PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαßγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαßγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαßγ to a greater extent than for IL2Rßγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rßγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

[THE INDUCTION OF CD25 EXPRESSION IN Jurkat T CELLS].

The expression of an α-subunit of interleukin-2 receptor (IL-2Rα) was assessed by quantifying activation-induced upregulation of CD25 in IL-2-independent Jurkat leukemic cell line. It has been found that in growing Jurkat culture within 24 h, phytohemagglutinin (PHA, 5 µg/ml) or PHA in combination with 12,13-phorbol dibutirate (PDBu, 10(-8)M) increase the number of CD25+ cells to 32.3 ± 3.4% (n = 11) and 44.8 ± 8.6% (n = 6) respectively. Interleukin-2 (IL-2, 200 U/ml) alone or in combination with PDBu did not induce CD25 expression in Jurkat cells. All the tested stimulatory agencies affected neither the proliferation status no the growth of Jurkat cell cultures. In contrast to human blood T cells, WHI-P131, a selective pharmacological inhibitor of JAK/STAT signaling and CD25 expression, did not decrease the number of induced CD25+ cells in Jurkat culture. Flow cytometry analysis revealed a dose-dependent decrease in the proportion of cells in G1 phase and an increase in the proportion of cells in G2/M phase in WHI-P131-treated Jurkat cultures. It has been also found that WHI-P131 induces G2/M arrest in the absence of PHA or PDBu. We have concluded that (1) the IL-2-independent T cells of Jurkat line had not loss the mechanism for IL-2Rα expression in response to T cell receptor activation, (2) in the transformed T cells, WHI-P131 can arrest cell cycle at G2/M phase and has effects on targets other than IL-2 receptor-associated tyrosine kinase JAK3.

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells.

Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25(+) B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25(+) cells for studying immune regulation.

Porin of Shigella dysenteriae directly promotes toll-like receptor 2-mediated CD4+ T cell survival and effector function.

Porin of Shigella dysenteriae type 1 up-regulated Toll-like receptor (TLR)2 on CD3-stimulated CD4(+) T cells but could not induce the expression of other TLRs. TLR2 in association with myeloid differentiating factor 88 (MyD88) triggered the downstream signal transduction pathway leading to activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB), and degradation of IkappaB, the NF-kappaB inhibitor. TLR2 co-stimulation by porin resulted in T cell expansion by inducing both proliferation and survival of the CD4(+) T cells. Extracellular signal-regulated kinase (ERK)1/2 activation inhibitor U0126 and NF-kappaB translocation inhibitor SN-50 significantly inhibited proliferation of T cells, highlighting a direct role of ERK and NF-kappaB in the process. However, cell survival involving Bcl-X(L) induction was found to be regulated essentially by ERK with no significant role of NF-kappaB. Porin-induced proliferation was supported by induction of IL-2 and CD25 that are known to play a pivotal role in T cell expansion. Apart from inducing T cell proliferation, porin triggered effector functions of the cells, evident from TLR2- and MyD88-dependent release of type 1 cytokines tumor necrosis factor (TNF) and interferon (IFN)-gamma along with the induction of type 1 chemokines macrophage-inflammatory protein (MIP)-1alpha and MIP-1beta and their receptor CCR5. The proliferation, survival and effector function of CD4(+) T cells through TLR2 co-stimulation show the capability of porin to directly turn adaptive immunity into action.

Apolipoproteins inhibit the innate immunity activated by necrotic cells or bacterial endotoxin.

We suggested earlier that the hydrophobic portions (Hyppos) of molecules, which are normally embedded in the membranes of cells or the core of molecular structures so as to be separated from the aqueous environment, might serve as evolutionarily ancient alarm signals of injury or stress to initiate innate immune responses when they are exposed on the surface. Under normal physiological conditions, the Hyppos released from endogenous or exogenous sources might be handled by 'Hyppo-quenchers'in vivo to maintain the tissue homeostasis and immune modulation. To test this idea, we selected apolipoproteins, which have been known to transport blood lipids and play a role in a number of pathological inflammatory conditions. We examined their role as Hyppo-quenchers in early immune responses and found that apolipoproteins showed significant inhibition of the nuclear factor-kappaB-dependent gene expression in recombinant Chinese hamster ovary (CHO) cells and dendritic cells stimulated by necrotic cells or bacterial endotoxin. In addition, our results indicate that apolipoproteins could dramatically abrogate complement fixation on the surface of necrotic cells. These findings suggest that apolipoproteins, besides having known functions in lipid metabolism, also have a role in preventing the initiation of innate immunity, potentially through neutralizing Hyppos from injured cells or exogenous endotoxin.

[Correlation of BrdU incorporation with activation and cytokine expression of T cells].

AIM: To investigate the correlation of BrdU incorporation with the activation and cytokine expression of T cells. METHODS: PBMCs from healthy persons were isolated and stimulated by PMA plus Ionomycin at different periods of time, BrdU was then added to the cells one hour before the end of culture. The cells were harvested and stained with anti-BrdU, anti-cell surface and intracellular antibodies. Then the cells were washed and analyzed by flow cytometer. RESULTS: The peak of BrdU incorporation was observed in T cells after they were stimulated for 48 hours in vitro, but no further increase of the peak of BrdU incorporation was found after incubated for a longer period of time. The comparison made between BrdU incorporation and cell activation indicated CD69 expression reached the peak after stimulated for 8 hours whereas CD25 was at the peak after stimulated for 24 hours. Furthermore, no correlation between BrdU incorporation and cytokine production was observed. High frequency of IFN-gamma producing cells was detected after stimulated for 8 hours but no obvious increase was observed for a longer period of time. When PBMC were stimulated with OKT3 plus antiCD28, the percentage of BrdU(+) T cells was higher than that stimulated by PMA plus Ionomycin. Similarly, the percentage of BrdU(+) CD8(+) T cells was higher than that of BrdU(+) CD4(+) T cells. CONCLUSION: The percentage of BrdU(+) cells can be detected by flow cytometer to evaluate the proliferation of T cells. Only a few T cells proliferate after polyclonal stimulation and BrdU incorporation is dependent on stimulants and time of stimulation. Therefore, BrdU incorporation is not correlated with activation markers and cytokine production.

Cutting edge: mechanism of enhancement of in vivo cytokine effects by anti-cytokine monoclonal antibodies.

Inhibitory anti-cytokine mAbs are used to treat cytokine-mediated disorders. Recently, however, S4B6, an anti-IL-2 mAb that blocks IL-2 binding to IL-2Ralpha, a receptor component that enhances affinity but is not required for signaling, was shown to enhance IL-2 agonist effects in vivo. We evaluated how S4B6 enhances IL-2 effects and whether a similar mechanism allows mAbs to IL-4 to enhance IL-4 effects. Induction of T cell proliferation by IL-2/S4B6 complexes did not require complex dissociation and was IL-2Ralpha independent. S4B6 increased IL-2 agonist effects by increasing in vivo half-life, not by focusing IL-2 onto cells through Fc receptors. In contrast to IL-2/S4B6 complexes, anti-IL-4 mAb enhancement of in vivo IL-4 effects required IL-4/anti-IL-4 mAb complex dissociation. Thus, agonist effects observed with high doses of anti-IL-2 mAb are most likely only applicable for mAbs that maintain cytokine half-life without blocking binding to receptor signaling components.

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha.

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.