Mario Assenmacher

Stat6-Independent GATA-3 Autoactivation Directs IL-4-Independent Th2 Development and Commitment

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA–peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 × 103/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.

Enrichment and detection of live antigen-specific CD4 + and CD8 + T cells based on cytokine secretion

Following appropriate antigen‐specific stimulation, CD4+ and CD8+ T lymphocytes rapidly express cytokines. Based on this stimulation‐induced cytokine secretion and using cell suface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen‐specific CD4+ and CD8+ T lymphocytes. The power of this technique is demonstrated here for HLA‐A0201‐restricted influenza matrix protein peptide 58‐66‐specific CD8+ cytotoxic T lymphocytes, influenza A virus‐ and recombinant tetanus toxin C fragment‐specific Th1 cells and tetanus toxoid‐specific Th2 cells.

Ex Vivo IFN-γ Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-γ. Magnetic cell sorting of IFN-γ-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-γ upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-γ-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-γ staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-γ upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RAlow Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-γ ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-γ is essential for tumor rejection, we isolated live T cells based on their IFN-γ production. IFN-γ secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-γ+ but not IFN-γ− T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-γ capture assay.

Regulation of Expression of IL-4 Alleles: Analysis Using a Chimeric GFP/IL-4 Gene

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG

Recent studies in mice have indicated that the long‐lasting specific antibody responses seen after vaccination are probably due to the existence of long‐lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long‐lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two‐step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen‐specific memory B cells, we show for tetanus toxin C‐fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom‐allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen‐specific IgG‐bearing memory B cells and the serum titers of antigen‐specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.

High-sensitivity immunofluorescence for detection of the pro- and anti-inflammatorycytokines gamma interferon and interleukin-10 on the surface of cytokine-secretingcells

High-sensitivity immunofluorescence for detection of the pro- and anti-inflammatory cytokines gamma interferon and interleukin-10 on the surface of cytokine-secreting cells

Purification of cytomegalovirus‐specific CD8 T cells from peripheral blood using HLA–peptide tetramers

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV‐specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV‐specific CD8+ T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I‐peptide tetramers and magnetic selection it is possible to select CMV‐specific T cells from CMV antibody‐positive individuals to high purity. Reliable purification of CMV‐specific T cells up to 99·8% of CD8+ cells was possible within hours, even when starting with a precursor frequency of < 0·1% of peripheral blood CD8+ T cells. CMV‐specific T cells remained functional after the selection process. This novel form of antigen‐specific T‐cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour‐specific antigens.

Detection and characterization of plasma cells in peripheral blood: correlation of IgE + plasma cell frequency with IgE serum titre

In atopic patients and patients with hyper‐IgE syndrome (HIE) highly elevated IgE serum levels can be detected. Due to their very low frequency little is known about IgE‐producing plasma cells (PC) in peripheral blood. We used CD138 MACS microbeads to enrich plasma cells from peripheral blood of normal donors, atopic patients and one HIE patient. CD138+ cells were mainly CD45+, CD44++, CD19dim, CD38++, CD27++, CD86+, HLA‐DR+/++, CD71dim, VLA‐4+, VLA‐5–, CD28–, CD25–, CD69–, CLA–, CD20–, CD21– and CD22–. They show weak expression of surface Ig but high levels of intracellular Ig and they secrete Ig in culture. Thus CD138+ cells from peripheral blood show characteristics of early plasma cells. IgE+ CD138+ plasma cells could be detected in 19 of 24 normal donors with an average frequency of 0·06% IgE+ cells among CD138+ cells. Higher frequencies were detected in atopic patients, atopic patients with markedly elevated serum IgE levels and the hyper‐IgE patient with an average of 0·32%, 7·21% and 6·54%, respectively. Additionally, using the recently developed cellular affinity matrix technology, we were able to detect IgE secreting plasma cells and thereby could demonstrate that most of the IgE secreting cells express CD138. The frequency of IgE+ CD138+ cells among PBMC correlated highly significantly with serum IgE titres (r = 0·8532***), indicating that IgE secreting CD138+ cells in peripheral blood are directly related to the plasma cell pool contributing to the IgE titre.