Background: Targeting long-lasting insulins to the liver may improve metabolic alterations that are not corrected with current insulin replacement therapies. However, insulin is only able to promote lipogenesis but not to block gluconeogenesis in the insulin-resistant liver, exacerbating liver steatosis associated with diabetes. Methods: In order to overcome this limitation, we fused a single-chain insulin to apolipoprotein A-I, and we evaluated the pharmacokinetics and pharmacodynamics of this novel fusion protein in wild type mice and in db/db mice using both recombinant proteins and recombinant adenoassociated virus (AAV). Results: Here, we report that the fusion protein between single-chain insulin and apolipoprotein A-I prolonged the insulin half-life in circulation, and accumulated in the liver. We analyzed the long-term effect of these insulin fused to apolipoprotein A-I or insulin fused to albumin using AAVs in the db/db mouse model of diabetes, obesity, and liver steatosis. While AAV encoding insulin fused to albumin exacerbated liver steatosis in several mice, AAV encoding insulin fused to apolipoprotein A-I reduced liver steatosis. These results were confirmed upon daily subcutaneous administration of the recombinant insulin-apolipoprotein A-I fusion protein for six weeks. The reduced liver steatosis was associated with reduced body weight in mice treated with insulin fused to apolipoprotein A-I. Recombinant apolipoprotein A-I alone significantly reduces body weight and liver weight, indicating that the apolipoprotein A-I moiety is the main driver of these effects. Conclusion: The fusion protein of insulin and apolipoprotein A-I could be a promising insulin derivative for the treatment of diabetic patients with associated fatty liver disease.

Background Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment. Methods In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic-polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry. Results The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8(+) T lymphocytes at day 8. Depletion of either myeloid cells or CD8(+) T lymphocytes diminished the anti-tumour efficacy of the combination. Conclusions The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their "master regulator". Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called "Foxp3 interactome", which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein-protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

Blocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immuno

Several studies have shown that some liver transplant recipients may tolerate immunosuppression withdrawal. Mechanisms and biomarkers of tolerance are not well known. Methods: Twenty-four LT patients with immunosuppression side-effects underwent progressive immunosuppression withdrawal. Peripheral lymphocyte populations and secretion of cytokines were analyzed at baseline and during withdrawal until tolerance (n = 15) or rejection (n = 9), as well as 3. months after tolerance achievement or rejection resolution (as follow-up). Immunological markers were compared among groups. Results: The percentages of CD3 + CD4 + cells progressively decreased in both groups. CD3 + CD8 + cells gradually increased in tolerant patients. B lymphocytes gradually decreased in tolerant and initially in non-tolerant patients, reverting at rejection. Regulatory T cells progressively increased until rejection in non-tolerants, decreasing to basal levels after renewing immunosuppression; no significant changes were found in tolerant patients. The percentages and absolute counts of natural killer cells significantly increased in both groups, being more evident in tolerant patients. The secretion of several cytokines was higher in non-tolerant patients when rejection was diagnosed. Conclusions: The greater increase of natural killer cells in tolerant patients suggests their potential role in the tolerance phenomenon

Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4+ T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-¿, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-ß. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

Peptide vaccines derived from CD8(+) T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8(+) T-cell responses, even in the absence of CD4(+) T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8(+) T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8(+) T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8(+) T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8(+) T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8(+) T cells. Taken together, these results suggest that CD40L expression induced by potent CD8(+) T-cell epitopes can activate antitumor CD8(+) T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8(+) T-cell epitopes and bypassing the requirement for CD4(+) helper T cells in vaccination protocols.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4+, CD8+, and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range¿=¿8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P¿=¿0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P¿=¿0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n¿=¿7 for an interval¿>¿10 years and an SI¿<¿20), 100% reached tolerance; in the second group (n¿=¿10 for an interval¿>¿10 years and an SI¿>¿20 or an interval¿<¿10 years and an SI¿<¿20), 60% reached tolerance; and in the third group (n¿=¿7 for an interval¿<¿10 years and an SI¿>¿20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables¿the time from transplantation and the SI¿may help to identify these patients.