The Quest for CD8+ Memory Stem Cells

In this issue of Immunity, Turtle et al. (2009) describe the identification of a distinct CD8+ memory T cell subset in humans, which could bring us closer to the identification of the enigmatic “memory stem cell.

Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion.

Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

Dynamic Imaging of the Effector Immune Response to Listeria Infection In Vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30–1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ [CD8 superscript +] T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ [CD8 superscript +] T cells in effector sites.National Institutes of Health (U.S.) (Grant P01AI-071195

Decreased Susceptibility of Mice to Infection with Listeria monocytogenes in the Absence of Interleukin-18▿

The induction of proinflammatory cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha is crucial for the early control of bacterial infections. Since interleukin-18 (IL-18) acts as a potent inducer of IFN-γ, it might play an important role in the induction of a protective immune response in listeriosis. We used a murine model of systemic Listeria monocytogenes infection to study the immune response to these intracellular bacteria in the absence of IL-18. For this purpose, IL-18-deficient mice and mice treated with anti-IL-18 neutralizing antibody were infected with L. monocytogenes, and their innate and adaptive immune responses were compared to those of control mice. Unexpectedly, we found that mice deficient in IL-18 were partially resistant to primary infection with L. monocytogenes. At day 3 after infection, the numbers of listeriae in the livers and spleens of control mice were up to 500 times higher than those in IL-18-deficient or anti-IL-18 antibody-treated mice. In addition, the level of proinflammatory cytokines was markedly reduced in IL-18-deficient mice. Enhanced resistance to L. monocytogenes infection in IL-18-deficient mice was accompanied by increased numbers of leukocytes and reduced apoptosis in the spleen 48 to 72 h after infection. In contrast, control and IL-18-deficient mice showed no significant differences in their abilities to mount a protective L. monocytogenes-specific T-cell response

Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells.

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT

Primary Cytomegalovirus Infection in Seronegative Kidney Transplant Patients Is Associated with Protracted Cold Ischemic Time of Seropositive Donor Organs.

Human Cytomegalovirus (CMV) can lead to primary infection or reactivation in CMV-seronegative or -seropositive kidney transplant recipients, respectively. Complications comprise severe end-organ diseases and acute or chronic transplant rejection. Risk for CMV manifestation is stratified according to the CMV-IgG-serostatus, with donor+/recipient- (D+/R-) patients carrying the highest risk for CMV-replication. However, risk factors predisposing for primary infection in CMV-seronegative recipients are still not fully elucidated. Therefore, we monitored D+/R- high-risk patients undergoing kidney transplantation in combination with antiviral prophylaxis for the incidence of CMV-viremia for a median follow-up time of 784 days (156-1155 days). In this period, we analyzed the functional CMV-specific T cell response by intracellular cytokine staining and CMV-serology by ELISA. Only four of eight D+/R- patients developed clinically relevant CMV-viremia followed by seroconversion. Viremia triggered expansion of functional CMV-specific T cells correlating with protection against secondary CMV-reactivations. In contrast, all other patients remained permanently aviremic and showed no immunological correlate of infection after discontinuation of antiviral prophylaxis for up to three years. Comparing cold ischemic times (CIT) of viremic (median = 1020 min; 720-1080 min) and aviremic patients (median = 335 min; 120-660 min) revealed significantly (p = 0.0286) protracted CIT in patients with primary CMV-infection. Taken together, primary CMV-infection affects only a subgroup of D+/R- patients correlating with length of CIT. Therefore, patients with extended CIT should be thoroughly monitored for CMV-replication well beyond discontinuation of antiviral prophylaxis. In contrast, patients with short CIT remained permanently uninfected and might benefit from shorter prophylactic treatment