31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Epidermal growth factor primes intestinal epithelial cells for proliferative effect of insulin-like growth factor I

Insulin-like growth factor I (IGF-I) synergistically enhances epidermal growth factor (EGF) -stimulated proliferation of intestinal epithelial cells. A possible mechanism of this synergy is that EGF acts as a competence factor increasing the fraction of proliferating cells by promoting transition from G0 to G1, thus allowing IGF-I, a progression factor, to act as a proliferative agent on the cycling population. Consistent with this hypothesis would be temporally distinct actions wherein initial brief exposure to EGF would permit synergy, whereas pretreatment with IGF-I would not. Rat intestinal epithelial cells of the IEC-18 crypt cell line were serum-deprived, then treated with EGF (5×10-9 M), IGF-I (5×10-9 M), or insulin (2×10-6 M) for a 30-min pulse and then media containing EGF, IGF-I, insulin, or no factor was substituted for 48 hr. IGF-I and EGF each stimulated enterocyte proliferation; together they synergistically promoted cell growth. A brief pulse of IGF-I neither induced cell proliferation nor enhanced the EGF effect. Initial brief exposure to EGF, however, was equally efficacious as continuous exposure and allowed full synergy with IGF-I. Insulin at supraphysiologic levels acted similarly to IGF-I. Thus, EGF acted as a competence factor priming the cells for subsequent action by IGF-I. The cell kinetic parameters of these growth factors may be important to both physiologic and pathologic enterocyte growth regulation. © 1994 Plenum Publishing Corporation

Autoradiographic mapping and characterization of insulin-like growth factor-I receptor binding in human greater saphenous vein

Purpose: The proliferation of vascular smooth muscle cells is an important step in the process of intimal hyperplasia. Veins exposed to arterial pressure develop intimal hyperplastic lesions that lead to failure of vein bypasses. Insulin-like growth factor-I is a polypeptide hormone structurally related to insulin with insulin-like metabolic effects. Insulin-like growth factor-I has been found to work in concert with other growth factors, including platelet-derived growth factor, to promote the growth of vascular smooth muscle cells in culture. Insulin-like growth factor-I exerts its effects via specific receptors located on the cell surface. We studied the in situ distribution of insulin-like growth factor-I receptor binding using autoradiography and examined insulin-like growth factor-I binding characteristics in normal human greater saphenous vein. Methods: Frozen sections 20 μm thick were prepared from the greater saphenous vein specimens. The sections were incubated in a buffer containing 125I-insulin-like growth factor-I in the presence of increasing concentrations of the unlabeled peptide. Autoradiograms were obtained by apposing the treated sections to autoradiography film. Results: Analysis of the autoradiographs showed that insulin-like growth factor-I binding was consistently present in the wall of human greater saphenous vein. To characterize these binding sites binding inhibition studies were performed. High-affinity insulin-like growth factor-I receptor binding was found with dissociation constant of 1.0 ± 0.32 nmol/L and maximum binding capacity of 0.46 ± 0.23 pmol/mg protein. These values are consistent with a physiologic role for insulin-like growth factor-I in the tissue examined. Conclusions: The presence of high-affinity (dissociation constant = 1.0 ± 0.32) insulin-like growth factor-I binding sites in the wall of saphenous vein suggests that insulin-like growth factor-I plays an important role in regulating the proliferation of venous wall cellular components, an essential step in the process of venous intimal hyperplasia. © 1993

VIP receptors and content after bowel transplantation

Advances in immunosuppressive therapy have renewed interest in small bowel transplantation. Little is known, however, about the functional capacity of transplanted intestine. To clarify the potential for normal function, we investigated whether elements of the enteric nervous system are preserved after denervation in our rat model of intestinal transplantation. We investigated whether VIP, a major peptide neurotransmitter of the enteric nervous system, and its receptors are preserved in the bowel after transplantation. In our model of transplantation, avascular fetal jejunum from term Fisher rats is transplanted to the subcutaneous tissues of host syngeneic rats. This neogut becomes vascularized and develops characteristics of native small bowel. VIP content was measured by RIA and the in situ distribution of VIP receptors was determined by the technique of receptor autoradiography. Neogut was studied 1 and 3 weeks after transplantation and compared with age-matched rat pup jejunum. Autoradiographs showed high silver grain density, representing VIP binding sites, in the mucosal layers of all tissues studied. VIP content in the transplanted bowel was comparable to that of native gut and showed a rise with developmental age similar to that of native gut. VIP levels (pmole/mg protein, x ± SEM) were neogut 1 week, 0.26 ± 0.14; jejunum 1 week, 0.25 ± 0.07; neogut 3 weeks, 0.60 ± 0.21; and jejunum 3 weeks, 0.69 ± 0.16. These results show that VIP receptors and content are preserved in this model of transplantation. This suggests that the enteric nervous system and receptors for peptide neurotransmitters remain intact after transplantation and may retain the potential for regulatory function. © 1989

Distribution of vasoactive intestinal polypeptide and substance P receptors in human colon and small intestine

Vasoactive intestinal polypeptide (VIP) and substance P are found in neurons in the lamina propria and submucosa and muscularis propria of human small intestine and colon. VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle, and mononuclear cells. This study analyzes the distribution of[125I]VIP binding and [125]substance P in human colon and small intestine using autoradiographic techniques. [125I]VIP binding was present in high density in the mucosal layer of colon and small intestine. [125I]VIP binding was not significantly greater than nonspecific binding in smooth muscle layers or the lymphoid follicles. In contrast, [125I]substance P binding was present in high density over the colonic muscle but was not present over the mucosal layer. In human colon cancer, [125I]VIP grain density over the malignant tissue was only slightly higher than background. These autoradiographic studies of [125I]VIP binding indicate that the highest density of VIP receptors was found in the small intestine and superficial colonic mucosa, whereas the density of substance P receptors was highest over the smooth muscle layers. These findings suggest a mismatch between immunochemical content of the peptide and autoradiographic density of the receptor. © 1989 Plenum Publishing Corporation

Primary culture of the enteric nervous system from neonatal hamster intestine. Selection of vasoactive intestinal polypeptide-containing neurons

The enteric nervous system is a major division of the autonomic nervous system and is responsible for the regulation of gastrointestinal function. The objective of the present study was to develop a simple and effective technique for isolating and culturing neurons of the enteric nervous system that would permit characterization of their development and regulatory peptide content. This was accomplished using a dispersed intestinal cell preparation cultured under conditions designed to support the growth and differentiation of neurons and neuroendocrine cells. Newborn hamster intestine was digested in 0.1% collagenase, mechanically dispersed, and cultured in RPMI 1640 supplemented with 2.5% serum and other additives. Phase and bright-field microscopy demonstrated neuronal cells and fibers after the second day in culture. This was confirmed by immunohistochemistry using antibodies directed against neurofilament and vasoactive intestinal polypeptide. Acetic acid extracts of the culture indicated that during the first 4 days of the culture the content of vasoactive intestinal polypeptide increased, whereas the content of substance P, mammalian bombesin, and neurotensin declined. High-performance liquid chromatography and fast protein liquid chromatography confirmed that the immunoreactive vasoactive intestinal polypeptide coeluted with synthetic and iodinated forms of the peptide. This study describes a technique for primary culture of intestinal tissue that supports the survival of enteric neurons and permits analysis of the development and synthetic and secretory characteristics of the enteric nervous system. © 1988

Increased specific binding of insulin‐like growth factor‐I in healing cutaneous wounds

Insulin‐like growth factor‐I is a polypeptide hormone structurally related to insulin. It is a potent mitogen that promotes growth and differentiation in many tissues. A role for insulin‐like growth factor‐I in wound healing is suggested by its rapid rise in levels and increased insulin‐like growth factor‐I messenger RNA expression in tissue after wounding. We designed our study to characterize possible changes in insulin‐like growth factor‐I receptor binding during wound healing. Surgical wounds created on the abdominal skin of anesthetized New Zealand White rabbits were either left open or closed primarily. Size‐ and weight‐matched specimens were harvested at wounding time (day 0), and at 1, 4, 7, 38, and 50 days after wounding. Preliminary experiments showed that the greatest difference in specific binding occurred between day 0 and day 7. 125I‐insulin‐like growth factor‐I binding studies were performed on frozen tissue specimens and autoradiography was performed and analyzed by computerized densitometry. Scatchard analysis of the binding data showed a single class of insulin‐like growth factor‐I binding sites whose affinity that is, binding constant (Kd = 0.6 × 10−9) did not change significantly over time; in contrast there was a threefold increase in the number of receptors per milligram tissue in day 7 wound tissue versus normal skin harvested at day 0 (17.3 ± 2.6 × 1010 versus 4.7 ± 2.5 × 1010, respectively, p \u3c 0.05). Binding inhibition experiments showed that 125I‐insulin‐like growth factor‐I binding was most specific to insulin‐like growth factor‐I with insulin‐like growth factor‐I \u3e insulin‐like growth factor‐II \u3e insulin. This increase in binding was due to upregulation of insulin‐like growth factor‐I receptors rather than increased levels of insulin‐like growth factor‐I binding protein as less than 20% of the threefold increase in binding at day 7 could be attributed to insulin‐like growth factor‐I binding protein in membrane‐free extracts. The presence of specific, high‐affinity insulin‐like growth factor‐I receptors in the skin and their upregulation at day 7 after wounding suggest that insulin‐like growth factor‐I plays an important role during wound healing. Copyright © 1995, Wiley Blackwell. All rights reserve