NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.

Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Monocyte infiltration is an independent positive prognostic biomarker in vulvar squamous cell carcinoma.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) arises after an HPV infection or the mutation of p53 or other driver genes and is treated by mutilating surgery and/or (chemo) radiation, with limited success and high morbidity. In-depth information on the immunological make up of VSCC is pivotal to assess whether immunotherapy may form an alternative treatment. METHODS: A total of 104 patient samples, comprising healthy vulva (n = 27) and VSCC (n = 77), were analyzed. Multispectral immunofluorescence (15 markers) was used to study both the myeloid and lymphoid immune cell composition, and this was linked to differences in transcriptomics (NanoString nCounter, 1258 genes) and in survival (Kaplan-Meier analyses). RESULTS: Healthy vulva and VSCC are both well infiltrated but with different subpopulations of lymphoid and myeloid cells. In contrast to the lymphoid cell infiltrate, the density and composition of the myeloid cell infiltrate strongly differed per VSCC molecular subtype. A relative strong infiltration with epithelial monocytes (HLADR-CD11c-CD14+CD68-CD163-CD33-) was prognostic for improved survival, independent of T cell infiltration, disease stage or molecular subtype. A strong infiltration with T cells and/or monocytes was associated with drastic superior survival: 5-year survival > 90% when either one is high, versus 40% when both are low (p < 0.001). CONCLUSION: A hot myeloid and/or lymphoid infiltrate predicts excellent survival in VSCC. Based on the response of similarly high-infiltrated other tumor types, we have started to explore the potential of neoadjuvant checkpoint blockade in VSCC.

APOLLO: neo-adjuvant pembrolizumab for primary vulvar squamous cell carcinoma-a multicenter, single-arm, phase II, clinical proof-of-concept study.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) is a rare cancer for which the cornerstone of treatment is surgery with high complication rates. The unmet need is a less radical and more effective treatment for VSCC. PRIMARY OBJECTIVES: To investigate the impact of mono-immunotherapy pembrolizumab as neoadjuvant treatment for primary resectable VSCC patients. STUDY HYPOTHESIS: Some primary VSCC patients display a specific immune profile which is associated with better survival. In other tumors, this profile is associated with a better response to programmed cell death protein 1 (PD-1) checkpoint blockade which may reinvigorate tumor-specific T cells. This potentially results in a reduced tumor load and less radical surgery and/or adjuvant treatment in patients with this immune profile. TRIAL DESIGN: This is an investigator-initiated, prospective, single arm, multicenter, phase II clinical trial. INCLUSION CRITERIA: Patients with VSCC clinical stage International Federation of Gynecology and Obstetrics (FIGO) I-III (2021) eligible for primary surgery, with at least one measurable lesion of at least one dimension ≥10 mm in the largest diameter, are included in this study. MAIN EXCLUSION CRITERIA: Patients not suitable for surgery and/or previously treated with immunomodulatory agents, and/or who suffer from comorbidities that may interfere with PD-1 blockade, are excluded from the study. ENDPOINTS: The clinical efficacy of neoadjuvant pembrolizumab in VSCC is measured by an objective change in tumor size according to the Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST 1.1) and documented by calipers using standardized digital photography with a reference ruler. In addition, the activation, proliferation, and migration of T cells in the tumor will be studied. The secondary endpoints are pathological complete responses at the time of surgery, feasibility, and safety. SAMPLE SIZE: 40 patients with FIGO I-III (2021) primary VSCC will be enrolled. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: The intervention phase started in July 2023 and will continue until July 2025. The expected completion of the entire study is July 2026. TRIAL REGISTRATION NUMBER: NCT05761132.

The common HLA class I-restricted tumor-infiltrating T cell response in HPV16-induced cancer.

Immunotherapies targeting truly tumor-specific targets focus on the expansion and activation of T cells against neoantigens or oncogenic viruses. One target is the human papilloma virus type 16 (HPV16), responsible for several anogenital cancers and oropharyngeal carcinomas. Spontaneous and vaccine-induced HPV-specific T cells have been associated with better clinical outcome. However, the epitopes and restriction elements to which these T cells respond remained elusive. To identify CD8+ T cell epitopes in cultures of tumor infiltrating lymphocytes, we here used multimers and/or a functional screening platform exploiting single HLA class I allele-engineered antigen presenting cells. This resulted in the detection of 20 CD8+ T cell responses to 11 different endogenously processed HLA-peptide combinations within 12 HPV16-induced tumors. Specific HLA-peptide combinations dominated the response in patients expressing these HLA alleles. T cell receptors (TCRs) reactive to seven different HLA class I-restricted peptides could be isolated and analysis revealed tumor reactivity for five of the six TCRs analyzed. The tumor reactive TCRs to these dominant HLA class I peptide combinations can potentially be used to engineer tumor-specific T cells for adoptive cell transfer approaches to treat HPV16-induced cancers.

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation.

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vß antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vß repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27- and/or CD28- memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.

HPV16 E7 DNA tattooing: safety, immunogenicity, and clinical response in patients with HPV-positive vulvar intraepithelial neoplasia.

BACKGROUND: Usual type vulvar intraepithelial neoplasia (uVIN) is caused by HPV, predominantly type 16. Several forms of HPV immunotherapy have been studied, however, clinical results could be improved. A novel intradermal administration route, termed DNA tattooing, is superior in animal models, and was tested for the first time in humans with a HPV16 E7 DNA vaccine (TTFC-E7SH). METHODS: The trial was designed to test safety, immunogenicity, and clinical response of TTFC-E7SH in twelve HPV16+ uVIN patients. Patients received six vaccinations via DNA tattooing. The first six patients received 0.2 mg TTFC-E7SH and the next six 2 mg TTFC-E7SH. Vaccine-specific T-cell immunity was evaluated by IFNγ-ELISPOT and multiparametric flow cytometry. RESULTS: Only grade I-II adverse events were observed upon TTFC-E7SH vaccination. The ELISPOT analysis showed in 4/12 patients a response to the peptide pool containing shuffled E7 peptides. Multiparametric flow cytometry showed low CD4+ and/or CD8+ T-cell responses as measured by increased expression of PD-1 (4/12 in both), CTLA-4 (2/12 and 3/12), CD107a (5/12 and 4/12), or the production of IFNγ (2/12 and 1/12), IL-2 (3/12 and 4/12), TNFα (2/12 and 1/12), and MIP1ß (3/12 and 6/12). At 3 months follow-up, no clinical response was observed in any of the twelve vaccinated patients. CONCLUSION: DNA tattoo vaccination was shown to be safe. A low vaccine-induced immune response and no clinical response were observed in uVIN patients after TTFC-E7SH DNA tattoo vaccination. Therefore, a new phase I/II trial with an improved DNA vaccine format is currently in development for patients with uVIN.

A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17(+) non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p < 0.0001). In contrast, HPV-negative OPSCC contained significantly higher numbers of IL-17(+) non-T cells (p < 0.0001). Although a high number of intra-tumoral T cells showed a trend toward improved survival of all OPSCC patients, their prognostic effect in patients with a low number of intra-tumoral IL-17(+) non-T cells was significant with regard to disease-specific (p = 0.033) and disease-free survival (p = 0.012). This suggests that a high frequency of IL-17(+) non-T cells was related to a poor immune response, which was further supported by the observation that a high number of T cells was correlated with improved disease-free survival in the HPV-positive OPSCC (p = 0.008). In addition, we detected a minor Th17 cell population. However, T cells obtained from HPV-positive OPSCC produced significantly more IL-17 than those from HPV-negative tumors (p = 0.006). The improved prognosis of HPV-positive OPSCC is thus correlated with higher numbers of tumor-infiltrating T cells, more active Th17 cells and lower numbers of IL-17(+) non-T cells.

Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study.

There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.

Potential use of lymph node-derived HPV-specific T cells for adoptive cell therapy of cervical cancer.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer.

Local and systemic XAGE-1b-specific immunity in patients with lung adenocarcinoma.

XAGE-1b is a cancer/testis antigen aberrantly expressed in pulmonary adenocarcinoma. Systemic antibody and T cell responses have been demonstrated in adenocarcinoma patients, but so far, local antigen-specific immunity has not been reported. In this study, XAGE-1b expression by tumor cells as well as the presence of systemic and/or local XAGE-1b-specific immunity was assessed in peripheral blood, tumor tissue and tumor-draining lymph nodes of Caucasian patients with pulmonary adenocarcinoma. XAGE-1b protein expression was detected in 43.6% (17 of 39) of patients when at least two different parts of a resected tumor were assessed. In 20 patients, analysis of T cells isolated and expanded from the primary tumor and its draining lymph node demonstrated XAGE-1b-specific responses in two patients. XAGE-1b-specific immunoglobulin G antibodies were found in 3 of 40 patients. These three antibody-positive patients had also mounted a systemic T cell response to XAGE-1b, measured by proliferation, cytokine production and expression of T cell activation markers on peripheral blood mononuclear cells. The population of XAGE-1b-specific T cells comprised both CD4+ and CD8+ T cells secreting both type I and II cytokines. Epitope mapping showed that T cells predominantly targeted the N-terminal part of the XAGE-1b protein, while the B cell response was directed against the C-terminal domain. Our study for the first time provides evidence for the presence of XAGE-1b-specific T cells within adenocarcinoma tissue, which supports the concept that XAGE-1b acts as a genuine tumor antigen and, therefore, might form an attractive target for a vaccine-based approach of immunotherapy.

Data analysis as a source of variability of the HLA-peptide multimer assay: from manual gating to automated recognition of cell clusters.

Multiparameter flow cytometry is an indispensable method for assessing antigen-specific T cells in basic research and cancer immunotherapy. Proficiency panels have shown that cell sample processing, test protocols and data analysis may all contribute to the variability of the results obtained by laboratories performing ex vivo T cell immune monitoring. In particular, analysis currently relies on a manual, step-by-step strategy employing serial gating decisions based on visual inspection of one- or two-dimensional plots. It is therefore operator dependent and subjective. In the context of continuing efforts to support inter-laboratory T cell assay harmonization, the CIMT Immunoguiding Program organized its third proficiency panel dedicated to the detection of antigen-specific CD8(+) T cells by HLA-peptide multimer staining. We first assessed the contribution of manual data analysis to the variability of reported T cell frequencies within a group of laboratories staining and analyzing the same cell samples with their own reagents and protocols. The results show that data analysis is a source of variation in the multimer assay outcome. To evaluate whether an automated analysis approach can reduce variability of proficiency panel data, we used a hierarchical statistical mixture model to identify cell clusters. Challenges for automated analysis were the need to process non-standardized data sets from multiple centers, and the fact that the antigen-specific cell frequencies were very low in most samples. We show that this automated method can circumvent difficulties inherent to manual gating strategies and is broadly applicable for experiments performed with heterogeneous protocols and reagents.

Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

Human papillomavirus 16-specific cell-mediated immunity in children born to mothers with incident cervical intraepithelial neoplasia (CIN) and to those constantly HPV negative.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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 effects of short-term fasting on tolerance to (neo) adjuvant chemotherapy in HER2-negative breast cancer patients: a randomized pilot study.

BACKGROUND: Preclinical evidence shows that short-term fasting (STF) protects healthy cells against side effects of chemotherapy and makes cancer cells more vulnerable to it. This pilot study examines the feasibility of STF and its effects on tolerance of chemotherapy in a homogeneous patient group with early breast cancer (BC). METHODS: Eligible patients had HER2-negative, stage II/III BC. Women receiving (neo)-adjuvant TAC (docetaxel/doxorubicin/cyclophosphamide) were randomized to fast 24 h before and after commencing chemotherapy, or to eat according to the guidelines for healthy nutrition. Toxicity in the two groups was compared. Chemotherapy-induced DNA damage in peripheral blood mononuclear cells (PBMCs) was quantified by the level of γ-H2AX analyzed by flow cytometry. RESULTS: Thirteen patients were included of whom seven were randomized to the STF arm. STF was well tolerated. Mean erythrocyte- and thrombocyte counts 7 days post-chemotherapy were significantly higher (P = 0.007, 95 % CI 0.106-0.638 and P = 0.00007, 95 % CI 38.7-104, respectively) in the STF group compared to the non-STF group. Non-hematological toxicity did not differ between the groups. Levels of γ-H2AX were significantly increased 30 min post-chemotherapy in CD45 + CD3- cells in non-STF, but not in STF patients. CONCLUSIONS: STF during chemotherapy was well tolerated and reduced hematological toxicity of TAC in HER2-negative BC patients. Moreover, STF may reduce a transient increase in, and/or induce a faster recovery of DNA damage in PBMCs after chemotherapy. Larger studies, investigating a longer fasting period, are required to generate more insight into the possible benefits of STF during chemotherapy. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01304251 , March 2011.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

Human papillomavirus 16 E2-, E6- and E7-specific T-cell responses in children and their mothers who developed incident cervical intraepithelial neoplasia during a 14-year follow-up of the Finnish Family HPV cohort.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.