Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand.

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation.

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D- and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D- and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.

Understanding the functional relevance of oral neutrophils, phenotype and properties in OSCC.

Neutrophils are the predominant white blood cells (WBC) that are recruited to the sites of inflammation and infection. They are acknowledged to perform dual roles by promoting (pro-tumor) or by exhibiting anti-cancer properties (anti-tumor). Neutrophils are characterized based on the changes in phenotype and functional properties. To this context, circulating polymorphonuclear neutrophils (cPMN) and tumor-associated neutrophils (TANs) in cancer biology has been well explored but limited to oral polymorphonuclear neutrophils (oPMNs) in oral squamous cell carcinoma (OSCC). However, oPMNs are eminent in maintaining the healthy oral ecosystem by neutralizing microorganisms. Neutralization process enhances the expression of cell surface markers (CD11b, CD63, CD66, CD66b, CD66c, and CD66e) and inflammatory cytokines (TNF-α, IFN-γ, GM-CSF, and IL-8) and increases the recruitment of neutrophils. Along with the inflammation, it has been reported that CEACAM1 and chemerin also favors the infiltration of neutrophils to the cancer site. This indicates that oPMN might contribute to the aetiology of OSCC. The main objective of this review is to explore, the production and migration of oPMNs to the oral cavity, their phenotypes and possible role in OSCC.

Cytomegalovirus-specific CD8+ T-cells are associated with a reduced incidence of early relapse after allogeneic stem cell transplantation.

Leukemia relapse is the main cause for mortality after allogeneic stem cell transplantation (allo-SCT). Donor-derived allo-immune responses eliminate the residual host hematopoiesis and protect against relapse. Cytomegalovirus (CMV) reactivation (CMV-R) after allo-SCT may trigger anti-leukemic effects. The impact of CMV-specific CD8+ T-cells (CMV-CTLs) on the outcome after allo-SCT is currently unknown. Here, we studied the relationship between CMV-CTLs, overall T-cell reconstitution and relapse incidence in 103 patients with acute leukemia (n = 91) or myelodysplastic syndrome (n = 12) following CMV-seropositive recipient/donor (R+/D+) allo-SCT. Patients were subdivided based on the presence or absence of CMV-CTLs at 3 months after allo-SCT. Presence of CMV-CTLs was associated with preceding CMV-R and a fast T-cell reconstitution. Univariate analysis showed a significantly lower 1-, 2- and 5-year cumulative incidence of relapse (CIR) in patients with CMV-CTLs compared to those without CMV-CTLs. Multivariable regression analysis of the outcome performed with other relevant parameters chosen from univariate analysis revealed that presence of CMV-CTLs and chronic graft-versus-host disease (cGvHD) were the only independent factors associated with a low CIR. Onset of relapse was significantly later in patients with CMV-CTLs (median 489 days) than in in those without (median 152 days, p = 0.041) during a five-year follow-up. Presence of CMV-CTLs was associated with a lower incidence of early relapses (1 and 2-years), while cGvHD lead to a lower incidence of late relapses (2 to 5-years). In conclusion, our data show that CMV-CTLs indicate a functional immune-reconstitution protective against early relapse.

Human CD8+ CD57- TEMRA cells: Too young to be called "old".

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed "young" CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.

Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.

The timely reconstitution and regain of function of a donor-derived immune system is of utmost importance for the recovery and long-term survival of patients after allogeneic hematopoietic stem cell transplantation (HSCT). Of note, new developments such as umbilical cord blood or haploidentical grafts were associated with prolonged immunodeficiency due to delayed immune reconstitution, raising the need for better understanding and enhancing the process of immune reconstitution and finding strategies to further optimize these transplant procedures. Immune reconstitution post-HSCT occurs in several phases, innate immunity being the first to regain function. The slow T cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse. Here we aim to summarize the major steps of the adaptive immune reconstitution and will discuss the importance of immune balance in patients after HSCT.

Multimer monitoring of CMV-specific T cells in research and in clinical applications.

Multimer monitoring has become a standard technique for detection of antigen-specific T cells. The term "multimer" refers to a group of reagents based on the multimerisation of molecules in order to raise avidity and thus stabilize binding to their ligand. Multimers for detection of antigen-specific T-cell responses are based on major histocompatibility complex class I peptide complexes. Multimer staining enables fast and direct visualization of antigen-specific T cells; thus, it is widely applied to assess antiviral immunity, e.g., monitor patients in vaccination trials or confirm purity of cell products for adoptive transfer. Assessment of T-cell immunity against persistent pathogens like cytomegalovirus (CMV) is of major importance in immunosuppressed patients. Recent advancements of multimers facilitate reversible labeling and allow isolation of epitope-specific T cells for adoptive transfer. Here, we give an overview on the different multimers and their applications, with an emphasis on CMV-specific T-cell responses.

Impaired functionality of antiviral T cells in G-CSF mobilized stem cell donors: implications for the selection of CTL donor.

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.