ (IFNγ) and Interleukin 2 (IL2) for use in clinical trial immune monitoring.

We validated an IFNγ/IL2 FluoroSpot assay to determine Epstein-Barr virus (EBV)-specific cellular immune responses (IFNγ, IL2 and double positive IFNγ + IL2 responses), using overlapping peptide pools corresponding to EBV-proteins BZLF1 and EBNA3A. Assay validation was performed using cryopreserved PBMC of 16 EBV-seropositive and 6 EBV-seronegative donors. Precision was assessed by (i) testing 16 donors using three replicates per assay (intra-assay precision/repeatability) (ii) using two plates in parallel (intermediate precision/plate-to-plate variability) and (iii) by performing the assays on three different days (inter-assay precision/reproducibility). In addition, we determined specificity, linearity and quantification limits of the assay. Further we tested precision across the two assay systems, IFNγ/IL2 FluoroSpot and the corresponding enzymatic single cytokine ELISpot.

The validation revealed: (1) a high intra-assay precision (coefficient of variation (CV) 9.96, 8.85 and 13.05 %), intermediate precision (CV 6.48, 10.20 and 12.97 %) and reproducibility (CV 20.81 %, 12,75 % and 12.07 %) depending on the analyte and antigen used; (2) a specificity of 100 %; (3) a linearity with R² values from 0.93 to 0.99 depending on the analyte. The testing of the precision across the two assay systems, adduced a concordance correlation coefficient p_c = 0.99 for IFNγ responses and p_c = 0.93 for IL2 responses, indicating a large agreement between both assay methods.

The validated primary endpoint assay, an EBV peptide pool specific IFNγ/IL2 FluoroSpot assay was found to be suitable for the detection of EBV-specific immune responses subject to the requirement of standardized assay procedure and data analysis.

The online version of this article (doi:10.1186/s12967-016-0932-7) contains supplementary material, which is available to authorized users.

• Assay precision
• Assay validation
• Clinical trial monitoring
• EBV-specific T-cell responses
• FluoroSpot

• CTLA4
• Combination drug therapy
• PD-1
• PD-L1
• biomarkers
• clinical trials
• immunotherapy
• oncology

HPV infections are detected in sexually naive children. This has raised the question about the role of early HPV infections in either protecting or predisposing to further HPV infections. HPV16-specific cell-mediated immunity (CMI) was studied in 10 case-children born to mothers with an incident cervical intraepithelial neoplasia (CIN) diagnosed during their 14-year follow-up (FU), and in 21 children born to mothers, who remained constantly HPV-negative (controls). The mean age of children was 12.3 years.

Peripheral blood mononuclear cells were isolated from blood and stimulated with peptide pools covering HPV16 E2, E6 and E7. Proliferation of lymphocytes, their secretion of cytokines, and the frequency of regulatory T-cells were determined. The results were correlated with the HPV status and analyzed in a nested case–control setting.

All children, except two controls, displayed CMI against HPV16 E2, E6 and/or E7 peptides associated with type 1 and 2 cytokine secretion. Only two statistically significant differences were found in the nested case–control setting; (1) case-children had a higher TNF-α response to HPV16 E2 (p = 0.004) than controls and (2) controls had no response to HPV16 E7.2 peptide pool while 3/10 case-children had (p = 0.013). Totally, 50 and 57 % of the cases and controls, respectively, had HPV positive oral samples at some FU-visit. In addition, the children without any HPV antibodies before the age of 6 months showed proliferative responses of PBMC after HPV16 exposure more frequently than other children (p = 0.045).

HPV16-specific CMI is common in young, sexually inexperienced children. This suggests that oral HPV infections occur frequently in children. Our results might also explain the previous findings that half of healthy adults demonstrate HPV-specific CMI irrespective of their partner/sexual status.

The online version of this article (doi:10.1186/s12967-015-0733-4) contains supplementary material, which is available to authorized users.

• Cellular immunology
• Dendritic cells
• Neonate immunity
• Regulatory T cells
• T cells

Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.

To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.

Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.

We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.

• Anti-HIV Agents/adverse effects
• Anti-HIV Agents/therapeutic use
• Cross Reactions
• Dideoxynucleosides/adverse effects
• Dideoxynucleosides/therapeutic use
• Drug Hypersensitivity/immunology
• Epitopes, T-Lymphocyte/immunology
• HIV Infections/complications
• HIV Infections/drug therapy
• HLA-B Antigens/immunology
• Humans
• Immunologic Memory
• Immunophenotyping
• Leukocytes, Mononuclear/immunology
• Leukocytes, Mononuclear/metabolism
• Lymphocyte Count
• Phenotype
• T-Lymphocyte Subsets/immunology
• T-Lymphocyte Subsets/metabolism
• Time Factors
• Vaccination
• Yellow Fever Vaccine/immunology

• R01 AI103348 NIAID NIH HHS
• R01A1103348 PHS HHS
• Medical Research Council

• AAV vectors
• T cell responses
• gene therapy
• immune modulation
• immunogenicity

• Immune assays
• Immune monitoring
• MIATA
• Reporting

• Consensus
• Cooperative Behavior
• Guideline Adherence/standards
• Humans
• Immunologic Tests/standards
• Immunotherapy/methods
• International Cooperation
• Laboratories/standards
• Laboratory Proficiency Testing/standards
• Monitoring, Immunologic/standards
• Observer Variation
• Practice Guidelines as Topic/standards
• Predictive Value of Tests
• Quality Control
• Reproducibility of Results
• T-Lymphocytes/immunology
• Treatment Outcome

• 15951 Cancer Research UK

Human papillomavirus (HPV) infection has traditionally been regarded as a sexually transmitted disease (STD), but recent evidence implicates that an infected mother can transmit HPV to her newborn during pregnancy, at delivery, perinatal period or later. Given the lack of any studies on HPV-specific immune responses in children, we conducted HPV16-specific cell-mediated immune (CMI) monitoring of the mother-child pairs with known oral and genital HPV follow-up (FU) data since the delivery. In the Finnish Family HPV Study, 10 out of 331 mothers developed incident cervical intraepithelial neoplasia (CIN) during their 14-year FU. Our hypothesis according to the common dogma is that there is no HPV16 specific immune response in offspring of the CIN mother as she/he has not started the sexual life yet.

We used overlapping 30–35 mer peptides covering the entire HPV16 E2, E6 and E7 protein sequences. Assays for lymphocyte proliferation capacity, cytokine production and HPV16-specific Foxp3 + CD25 + CD4+ regulatory T-cells were performed.

HPV16-specific proliferative T-cell responses were broader in children than in their mothers. Nine of 10 children had responses against both E2 peptide pools compared to only 4 of the 10 mothers. Six of the 10 children and only 2 mothers displayed reactivity to E6 and/or E7. The cytokine levels of IL-2 (p = 0.023) and IL-5 (p = 0.028) induced by all peptide pools, were also higher among children than their mothers. The children of the mothers with incident CIN3 had significantly higher IFN-γ (p = 0.032) and TNF-α (p = 0.008) levels than other children.

Our study is the first to show that also children could have HPV-specific immunity. These data indicate that the children have circulating HPV16-specific memory T-cells which might have been induced by previous HPV16 exposure or ongoing HPV 16 infection.

• Active immunotherapy
• Angiogenesis
• Cancer
• Clinical trial
• VEGF
• Vaccine

• cin
• hpv16
• immunotherapy
• vaccination
• memory response

• Adult
• Aged
• Double-Blind Method
• Female
• Human papillomavirus 16/immunology
• Humans
• Immunologic Memory
• Middle Aged
• Neoplasm Grading
• Papillomavirus Vaccines/immunology
• Precancerous Conditions/immunology
• T-Lymphocytes/immunology
• Uterine Cervical Neoplasms/immunology
• Uterine Cervical Neoplasms/pathology
• Uterine Cervical Neoplasms/virology
• Vaccination/adverse effects
• Vaccines, Subunit/immunology

Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37°C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.

• Adult
• Aged
• Antigen-Presenting Cells/immunology
• Antigens/immunology
• CD4-Positive T-Lymphocytes/immunology
• CD4-Positive T-Lymphocytes/virology
• CD8-Positive T-Lymphocytes/immunology
• CD8-Positive T-Lymphocytes/virology
• Cell Survival
• Chemokines/metabolism
• Clinical Trials as Topic
• Cryopreservation
• Epitopes/immunology
• Epitopes, T-Lymphocyte/immunology
• Female
• Humans
• Inflammation Mediators/metabolism
• Intracellular Space/metabolism
• Lymphocyte Count
• Male
• Middle Aged
• Monitoring, Immunologic
• Staining and Labeling
• Time Factors

• MIATA
• T cell assay

• Data Collection/methods
• Data Collection/standards
• Data Display
• Guidelines as Topic
• Humans
• Immunoassay/methods
• Immunoassay/standards
• Neoplasms/immunology
• T-Lymphocytes/immunology

Recent technical improvements in evaluations of immune cells in situ and immune monitoring of patients with cancer have provided a wealth of new data confirming that immune cells play a key role in human cancer progression. This, in turn, has revived the expectation that immune endpoints might serve as reliable biomarkers of outcome or response to therapy in cancer. The recent successes in linking the T-cell signature in human colorectal carcinoma (CRC) with prognosis have provided a strong motive for searching for additional immune biomarkers that could serve as intermediate endpoints of response to therapy and outcome in human cancers. A number of potentially promising immune biomarkers have emerged, but most remain to be validated. Among them, the B-cell signature, as exemplified by expression of the immunoglobulin G kappa chain (IGKC) in tumor-infiltrating lymphocytes (TIL), has been validated as a biomarker of response to adjuvant therapy and better survival in patients with breast carcinoma and several other types of human solid tumors. Additional immune endpoints are being currently tested as potentially promising biomarkers in cancer. In view of currently growing use of immune cancer therapies, the search for immune biomarkers of prognosis are critically important for identifying patients who would benefit the most from adjuvant immunotherapy.

• B-cell signature
• T-cell signature
• cancer
• immune system
• prognosis

• Algorithms
• Computer Security
• Cooperative Behavior
• HIV-1/immunology
• Humans
• Immunoassay/methods
• Immunoassay/standards
• Quality Control
• Software

• P30 AI 64518 NIAID NIH HHS
• 5U01 AI46725-05 NIAID NIH HHS
• UM1 AI068635 NIAID NIH HHS
• U01-AI-068635 NIAID NIH HHS
• UM1-AI-068618 NIAID NIH HHS

• immunomonitoring
• hla/peptide multimer staining
• t cells
• immunotherapy
• assay controls

• Antigens, Neoplasm/immunology
• Biological Assay/methods
• Biological Assay/standards
• CD8-Positive T-Lymphocytes/immunology
• HLA Antigens/immunology
• HLA-A2 Antigen/immunology
• Humans
• Leukocytes, Mononuclear/immunology
• Monitoring, Immunologic/standards
• Observer Variation
• Receptors, Antigen, T-Cell/immunology
• Reference Values
• Staining and Labeling
• Transduction, Genetic
• Transgenes

• 15951 Cancer Research UK

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA_27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2⁺ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2⁻ control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4⁺ and PSMA₂₇-specific CD8⁺ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4⁺ T-cell response and PSMA₂₇-specific CD8⁺ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4⁺ and CD8⁺ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA₂₇ vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.

The online version of this article (doi:10.1007/s00262-012-1263-z) contains supplementary material, which is available to authorized users.

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

• Antigen Presentation/immunology
• Antigens/immunology
• CD4-Positive T-Lymphocytes/immunology
• CD8-Positive T-Lymphocytes/immunology
• Cells, Cultured
• Enzyme-Linked Immunospot Assay
• Epitopes, T-Lymphocyte/analysis
• Epitopes, T-Lymphocyte/immunology
• Flow Cytometry/methods
• Humans
• Interferon-gamma/immunology
• Lymphocyte Activation/immunology
• Peptides/immunology
• Poly I-C/immunology

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4^th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.

• immunotherapy
• cancer vaccines/immunology
• cancer vaccines/therapeutic use
• human

• Antigens, Neoplasm/immunology
• Biomarkers, Tumor/immunology
• Cancer Vaccines/immunology
• Hematologic Neoplasms/immunology
• Hematologic Neoplasms/therapy
• Humans
• Immunologic Factors/therapeutic use
• Immunotherapy/methods

• T32 AI007247 NIAID NIH HHS
• T32 AI007247-30 NIAID NIH HHS

• Biomarkers/metabolism
• CD8-Positive T-Lymphocytes/cytology
• CD8-Positive T-Lymphocytes/immunology
• Cell Survival
• Coloring Agents/metabolism
• Computational Biology
• HLA Antigens/metabolism
• Humans
• Lymphocyte Count
• Peptides/metabolism
• Protein Binding
• Protein Multimerization
• Reproducibility of Results
• Staining and Labeling/methods
• Subcellular Fractions/metabolism

• U54 HG004028 NHGRI NIH HHS