Bioinformatory-assisted analysis of next-generation sequencing data for precision medicine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with an extremely poor prognosis, predominantly as a result of chemotherapy resistance and numerous somatic mutations. Consequently, PDAC is a prime candidate for the use of sequencing to identify causative mutations, facilitating subsequent administration of targeted therapy. In a feasibility study, we retrospectively assessed the therapeutic recommendations of a novel, evidence-based software that analyzes next-generation sequencing (NGS) data using a large panel of pharmacogenomic biomarkers for efficacy and toxicity. Tissue from 14 patients with PDAC was sequenced using NGS with a 620 gene panel. FASTQ files were fed into treatmentmap. The results were compared with chemotherapy in the patients, including all side effects. No changes in therapy were made. Known driver mutations for PDAC were confirmed (e.g. KRAS, TP53). Software analysis revealed positive biomarkers for predicted effective and ineffective treatments in all patients. At least one biomarker associated with increased toxicity could be detected in all patients. Patients had been receiving one of the currently approved chemotherapy agents. In two patients, toxicity could have been correctly predicted by the software analysis. The results suggest that NGS, in combination with an evidence-based software, could be conducted within a 2-week period, thus being feasible for clinical routine. Therapy recommendations were principally off-label use. Based on the predominant KRAS mutations, other drugs were predicted to be ineffective. The pharmacogenomic biomarkers indicative of increased toxicity could be retrospectively linked to reported negative side effects in the respective patients. Finally, the occurrence of somatic and germline mutations in cancer syndrome-associated genes is noteworthy, despite a high frequency of these particular variants in the background population. These results suggest software-analysis of NGS data provides evidence-based information on effective, ineffective and toxic drugs, potentially forming the basis for precision cancer medicine in PDAC.

A Preliminary Report: Radical Surgery and Stem Cell Transplantation for the Treatment of Patients With Pancreatic Cancer.

We examined the immunologic effects of allogeneic hematopoietic stem cell transplantation (HSCT) in the treatment of pancreatic ductal adenocarcinoma, a deadly disease with a median survival of 24 months for resected tumors and a 5-year survival rate of 6%. After adjuvant chemotherapy, 2 patients with resected pancreatic ductal adenocarcinoma underwent HSCT with HLA-identical sibling donors. Comparable patients who underwent radical surgery, but did not have a donor, served as controls (n=6). Both patients developed humoral and cellular (ie, HLA-A*01:01-restricted) immune responses directed against 2 novel tumor-associated antigens (TAAs), INO80E and UCLH3 after HSCT. Both TAAs were highly expressed in the original tumor tissue suggesting that HSCT promoted a clinically relevant, long-lasting cellular immune response. In contrast to untreated controls, who succumbed to progressive disease, both patients are tumor-free 9 years after diagnosis. Radical surgery combined with HSCT may cure pancreatic adenocarcinoma and change the cellular immune repertoire capable of responding to clinically and biologically relevant TAAs.

IL-7δ5 protein is expressed in human tissues and induces expression of the oxidized low density lipoprotein receptor 1 (OLR1) in CD14+ monocytes.

OBJECTIVES: The 6-exon-spanning 'canonical' Interleukin-7 (IL-7c) is a non-redundant cytokine in human T-cell homeostasis that undergoes extensive alternative pre-mRNA splicing. The IL-7 gene variant lacking, exon 5 (IL-7δ5), exhibits agonistic effects as compared to IL-7c. We studied in this report for the first time the protein expression of IL-7δ5 variant in tissues and its role in monocyte activation. METHODS: We visualized the expression of IL-7δ5 protein by immunohistochemistry in both healthy and malignant (human) tissues and investigated the impact of IL-7δ5 stimulation on CD14+ monocytes using gene expression analysis and flow cytometry. RESULTS: IL-7δ5 is largely expressed by human epithelial cells, yet also by stromal cells in malignant lesions. Gene expression analysis in CD14+ monocytes, induced by the 6-exon spanning IL-7 or IL-7δ5 showed similar changes resulting in a pro-inflammatory phenotype and increased expression of genes involved in lipid metabolism. IL7δ5 was superior in inducing upregulation of the oxidised low density lipoprotein receptor (OLR), measured by flow cytometry, in CD14+ cells. CONCLUSION: IL-7δ5, produced from non-transformed and transformed cells, may contribute to chronic inflammatory responses and development of 'foamy' cells by increased OLR1 expression that mediates increased oxLDL uptake.

Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8(+) V[beta]13.2) reacting strongly against autologous tumor defined by INF-[gamma] production or by cytotoxicity. We have optimized methods for generating pancreatic cancer­specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis.

BACKGROUND: Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). METHODS: Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. RESULTS: 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. CONCLUSION: We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

Whole CMV proteome pattern recognition analysis after HSCT identifies unique epitope targets associated with the CMV status.

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/- for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the 'exclusive recognition analysis (ERA)' to identify unique CMV epitope responses for each patient group. The 'exclusive recognition analysis' of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D-/R-). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution.

Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial.

BACKGROUND: Novel treatment options are urgently needed for multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, which are associated with immune dysfunction and poor treatment outcomes. Mesenchymal stromal cells (MSCs) are immunomodulatory and adjunct autologous treatment with bone marrow-derived MSCs might improve clinical outcome by transforming chronic inflammation into productive immune responses. Our aim was to assess the safety of infusion of autologous MSCs as an adjunct treatment in patients with tuberculosis. METHODS: 30 patients with microbiologically confirmed MDR or XDR tuberculosis were treated with single-dose autologous bone marrow-derived MSCs (aimed for 1×10(6) cells per kg), within 4 weeks of the start of antituberculosis-drug treatment in a specialist centre in Minsk, Belarus. Inclusion patients were those with pulmonary tuberculosis confirmed by sputum smear microscopy, culture, or both; MDR or XDR tuberculosis confirmed by drug-susceptibility testing to first-line and second-line drugs; age older than 21 years to 65 years or younger; and absence of lesion compatible with a malignant process or ongoing tuberculosis in organs other than the lungs and pleura. In addition to the inclusion criteria, patients were excluded if they were pregnant, coinfected with HIV, or infected with hepatitis B, C, or both. The primary endpoint was safety measured by MSC-infusion related events; any tuberculosis-related event within the 6 month observation period that related to a worsening of the underlying infectious disease, measured by conversion of Mycobacterium tuberculosis culture or microscopic examination; or any adverse event defined clinically or by changes in blood haematology and biochemistry variables, measured monthly for 6 months after MSC infusion per protocol. This study is registered with the German Clinical Trials Registry, number DRKS00000763. FINDINGS: The most common (grade 1 or 2) adverse events were high cholesterol levels (14 of 30 patients), nausea (11 of 30 patients), and lymphopenia or diarrhoea (ten of 30 patients). There were no serious adverse events reported. We recorded two grade 3 events that were transitory-ie, increased plasma potassium ion concentrations in one patient and a transitory grade 3 γ-glutamyltransferase elevation in another patient. INTERPRETATION: MSCs as an adjunct therapy are safe and can now be explored further for the treatment of patients with MDR or XDR tuberculosis in combination with standard drug regimens. Adjunct treatment with MSCs needs to be evaluated in controlled phase 2 trials to assess effects on immune responses and clinical and microbiological outcomes.

Decreased IL-7 signaling in T cells from patients with PTLD after allogeneic HSCT.

Interleukin (IL)-7, a nonredundant cytokine for B and T cells, plays a central role in cell survival and immune memory formation. Peripheral blood mononuclear cells (PBMCs) from 7 patients after hematopoietic stem cell transplantation (HSCT) diagnosed with posttransplantation lymphoproliferative disease (PTLD) and from 10 Epstein-Barr virus (EBV) polymerase chain reaction-positive HSCT patients (controls) were evaluated for IL-7- and IL-2 induced Stat5 phosphorylation in CD4+ and CD8+ T cells. PBMCs from PTLD+ and control patients exhibited detectable EBV specific CD8+ T cells defined by tetramer analysis. CD4+ and CD8+ T cells from patients with PTLD showed statistically significant reduction in responsiveness to IL-7 compared with PBMCs obtained from controls defined by Stat5 phosphorylation. CD20+ B cells from patients with PTLD and from some EBV+ polymerase chain reaction control individuals exhibited IL-7R expression. Dysregulated immune surveillance, reflected by deficient Stat5 phosphorylation, may facilitate PTLD development despite the presence of EBV-reactive CD8+ T cells. Reduced IL-7 responsiveness will aid to monitor patients after HSCT for increased risk to develop EBV-associated PTLD.

High content cellular immune profiling reveals differences between rhesus monkeys and men.

A better understanding of similarities and differences in the composition of the cellular immune system in non-human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8alpha, CD8beta, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin-7 receptor alpha-chain (IL-7Ralpha) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti-CD3, -CD4, -CD25, -FoxP3, and -IL-7Ralpha monoclonal antibodies. Responsiveness to IL-7 was gauged in a signal transducer and activation of transcription 5 (STAT-5) phosphorylation assay. Human and NHP PBMCs showed a similar T-cell composition pattern with some remarkable differences. Similarities: human and NHP CD4(+) and CD8(+) cells showed a similar STAT-5 phosphorylation pattern in response to IL-7. Multicolour flow cytometric analysis identified a CD4(+) CD8alphaalpha(+) CD8alphabeta(+) T-cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4(+) T-cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5.16% in CD3(+) T cells) of CD8alphaalpha(+) T cells when compared with human donors (1.22% in CD3(+) T cells). NHP CD8alphaalpha(+) T cells produced tumour necrosis factor-alpha / interferon-gamma (TNF-alpha/IFN-gamma) or TNF-alpha, whereas human CD8alphaalpha(+) T cells produced simultaneously TNF-alpha/IFN-gamma and IL-2. A minor percentage of human CD8(+) T cells expressed CD25(bright) and FoxP3 (0.01%). In contrast, 0.07% of NHP CD8(+) T cells exhibited the CD25(bright) FoxP3(+) phenotype. PBMCs from NHPs showed less IL-7Ralpha-positive events in all T-cell subsets including CD4(+) Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long-lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)-B*0702 and HLA-B*0801 alleles in TB10.4 CD8 T-cell responses.

The molecular definition of major histocompatibility complex (MHC) class I-presented CD8(+) T-cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T-cell-based diagnostics of tuberculosis (TB) and the measurement of TB vaccine-take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)-A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I-binding peptides from overlapping 9-mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I-binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N- and C-termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off-rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8(+) T-cell interaction with their nominal MHC class I-peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8(+) T cells from patients with active pulmonary TB. HLA-B alleles served as the dominant MHC class I restricting molecules for anti-Mtb TB10.4-specific CD8(+) T-cell responses measured in CD8(+) T cells from patients with pulmonary TB.

T-cell epitope mapping.

Identification of epitopes defined by T-cell responses aids to (1) monitor antigen-specific cellular immune responses (2) guide rational vaccine design, and (3) understand the nature of protective or harmful T-cell responses in diseases with defined target antigens. The 6-h intracellular cytokine staining (ICS) assay preferentially identifies effector T cells that are readily detectable in the peripheral circulation. In contrast, the whole blood assay (WBA) allows to gauge expansion of antigen-specific T cells over time (7 days), i.e., T cells with lower frequencies (e.g., memory T cells) defined by proliferation and cytokine production. Any cellular immune profile can be measured in the WBA (using the 7 days cell culture supernatants) or directly in responder T cells after antigenic stimulation (in the ICS) with appropriate cytokine-specific detection systems. The choice of the cytokine test panel depends on the nature of the expected immune response. A broad panel of candidate peptides can be tested for T-cell recognition in the WBA due to its simplicity and the low input of (unprocessed, heparinized) blood.

Pattern recognition in pulmonary tuberculosis defined by high content peptide microarray chip analysis representing 61 proteins from M. tuberculosis.

BACKGROUND: Serum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M. tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations. METHOD: We constructed a high-content peptide microarray with 61 M. tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/-. FINDINGS: Three distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB-, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB- does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope 'hotspots' at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background. CONCLUSIONS: A uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize biologically relevant targets useful for development of novel TB diagnostics and vaccines.

Tumor antigen-specific T-cells are Present in the CD8alphaalpha+ T-cell effector-memory pool.

CD8+ T-cell memory formation has recently been demonstrated to be associated with CD8alphaalpha homodimer expression by T-cells in mice. Up to now, the knowledge about the clinical significance of CD8alphaalpha+ T-cells in humans is limited. We assessed in longitudinally collected blood samples from patients with melanoma, who underwent a peptide-based vaccination, the role of CD8alphaalpha+ T-cells in tumor-specific cellular immune responses. Phenotypic analysis showed that the expression of CD8alphaalpha+ by T-cells was stable over time and associated with a CD45RA+/-CCR7- effector-memory profile. Melan-A/MART-1-specific T-cells were identified in the CD8alphaalpha+ T-cell compartment by tetramer technology. Detection of intracellular cytokine production (interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) upon phorbol 12-myristate 13-acetate-ionomycin stimulation in CD8alphaalpha+ and CD8alphabeta+ T-cells revealed that CD8alphaalpha+ T-cells show a unique cytokine production pattern (tumor necrosis factor-alpha and interferon-gamma production) as compared with CD8alphabeta+ T-cells. T-cell receptor-CDR3 length analysis revealed that Melan-A/MART-1-specific CD8alphaalpha+ T-cells showed a similar T-cell receptor-repertoire as compared with Melan-A/MART-1-specific CD8alphabeta+ T-cells. Our results show that CD8alphaalpha+ T-cells represent a compartment of CD45RA+/- effector-memory cells in the peripheral circulation of patients with melanoma and suggest that CD8alphaalpha T-cells may originate from CD8+ T-cells that have down-regulated the expression of the CD8beta chain. CD8alphaalpha+ and tetramer-specific T-cells may represent a valuable marker to gauge long-term antigen-specific T-cell memory.

Reduced numbers of IL-7 receptor (CD127) expressing immune cells and IL-7-signaling defects in peripheral blood from patients with breast cancer.

Interleukin-7-receptor-signaling plays a pivotal role in T-cell development and maintenance of T-cell memory. We studied IL-7Ralpha (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL-7 receptor-mediated downstream effects defined by STAT5 phosphorylation (p-STAT5). Reduced numbers of IL-7Ralpha-positive cells were identified in CD4+ T-cells as well as in a CD8+ T-cell subset defined by CD8alpha/alpha homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p-STAT5 expression in the range of 0-6.4% in CD4+ T-cells and 0-4% in CD8+ T-cells. Stimulation with recombinant human IL-7 for 15 min increased p-STAT5 expression up to 36-97% in CD4+T-cells and to 26-90% in CD8+T-cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL-7 as defined by STAT5 phosphorylation, despite expression of IL-7Ralpha on T-lymphocytes. T-cells were further characterized for IL- 2 and IFN-gamma production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL-2 and IFN-gamma production combined with IL-7-signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL-7-signaling, yet intact cytokine production. Reduced numbers of IL-7Ralpha-positive cells and nonresponsiveness to IL-7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T-cells from patients with breast cancer.

Human papillomavirus 16 E6-specific CD45RA+ CCR7+ high avidity CD8+ T cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer.

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201-restricted and HPV16 E6-reactive T cells. CD8 T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201-restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-gamma production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201-matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8 T cells were of high avidity defined by blocking with an anti-CD8-alpha specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-gamma production in response to HPV antigens.

Differential MHC class II component expression in HPV-positive cervical cancer cells: implication for immune surveillance.

Effective eradication of human papillomavirus (HPV)-positive tumors may require CD8+ and CD4+ T-cell-mediated immune responses. Ectopic expression of MHC class II surface molecules has been described in the context of cervical cancer, but coexpression with other components of the MHC class II antigen presentation pathway has not been addressed. We have evaluated the MHC class II antigen presentation pathway in malignant squamous epithelium of HPV+ cervical cancer lesions by in situ costaining HLA-DR with CLIP or DMA/DMB. Cervical cancer cells exhibit 3 MHC class II phenotypes: (i) DR+/CLIP+ or DM+; (ii) DR+/CLIP- or DM-; and (iii) DR-/CLIP+ or DM+. The identical profile has been identified in HPV+ ME180 cells, which serve as a target for HLA-DR4-restricted and HPV68, E7-specific CD4+ T cells. IFN-gamma pretreatment of ME180 cells, associated with differential trafficking of MHC class II molecules, is necessary for effective T-cell recognition. Although proinflammatory cytokines may facilitate MHC class II-restricted antigen recognition in tumor cells, different phenotypes of the MHC class II antigen presentation pathway may be associated with evasion from CD4+-mediated cellular immune responses.

Diagnosing Helicobacter pylori in vivo by confocal laser endoscopy.

BACKGROUND & AIMS: Confocal laser endomicroscopy enables subsurface microscopic imaging of living tissue during ongoing endoscopy. This case report describes the in vivo detection of Helicobacter pylori by endomicroscopy. METHODS: Endomicroscopy (Pentax, Tokyo, EC-3870CIFK) was performed by using two different contrast stains: Topical Acriflavine in addition to intravenously applied fluorescein netted the surface and allowed identification of focal accumulation of Helicobacter pylori at the surface and in deeper layer of the gastric epithelium. Biopsies were performed at the antrum and corpus for urease testing and histology. In addition, biopsies were cultured for Helicobacter pylori. Cultured bacteria were re-assessed ex vivo using confocal microscopy with and without acriflavine staining. RESULTS: Helicobacter pylori infection could be detected in a 70-year-old male by endomicroscopy. Accumulated, as well as single bacteria, could be observed and the distinct shape and flagella of Helicobacter pylori could be identified. Helicobacter pylori infection was proved by histology. Furthermore, ex vivo examination of cultures proved the presence of Helicobacter pylori and the active uptake of acriflavine into the bacteria. CONCLUSIONS: Endomicroscopy is a new diagnostic approach, which enables the immediate diagnosis of Helicobacter pylori in vivo during standard video endoscopy.

Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection.

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-gamma in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242-250, Ag85b199-207, early secreted antigenic target 6 (ESAT-6)28-36, 19-kDa Ag88-97, or the HLA-DR-presented ESAT-6(1-20) epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-6(1-20) could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.

Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer.

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA-B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA-B8-binding peptide TLHEYMLDL (HPV16 E7(7-15)), which is able to drive HPV16 E7-specific and MHC class I-restricted T-cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL-specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA-B8 gene defined by IFN-gamma production. This peptide epitope is also recognized by freshly harvested tumor-infiltrating T cells or T cells from tumor-draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA-B8-restricted HPV E7(7-15)-specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7- T-cell population.

Longitudinal analysis of Mycobacterium tuberculosis 19-kDa antigen-specific T cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from HIVenv gp120.

CD8(+) T cells play a central role in immune protection against infection with Mycobacterium tuberculosis. One of the target epitopes for anti-M. tuberculosis directed CD8(+) T cells is the HLA-A2-restricted 19-kDa lipoprotein peptide VLTDGNPPEV. T cell clones directed against this epitope recognized not only the nominal peptide ligand, but also a closely related peptide (VPTDPNPPEV) from the HIV envelope gp120 (HIV(env) gp120) protein characterized by IFN-gamma release. This cross-reactivity was confirmed in ex vivo in M. tuberculosis 19-kDa tetramer-sorted T cells from patients with tuberculosis and in HIVgp120 tetramer-reactive T cells sorted from HIV(+) patients. M. tuberculosis 19-kDa antigen-reactive T cells were present in HLA-A2(+) patients (10/10) with HIV infection with no evidence of M. tuberculosis infection, but they are absent in peripheral blood lymphocytes from healthy HLA-A2(+) individuals (10/10). M. tuberculosis 19-kDa antigen-reactive T cells were elevated in acute pulmonary tuberculosis, declined with response to therapy (7/10 patients) and resided in the terminally differentiated CD8(+) T cell subset. CD8(+) cross-reactive T cells recognizing HIV(env) or M. tuberculosis 19-kDa antigens may contribute to pathogenesis in individuals co-infected with both pathogens and may also present a marker for active tuberculosis.