Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated cord blood-derived NKcells facilitates CAR-like responses against CD30+ malignancies
Purpose: Natural killer (NK) cell recognition and function against NK-resistant cancers remains substantial barriers to the broad application of NK cell immunotherapy. Potential solutions include bispecific engagers that target NK cell activity via an NK activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL-12/15/18 cytokine pre-activation.
Experimental design: We assessed single-cell NK cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL-12/15/18 pre-activation of blood and cord-blood-derived NK cells was investigated in vitro and in vivo.
Results: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood NK cells pre-activated with IL-12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo.
Conclusions: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.
Description: A polyclonal antibody against CDK1 (Ab-161). Recognizes CDK1 (Ab-161) from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IF;WB:1:500-1:1000, IF:1:100-1:200
Description: A polyclonal antibody against CDK1 (Ab-161). Recognizes CDK1 (Ab-161) from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IF;WB:1:500-1:1000, IF:1:100-1:200
Description: A polyclonal antibody against LAT (Ab-255). Recognizes LAT (Ab-255) from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, WB;WB:1:500-1:3000
×
Low-dose interleukin-2-loaded nanoparticle effect on NK and T-reg cell expression in experimentally induced type 1 diabetes mellitus
Introduction: Type 1 diabetes mellitus is an autoimmune disorder characterized by inflammatory damage to pancreatic β cells resulting in loss of insulin secretion. In autoimmune type 1 diabetes mellitus (T1D) natural killer cells (NK) initiate pancreatic islets cell lyses in autoimmune T1D. Loss of T regulatory cells (Treg) at disease onset facilitates the activation and accumulation of NKs in the pancreatic microenvironment. A proper low-dose interleukin 2 (IL-2) could enhance Tregs and enforce control and regulation of pro-inflammatory NKs.
Aim: This relation needs to be studied to improve therapeutic strategies aimed at resetting the balance between Tregs and proinflammatory cells.
Material and methods: We used novel formulations of low-dose IL-2 loaded on chitosan nanoparticles. The study included 116 T1D BALB/c mice experimentally induced by streptozotocin, divided into groups. Their splenocytes were maintained in a short-term culture for assessment of expression of CD4+FOXP3+ Treg and NKp46+NK by both flow cytometry and enzyme-linked immunoassay (ELISA). Morphological, immunohistochemical, and morphometrical analyses were done. In vitro suppressor assay was used to assess the suppressor effect of Treg cells after exogenous IL-2 treatment.
Results: NK cell expression, NKp46 level, and NK cell functions were modulated more in mice injected with IL-2-loaded chitosan nanoparticles than in other groups. A statistical inverse correlation was found between Treg and NK cell expression in IL-2-loaded chitosan with 0.3 µIU (p = 0.047), and this correlation was related to FOXP3 expression on Treg cells. The modified expression of NK and NKp46 was noticed in mice injected with 0.3 µIU for longer duration (3 weeks) (p < 0.001), but the NK functions did not show any significant changes with prolonged treatment.
Conclusions: Prolonged administration of low-dose IL-2 results in the vigorous expression of NKp46, indicating a significant role of Tregs in NK stimulation and motivation. Low-dose IL-2 selectively modulates NKp46 NK and FOXP3+ Tregs and increases their expression.
NKCells and PMN-MDSCs in the Graft From G-CSF Mobilized Haploidentical Donors Display Distinct Gene Expression Profiles From Those of the Non-Mobilized Counterpart
A recent approach of hematopoietic stem cell (HSC) transplantation from haploidentical donors “mobilized” with G-CSF is based on the selective depletion of αβ T and B lymphocytes from the graft. Through this approach, the patient receives both HSC and mature donor-derived effector cells (including NK cells), which exert both anti-leukemia activity and protection against infections. We previously showed that donor HSC mobilization with G-CSF results in accumulation in the graft of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), capable of inhibiting in vitro the anti-leukemia activity of allogeneic NK cells. Here, we performed a detailed gene expression analysis on NK cells and PMN-MDSCs both derived from mobilized graft.
Cytotoxicity assays and real time PCR arrays were performed in NK cells. Microarray technology followed by bioinformatics analysis was used for gene expression profiling in PMN-MDSCs. Results indicate that NK cells from the graft have a lower cytolytic activity as compared to those from non-mobilized samples. Further, mobilized PMN-MDSCs displayed a peculiar transcriptional profile distinguishing them from non-mobilized ones. Differential expression of pro-proliferative and immune-modulatory genes was detected in mobilized PMN-MDSCs. These data strengthen the concept that G-CSF-mobilized PMN-MDSCs present in the graft display unique molecular characteristics, in line with the strong inhibitory effect on donor NK cells.
Modulation of CXCR1 and CXCR3 expression on NKcells via Tim-3 in a murine model of primary biliary cholangitis
Tim-3, which is expressed on a variety of innate immune cells including NK cells, plays a key role in many autoimmune diseases. However, the immunomodulatory actions of Tim-3 on NK cells in primary biliary cholangitis (PBC) remain uncertain. Using a murine model of PBC we evaluated the expression of Tim-3 and its ligand Gal-9 in peripheral blood, liver, and spleen. Additionally, we studied Tim-3 regulation of chemokine receptors (CXCR1 and CXCR3) in vitro. Flow cytometric analysis indicated large numbers of infiltrating NK cells in the liver which exhibited high expression of Tim-3 and CXCR3. Moreover, we found overexpression of CXCR1 in liver tissue and liver-derived NK cells in PBC mice.
We also observed lower levels of soluble Tim-3 in the serum of PBC mice. In vitro experiments with liver-derived NK cells from PBC mice indicated that CXCR3 was up-regulated by treatment with recombinant mouse TIM-3 Fc (rmTim-3 Fc) to activate the Tim-3 pathway. Furthermore, stimulating normal mouse spleen NK cells with poly I:C resulted in elevated expression of CXCR1 and interferon-γ release. Nonetheless, adding rmTim-3 Fc or rmGal-9 significantly down-regulated CXCR1 expression and IFN-γ release in NK cells activated by poly I:C, proposing a means to exploit the Tim-3 pathway to reverse responses in NK cells. In conclusion, our data demonstrate that dysregulation of Tim-3/Gal-9 is involved in modulating the local immune microenvironment in PBC mice. Our findings highlight the potential of Tim-3 pathway to modulate chemokine responses in NK cells during autoimmunity.
