Dynamic of systemic immunity and its impact on tumor recurrence after radiofrequency ablation of hepatocellular carcinoma.

Background: Percutaneous radiofrequency ablation (RFA) is one of the main treatments of small hepatocellular carcinoma (HCC). However, it remains unclear whether this local treatment can induce systemic immune variations. Methods: We conducted a prospective study in a tertiary center including consecutive cirrhotic patients with unifocal HCC<5 cm treated by a first RFA between 2010 and 2014. Peripheral blood mononuclear cells were isolated on the day before (D0), day after (D1) and month after RFA (M1). Frequencies and phenotypes of myeloid cells, T cells, and NK cells were compared between timepoints. Overall recurrence and associated variables were estimated using Kaplan-Meier, log-rank and Cox proportional-hazards models. Results: 80 patients were included (69% male, median age: 67 years old). Main aetiologies of HCC were alcohol (51%), hepatitis C virus (45%), non-alcoholic steatohepatitis (36%) and hepatitis B virus (9%). Median overall survival was 55 months (M); median progression-free survival was 29.5M. Among innate immune populations, we observed variations between D0, D1 and M1 in NKp30+ NK cells (p < .0001) and in plasmacytoid dendritic cells (pDC, p < .01). Concerning adaptive immunity, we observed variations in CD8 Central Memory (p < .05) and CD28+ CD8 Central Memory (p < .01). An early dynamic (D0/D1) of activated NKp30+ NK cells was associated with a decreased overall recurrence (log-rank, p = .016, median delay 25.1 vs 40.6 months). In contrast, a late dynamic (D1/M1) of immature NK cells (CD56bright) and altered myeloid DC (PDL1+) was associated with an increased overall recurrence (log-rank, p = .011 and p = .0044, respectively). In multivariate analysis, variation of immature NK cells predicts tumor recurrence independently of classical clinical prognostic features (HR = 2.41, 95% CI: 1.15-5.057), p = .019). Conclusions: Percutaneous RFA of small HCC leads to systemic modifications of innate and adaptive immunity closely linked with overall tumor recurrence.

NKG2C+ memory-like NK cells contribute to the control of HIV viremia during primary infection: Optiprim-ANRS 147.

Natural-killer (NK) cells are important immune effectors during a viral infection. Latent CMV infection is widely spread and was demonstrated to shape the NK cell repertoire through the NKG2C receptor. An expansion of NKG2C+ NK cells has been reported during primary HIV infection (PHI), but their role is not known. We previously found a correlation between the maturation state of the NK cell compartment and a lower viral load by studying patients from the ANRS 147 Optiprim trial. We investigated here extensively the NKG2C+ NK cells at the time of PHI and its evolution after 3 months of early antiretroviral therapy (combination antiretroviral therapy (cART)). Multiparametric cytometry combined with bioinformatics was used to determine subsets. NKbright NKG2C+ progenitor, NKdim NKG2C+ effector and NKdim NKG2C+CD57+ memory-like populations were identified. Two groups of patients were unraveled according to the distribution of the NKG2C+ subsets skewed toward either progenitor/effector or memory-like phenotype. Patients with high NKG2C+CD57+ NK cell frequencies showed lower HIV-RNA, lower immune activation, higher pDC counts and reached more rapidly undetectable levels of HIV-RNA at M1 under cART. NKG2C+CD57+ NK cell frequency was the only factor strongly correlated to low viral load among other clinical features. While the patients were cytomegalovirus (CMV) infected, there was no sign of reactivation of CMV during PHI suggesting that memory-like NK cells were already present at the time of HIV infection and constituted a preexisting immune response able to contribute to natural control of HIV. This parameter appears to be a good candidate in the search of predictive markers to monitor HIV remission.

A Mature NK Profile at the Time of HIV Primary Infection Is Associated with an Early Response to cART.

Natural killer (NK) cells are major effectors of the innate immune response. Despite an overall defect in their function associated with chronic human immunodeficiency virus (HIV) infection, their role in primary HIV infection is poorly understood. We investigated the modifications of the NK cell compartment in patients from the ANRS-147-Optiprim trial, a study designed to examine the benefits of intensive combination antiretroviral therapy (cART) in patients with acute or early primary HIV infection. Multiparametric flow cytometry combined with bioinformatics analyses identified the NK phenotypes in blood samples from 30 primary HIV-infected patients collected at inclusion and after 3 months of cART. NK phenotypes were revealed by co-expression of CD56/CD16/NKG2A/NKG2C and CD57, five markers known to delineate stages of NK maturation. Three groups of patients were formed according to their distributions of the 12 NK cell phenotypes identified. Their virological and immunological characteristics were compared along with the early outcome of cART. At inclusion, HIV-infected individuals could be grouped into those with predominantly immature/early differentiated NK cells and those with predominantly mature NK cells. Several virological and immunological markers were improved in patients with mature NK profiles, including lower HIV viral loads, lower immune activation markers on NK and dendritic cell (DC), lower levels of plasma IL-6 and IP-10, and a trend to normal DC counts. Whereas all patients showed a decrease of viremia higher than 3 log10 copies/ml after 3 months of treatment, patients with a mature NK profile at inclusion reached this threshold more rapidly than patients with an immature NK profile (70 vs. 38%). In conclusion, a better early response to cART is observed in patients whose NK profile is skewed to maturation at inclusion. Whether the mature NK cells contributed directly or indirectly to HIV control through a better immune environment under cART is unknown. The NK maturation status of primary infected patients should be considered as a relevant marker of an immune process contributing to the early outcome of cART that could help in the management of HIV-infected patients.

Early recovery of immunity after cord-blood transplantation in children: qualitative features and impact of cytomegalovirus infection.

This study investigated immune recovery and the effect of cytomegalovirus (CMV) infection on the early stages of T-lymphocyte recovery after cord-blood transplantation (CBT) from unrelated donors to 24 children. In addition, 15 children who were recipients of a bone-marrow transplant (BMT) were studied concomitantly. The main objectives of the study were whether lymphocytes from CBT recipients were capable of rapid qualitative differentiation in the effector memory (EM) stage, of regenerating de novo naïve cells and of responding to CMV infection. Results showed that CB lymphocytes were mainly differentiated into the EM stage at 3 months and into the naïve stage at 6 months. CMV infection induced a dramatic increase in CD8 but not CD4 T-cell counts, and differentiation into the EM stage with high perforin contents. Our data suggest that CB lymphocytes are capable of rapid differentiation in children, but stabilization of lymphocyte counts in children is associated with effective naïve subset regeneration at 6 months. Moreover, the T-cell repertoire is capable of rapid adjustment when CMV infection occurs.

Primary B-CLL resistance to NK cell cytotoxicity can be overcome in vitro and in vivo by priming NK cells and monoclonal antibody therapy.

Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy.

Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction.

Natural killer (NK) cells play an important role in tumor-cell clearance, particularly against leukemia, as shown by killer cell inhibitory receptor (KIR)-mismatched allogeneic stem cell transplantation. Analysis of in vitro IL-2-expanded NK cells from patients with myelocytic/monocytic acute myeloid leukemia (AML-NK cells) has revealed poor cytolytic functions because of deficient expression of pivotal activation molecules-the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46. To exclude the possibility that this observation was caused by the in vitro amplification of a small NCR(dull) population, we analyzed the AML-NK phenotype directly, without any in vitro expansion. We first confirmed that the NCR(dull) phenotype was not an in vitro artifact. Moreover, analysis of a large population of AML patients allowed us to demonstrate that phenotype was not restricted to a French-American-British (FAB) subtype and was not associated with a particular cytogenetic abnormality. Our longitudinal study of AML patients showed that the NCR(dull) phenotype was acquired during leukemia development because we observed its complete (for NKp46) or partial (for NKp30) reversibility in patients achieving complete remission (CR). Reversibility of the NCR(dull) phenotype after CR suggested that leukemia cells might be involved in NCR down-regulation. In agreement with this hypothesis, direct contact between leukemic blasts and NK cells (but not leukemia-cell supernatants) induced loss or decrease in NKp30 and NKp46 expression while impeding NKp44 induction by IL-2. We excluded the major implication of TGF-beta in NCR down-regulation. Although the clinical antitumor value of NK cells is clearly demonstrated in allogeneic stem cell transplantation, the role of NK cells in autologous transplantation is not proved. Interestingly, we observed a correlation between the NCR(dull) phenotype and poor survival in AML patients, suggesting that NK-deficient activation caused by NCR down-regulation could play a role in patient outcome. The prognostic value of NCR expression is discussed, and pathophysiologic implication of the NCR phenotype will be further investigated in a larger study.

The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function.

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.

LIGHT costimulates CD40 triggering and induces immunoglobulin secretion; a novel key partner in T cell-dependent B cell terminal differentiation.

The T cell-dependent differentiation and function of B lymphocytes are tightly regulated by TNF ligands (L) and receptors interactions, such as CD40/CD40L, CD27/CD70 and CD134/CD314L. The LIGHT/HVEM system [homologous to lymphotoxin, inducible expression, competing for GpD of herpes virus, that binds to the herpes virus entry mediator (HVEM), and is expressed on activated T lymphocytes) focused our attention since HVEM has a large distribution that, in addition to T cells, DC or NK, includes tumor and normal B lymphocytes. HVEM was expressed on memory and naive B cells from peripheral blood or tonsils, but not on germinal center (GC) B cells. Costimulation by CD40L+LIGHT induced LIGHT expression at the B lymphocyte surface by a transcriptional mechanism since we detected de novo expression of LIGHT-specific mRNA. LIGHT expression was further enhanced by triggering of surface IgM, a stimulus that mimics a normal step of B cell physiology, i.e. specific antigen encounter. Stimulation by LIGHT increased the B cell proliferation induced by CD40L, and induced IgG and IgM (but not IgA) secretion. We conclude that LIGHT costimulation, that mimics the B cell encounter with activated LIGHT-expressing T lymphocytes, enhances both B cell proliferation and Ig production, and thus has a central importance for humoral immunity development.