Walter E. Hitzler

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-γ 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-61–20 epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-61–20 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.

Quantification of Thrombocyte Growth Factors in Platelet Concentrates Produced by Discontinuous Cell Separation

Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean - SD platelet count in whole blood from these donors was 262,000 - 58,000/ w l, while in PC produced by discontinuous cell separation it was 1,419,000 - 333,000/ w l. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125 - 55 ng/ml; TGF- g 1 221 - 92 ng/ml; IGF-I 85 - 25 ng/ml; PDGF-BB 14 - 9 ng/ml; TGF- g 2 0.4 - 0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277 ng/ml; PDGF-BB 2-33 ng/ml; TGF- g 1 32-397 ng/ml; TGF- g 2 0.1-1.2 ng/ml; IGF-I 40-138 ng/ml. Platelet counts in PC were slightly higher for women (Mann-Whitney Test all p <0.001 ) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance.

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function

Abstract Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV.

BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti‐HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated.

The quality of plasma collected by automated apheresis and of recovered plasma from leukodepleted whole blood

BACKGROUND: There exists a current lack of information about the composition of the different types of plasma. No direct comparisons between apheresis plasma (AP) and recovered plasma (RP) derived from in‐line–filtered whole blood (WB) have been published to date.

MHC class II Tetramer Guided Detection of Mycobacterium tuberculosis‐specific CD4+ T Cells in Peripheral Blood from Patients with Pulmonary Tuberculosis

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma‐based assays have recently been developed in addition to the more than 100‐year‐old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen‐specific T cells. We identified novel MHC class II‐restricted MTB epitopes and used HLA‐DR4 tetrameric complexes to visualize ex vivo CD4+ T cells directed against the antigens Ag85B and the 19‐kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4+ T cells which recognize the MTB‐associated ESAT‐6 antigen. MTB‐reactive CD4+ T cells reside predominantly in the CD45RA+ CD28+ and CD45− CD28+ T‐cell subset and recognize naturally processed and presented MTB epitopes. HLA‐DR4‐restricted, Ag85B or ESAT‐6‐specific CD4+ T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8+ T cells directed against the corresponding HLA‐A2‐presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T‐cell responses directed against the 19‐kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4+ T cells directed 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 E77–15), 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‐γ 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 E77–15‐specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7− T‐cell population.

Longitudinal analysis of the T-cell receptor (TCR)-VA and -VB repertoire in CD8+ T cells from individuals immunized with recombinant hepatitis B surface antigen.

Recent studies have suggested that vaccination induces alterations in the T cell receptor (TCR) repertoire. We investigate the diversity of the TCR repertoire after immunization with a recombinant hepatitis B surface vaccine in seven healthy subjects in CD8+ T cells in peripheral blood lymphocytes. Cellular immune responses were monitored over time by sorting CD8 T cells followed by TCR‐VA and ‐VB complementarity determining region 3 (CDR3) analysis. Frequency of individual VB families was determined by flow cytometry. TCR‐VA/VB repertoires obtained from CD8+ T cells drawn after vaccination were compared to the TCR repertoire determined prior to vaccination. Monoclonal TCR transcripts could be detected exclusively in CD8+, but not in CD4+ T cells. Such monoclonal TCR transcripts were either stable in some individuals, or could only be detected at certain time points after vaccination. Sorting of monoclonal TCR‐VB3+ T cells, which constituted up to 5% of the CD8+ T cell population from one individual, revealed that this T cell clone recognizes an epitope provided by the recombinant hepatitis B vaccine presented by MHC‐class I on autologous antigen‐presenting cells. Examination of the structural anatomy, defined by the TCR, and the frequency of T cells responding to the immunizing antigen may be helpful to provide surrogate markers to monitor cellular immune responses induced by protein antigens utilized for vaccination.

Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016

Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.

Comparison of intermittent- and continuous-flow cell separators for the collection of autologous peripheral blood progenitor cells in patients with hematologic malignancies.

BACKGROUND: The transplantation of autologous peripheral blood progenitor cells (PBPCs) after high‐dose chemotherapy is a valuable therapy for patients with hematologic and solid malignancies. Several methods are used for harvesting PBPCs. The efficiency of intermittent‐ and continuous‐flow blood cell separators in collecting progenitor cells from the blood of patients undergoing myeloablative treatment for cancer was compared.