Mechanism of leukemic cell killing by IL-2 activated natural killer cells : role of cell adhesion molecules

Natural killer (NK) cells and lymphokine activated NK (LAK) cells, contribute to the elimination and growth control of malignant and virally infected cells. The binding of killer cells to their targets is a prerequisite for the lysis of malignant cells by NK cells, which utilize cell adhesion molecules (CAMs) to establish initial attachment to target cells. This thesis examined the possibility that defective expression of CAMs on some leukemic cells may be the primary cause of resistance to NK cell-mediated killing. To elucidate the mechanisms by which some leukemic cells are resistant to NK cytotoxicity, a model system was established with the human NK cell line NK-92, and the NK resistant leukemic cell line SR-91 which were established and characterized. SR-91 cells express very low levels of ICAM-1 and they failed to bind to NK-92 cells. NK-92 is highly cytotoxic and kills virtually all leukemic cell lines with the only exception being SR-91. Pre-treatment of SR-91 cells with TNF-α or IFN-γ, two cytokines known to upregulate ICAM-1 expression, increased both ICAM-1 expression on SR-91 cells and binding to NK-92 cells. However, only TNF-α treated SR-91 cells became sensitive to killing by NK-92 cells. The increased binding to NK-92 cells and sensitivity to their killing were abrogated by anti-LFA-1 antibody or a combination of antibodies against ICAM-1, ICAM-2 and ICAM-3, indicating that LFA-1 interaction with the three ICAMs is essential for effector-target cell binding, which is a prerequisite for subsequent target cell lysis. These results underline the importance of ICAM-1 expression on the target cell SR-91 to allow adequate conjugate formation. However, this is, on its own, insufficient to allow target cell lysis by NK-92 cells. TNF-α, but not IFN-γ, also induced the activation of LFA-1, CD44 and β1 integrins on SR-91 cells. Based on these observations, it was hypothesized that the differential effect of TNF-α and IFN-γ could be due to the TNF-α activation of LFA-1 and CD44 on the surface of SR-91 cells that bind to their counter-receptors and activate NK-92 cells. Preliminary experiments showed that engagement of ICAM-3 and CD44 on NK-92 cells induced tyrosine phosphorylation of several proteins including the tyrosine kinase p56*. Further confirmation of these results would not only suggest a role for these adhesion molecules in signal transduction events in NK-92 cells, but perhaps implicates the protein tyrosine kinase p56fc* as an early intermediate in the subsequent lysis of SR-91 cells. These data suggest that NK resistance of leukemic cells can be overcome by some cytokines. Although increased conjugate formation is induced by both TNF-α and IFN-γ, only TNF-α functionally activates LFA-1 and CD44 on target cells that may, upon interaction with counter-receptors on NK-92 cells induce signal transduction events in the latter that lead to target cell lysis. Therefore, treatment of patients with cytokines to overcome NK cell resistance and to eradicate tumor cells may not only activate and stimulate immune effector cells function but may also have direct effects on leukemic cells to make them more susceptible to the lytic effects of NK cells.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Severe Altered Immune Status After Burn Injury Is Associated With Bacterial Infection and Septic Shock.

Introduction: Burn injury is associated with a high risk of death. Whether a pattern of immune and inflammatory responses after burn is associated with outcome is unknown. The aim of this study was to explore the association between systemic immune and inflammatory responses and outcome in severely-ill burn patients. Materials and Methods: Innate immunity, adaptive immunity, activation and stress and inflammation biomarkers were collected at admission and days 2, 7, 14, and 28 in severely-ill adult burn patients. Primary endpoint was mortality at day 90, secondary endpoint was secondary infections. Healthy donors (HD) served as controls. Multiple Factorial Analysis (MFA) was used to identify patterns of immune response. Results: 50 patients were included. Age was 49.2 (44.2-54.2) years, total burn body surface area was 38.0% (32.7-43.3). Burn injury showed an upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. High interleukin-10 (IL-10) at admission was associated with risk of death. However, no cluster of immune/inflammatory biomarkers at early timepoints was associated with mortality. HLA-DR molecules on monocytes at admission were associated with bacterial infections and septic shock. Later altered immune/inflammatory responses in patients who died may had been driven by the development of septic shock. Conclusion: Burn injury induced an early and profound upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. Immune and inflammatory responses were associated with bacterial infection and septic shock. Absence of immune recovery patterns was associated with poor prognosis

Severe Altered Immune Status After Burn Injury Is Associated With Bacterial Infection and Septic Shock

International audienceIntroduction: Burn injury is associated with a high risk of death. Whether a pattern of immune and inflammatory responses after burn is associated with outcome is unknown. The aim of this study was to explore the association between systemic immune and inflammatory responses and outcome in severely-ill burn patients.Materials and Methods: Innate immunity, adaptive immunity, activation and stress and inflammation biomarkers were collected at admission and days 2, 7, 14, and 28 in severely-ill adult burn patients. Primary endpoint was mortality at day 90, secondary endpoint was secondary infections. Healthy donors (HD) served as controls. Multiple Factorial Analysis (MFA) was used to identify patterns of immune response.Results: 50 patients were included. Age was 49.2 (44.2–54.2) years, total burn body surface area was 38.0% (32.7–43.3). Burn injury showed an upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. High interleukin-10 (IL-10) at admission was associated with risk of death. However, no cluster of immune/inflammatory biomarkers at early timepoints was associated with mortality. HLA-DR molecules on monocytes at admission were associated with bacterial infections and septic shock. Later altered immune/inflammatory responses in patients who died may had been driven by the development of septic shock.Conclusion: Burn injury induced an early and profound upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. Immune and inflammatory responses were associated with bacterial infection and septic shock. Absence of immune recovery patterns was associated with poor prognosis

Long-term immune reconstitution and T cell repertoire analysis after autologous hematopoietic stem cell transplantation in systemic sclerosis patients

International audienceThe determinants of clinical responses after autologous hematopoietic stem cell transplantation (aHSCT) in systemic sclerosis (SSc) are still unraveled. We analyzed long-term immune reconstitution (IR) and T cell receptor (TCR) repertoire diversity in 10 SSc patients, with at least 6 years simultaneous clinical and immunological follow-up after aHSCT. Patients were retrospectively classified as long-term responders (A, n = 5) or non-responders (B, n = 5), using modified Rodnan's skin score (mRSS) and forced vital capacity (FVC%). All patients had similar severe SSc before aHSCT. Number of reinjected CD34 + cells was higher in group B versus A (P = 0.02). Long-term mRSS fall >25% was more pronounced in group A (P = 0.004), the only to improve long-term FVC% >10% (P = 0.026). There was an overall trend toward increased of T cell reconstitution in group B versus A. B cells had a positive linear regression slope in group A (LRS = 11.1) and negative in group B (LRS = −11.6). TCR repertoire was disturbed before aHSCT and the percentage of polyclonal families significantly increased at long-term (P = 0.046), with no difference between groups. Despite improved skin score after aHSCT in all SSc patients, pretransplant B cell clonal expansion and faster post-transplant T cell IR in long-term non-responder/relapsing patients call for new therapeutic protocols guided by IR analysis to improve their outcome

CD158k Is a Reliable Marker for Diagnosis of Sézary Syndrome and Reveals an Unprecedented Heterogeneity of Circulating Malignant Cells

The diverse aspects of cutaneous T-cell lymphomas may impede the diagnosis of Sézary syndrome (SS) and mycosis fungoides (MF), in particular, at early stages of the disease. We defined the CD158k/KIR3DL2 molecule as a first positive cell surface marker for Sézary cells (SCs). Here, we designed an optimized flow cytometry gating strategy, allowing the definition of lymphocytes of different sizes and defects of cell surface markers. Quantification by cytomorphology, flow cytometry, or clonal evaluation, gave similar results at initial time points and during the evolution in a prospective study involving 64 consecutive cutaneous T-cell lymphoma or erythrodermic patients. We found that CD158k+ T cells and circulating CD4+ T cells from MF patients exhibited unexpected patterns of cell surface expression with a marked heterogeneity of circulating lymphocytes even at initial diagnosis. Taken together, our results show that a multistep gating of CD158k+ cells is reliable to assess tumor burden in case of SS and suggest that both circulating MF CD4+ T cells and CD158k+ T cells are not homogeneous distinct memory populations. Further phenotypic and functional characterizations of such subsets are needed to better understand the underlying molecular mechanisms leading to the development of these diseases

Long term Immune Reconstitution and infection burden after Mismatched Hematopoietic Stem Cell Transplantation

Mismatched unrelated donor (MMUD) or umbilical cord blood (UCB) can be chosen as alternative donors for allogeneic stem cell transplantation but might be associated with long lasting immune deficiency. Sixty-six patients who underwent a first transplantation from either UCB or 9/10 MMUD (n= 36) and who survived beyond 3 months were evaluated. Immune reconstitution was prospectively assessed at sequential time points after transplantation. NK, B, CD4+ and CD8+T cells and their subsets as well as regulatory T cells (Treg) were studied. Detailed analyses on infections occurring after 3 months were also assessed. The 18-month cumulative incidences of infection-related death were 8 and 3%, and of infections were 72 and 57% after MMUD and UCB transplantation, respectively. Rates of infection per 12 patient-month were roughly 2 overall (1 for bacterial, 0.9 for viral and 0.3 for fungal infections). Memory, naïve CD4+ and CD8+T cells, naïve B cells and Treg cells reconstitution between the 2 sources was roughly similar. Absolute CD4+T cells hardly reached 500 per μL by one year posttransplantation and most B cells were of naïve phenotype. Correlations between immune reconstitution and infection were then performed by multivariate analyses. Low CD4+ and high CD8+T cells absolute counts at 3 months were linked to increased risks of overall and viral (but not bacterial) infections. When assessing for the naïve/memory phenotypes at 3 months among the CD4+ T cell compartment, higher percentages of memory subsets were protective against late infections: central memory CD4+T cells protected against overall and bacterial infections; late effector memory CD4+T cells protected against overall, bacterial and viral infections. At the opposite, high percentage of effector- and late effector-memory subsets at 3 months among the CD8+ T cell compartment predicted higher risks for viral infections. Patients transplanted from alternative donors represent a population with very high risk of infection. Detailed phenotypic analysis of immune reconstitution may help to evaluate infection risk and to adjust infection prophylaxis