Roberta Carosio

NK cell-mediated lysis of autologous antigen-presenting cells is triggered by the engagement of the phosphatidylinositol 3-kinase upon ligation of the natural cytotoxicity receptors NKp30 and NKp46.

Interleukin‐2 (IL‐2)‐activated polyclonal or clonal NK cells lysed autologous antigen presenting cells (APC) through the engagement of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. NK cell‐mediated cytolysis of APC correlated with the surface density of these NCR. Indeed, NK cell clones bearing low amounts of NKp30 and NKp46 did not lyse autologous APC, whereas NK cell clones with bright expression of these NCR efficiently killed autologous APC. Upon masking of NKp30 or NKp46 by specific monoclonal antibodies a strong reduction (by 50%) of APC lysis could be detected and the complete inhibition was achieved by the simultaneous masking of these NCR. Interestingly, NK cell‐mediated APC lysis was impaired by the phosphatidylinositol 3‐kinase (PI‐3 K) inhibitors LY294002 or wortmannin. Similarly, these drugs strongly reduced NK cell activation triggered by NKp30 or NKp46 in a re‐directed killing assay as well as the activation of Akt/PKB, substrate of PI‐3 K, induced by the engagement of these receptors. Altogether, these findings strongly suggest that NCR are responsible for the killing of autologous APC through the activation of PI‐3 K.

NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase II: Inhibition by HIV-1 Tat C-Terminal Domain

In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-γ secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-γ release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.

Transendothelial Migratory Pathways of Vδ1+TCRγδ+ and Vδ2+TCRγδ+ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients: Involvement of Phosphatidylinositol 3 Kinase and Calcium Calmodulin-Dependent Kinase II

We have previously reported that the Vδ2+TCRγδ+ T lymphocyte subset, expressing the NK receptor protein 1a (NKRP1a; CD161), is expanded in patients with relapsing-remitting multiple sclerosis and uses this molecule to migrate through endothelium. In this work, we show that Vδ1+ and Vδ2+ γδ T lymphocytes use distinct signal transduction pathways to accomplish this function. Indeed, we have found that Vδ1+ cells lack NKRP1a and selectively express the platelet endothelial cell adhesion molecule 1 (PECAM1; CD31), which drives transendothelial migration of this cell subset, at variance with Vδ2+ T cells, which are PECAM1 negative and use NKRP1a for transmigration. Interestingly, when Vδ2+ T cells were pretreated with two specific inhibitors of the calcium calmodulin-dependent kinase II KN62 and KN93, but not with the inactive compound KN92, the number of migrating cells and the rate of transmigration were significantly decreased. In turn, the phosphatidylinositol 3 kinase blockers wortmannin and LY294002 exerted a dose-dependent inhibition of Vδ1+ cell migration. Finally, NKRP1a and PECAM1 engagement led to activation of different signal transduction pathways: indeed, oligomerization of NKRP1a on Vδ2+ T cells activates calcium calmodulin-dependent kinase II, while occupancy of PECAM1 on Vδ1+ cells triggers the phosphatidylinositol 3 kinase-dependent Akt/protein kinase Bα activation. These findings suggest that subsets of γδ T lymphocytes may migrate to the site of lesion in multiple sclerosis using two different signaling pathways to extravasate.

IFN‐γ production in human NK cells through the engagement of CD8 by soluble or surface HLA class I molecules

The engagement of CD8 on NK cell surface by either surface or soluble HLA class I (sHLA‐I) molecules induces synthesis and secretion of IFN‐γ. HLA‐I‐mediated effects were inhibited by the covering of CD8 with specific anti‐CD8 monoclonal antibodies, indicating a direct interaction of HLA‐I and CD8. That CD8 ligation induces IFN‐γ production was further supported by the finding that cross‐linking of CD8 led to release of IFN‐γ at similar levels to those obtained with HLA‐I. The sHLA‐I‐induced IFN‐γ production via CD8 was strongly down‐regulated by the engagement of the inhibitory isoforms of either CD94/NKG2 complex by sHLA‐I‐non‐(A,B,C,G) (putative sHLA‐E) or CD158b by sHLA‐I‐Cw3 allele. Ligation of CD8 did not elicit, different from other activating NK cell surface molecules such as CD16 or CD69, triggering of NK cell‐mediated cytolysis. Cyclosporin A, but not concanamycin A, an H+‐ATPase vacuolar inhibitor which affects perforin and granzyme release, strongly reduced the sHLA‐I‐mediated CD8‐dependent IFN‐γ production but did not affect cytolytic activity of NK cells, suggesting that different biochemical pathways are involved. Altogether, these findings indicate that CD8 engagement by sHLA‐I activates a cyclosporin A‐dependent pathway leading to production and secretion of IFN‐γ which may play a role in the regulation of innate immune responses in humans.

β3-Mediated engulfment of apoptotic tumor cells by dendritic cells is dependent on CAMKII: inhibition by HIV-1 Tat

In this paper, we show that the engulfment of apoptotic tumor cells by DC requires the activation of the calcium‐calmodulin kinase II (CAMKII). Indeed, DC phagocytosis of apoptotic lymphoma cells is consistently inhibited by KN62 and KN93, two blockers of CAMKII, but not by the inactive compound KN92. Wortmannin and LY294002, two inhibitors of the phosphatidyl‐inositol‐3 kinase, slightly decrease the phagocytosis of apoptotic cells, at variance with PD98059, an inhibitor of the mitogen‐activated protein kinase. It is interesting that the addition of synthetic HIV‐1 Tat, which we demonstrated to inhibit phagocytosis and calcium influx in DC, blocks the activation of CAMKII elicited via β3 integrin, which is involved in apoptotic body engulfment by DC. Experiments performed with Tat‐derived peptides showed that this inhibition is mediated by the C‐terminal domain of Tat. Finally, pertussis toxin can prevent HIV‐1 Tat‐mediated inhibition, suggesting the involvement of a guanosine triphosphate‐binding (G) protein in DC‐mediated phagocytosis.

Association of CTLA-4 gene variants with response to therapy and long-term survival in metastatic melanoma patients treated with ipilimumab: An Italian melanoma intergroup study

Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (−1661A>G, −1577G>A, −658C>T, −319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan–Meier method. Both −1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A “dose” (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, −1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying −1577G/A and CT60G/A, respectively. MM patients carrying −1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.

Immune Checkpoints and Innovative Therapies in Glioblastoma

Targeting the Immune Checkpoint molecules (ICs; CTLA-4, PD-1, PD-L1/2, and others) which provide inhibitory signals to T cells, dramatically improves survival in hard-to-treat tumors. The establishment of an immunosuppressive environment prevents endogenous immune response in glioblastoma; therefore, manipulating the host immune system seems a reasonable strategy also for this tumor. In glioma patients the accumulation of CD4+/CD8+ T cells and Treg expressing high levels of CTLA-4 and PD-1, or the high expression of PD-L1 in glioma cells correlates with WHO high grade and short survival. Few clinical studies with IC inhibitors (ICis) were completed so far. Notably, the first large-scale randomized trial (NCT 02017717) that compared PD-1 blockade and anti-VEGF, did not show an OS increase in the patients treated with anti-PD-1. Several factors could have contributed to the failure of this trial and must be considered to design further clinical studies. In particular the possibility of targeting at the same time different ICs was pre-clinically tested in an animal model were inhibitors against IDO, CTLA-4 and PD-L1 were combined and showed persistent and significant antitumor effects in glioma-bearing mice. It is reasonable to hypothesize that the immunological characterization of the tumor in terms of type and level of expressed IC molecules on the tumor and TIL may be useful to design the optimal ICi combination for a given subset of tumor to overcome the immunosuppressive milieu of glioblastoma and to efficiently target a tumor with such high cellular complexity.