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

Prolactin receptor gene diversity in Azara's owl monkeys (Aotus azarai) and humans (Homo sapiens) suggests a non-neutral evolutionary history among primates

Although paternal care is rare in mammals, males of several primate taxa exhibit high degrees of this behavior. Studies of many species of vertebrates found a positive correlation between prolactin (PRL) levels and paternal care. Studies of maternal care in knockoutmice indicate that the prolactin receptor (PRLR) plays a critical role in the neural regulation of parental care. To understand better the extent of PRLR genetic variation within primates, we characterized intraspecific coding variation in Azara?s owl monkeys (Aotus azarai) fromnorthern Argentina, a species with intensive paternal care. We then examined PRLR variation in 1088 humans (Homo sapiens) from the 1000 Genomes Project. Lastly, we assessed interspecific variation in PRLR in 4 different Aotus spp. and 12 phylogenetically (and behaviorally) disparate primate taxa. Our analyses revealed that the coding region of PRLR exhibits significant variation in all species of primates, with nonsynonymous amino acid substitutions being enriched in the intracellular domain, a region responsible for activation of downstream targets in thePRL pathway. In addition, several species exhibit entire codon deletions in this region. These results suggest a non-neutral evolutionary history of the PRLR locus within different primate lineages, and further imply that the translated PRLR protein has undergone considerable change throughout primate evolution. Such changes may be driven by selection for different behaviors and physiologies resulting from modulations of the pleiotropic prolactin pathway. Yet, the genetic variants in PRLR among primate taxa do not discretely cluster with species-level differences in paternal care behaviors, signifying that other mechanisms must be involved in the regulation of paternal care in primates.Fil: Babb, Paul L.. University of Pennsylvania; Estados Unidos de América;Fil: McIntosh, Annick M.. University Of Yale; Estados Unidos de América;Fil: Fernandez Duque, Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Centro de Ecologia Aplicada del Litoral (i); Argentina;Fil: Schurr, Theodore G.. University of Pennsylvania; Estados Unidos de América

An Optimized Microsatellite Genotyping Strategy for Assessing Genetic Identity and Kinship in Azara's Owl Monkeys (Aotus azarai)

In this study, we characterize a panel of 20 microsatellite markers that reproducibly amplify in Azara’s owl monkeys (Aotus azarai) for use in genetic profiling analyses. A total of 128 individuals from our study site in Formosa, Argentina, were genotyped for 20 markers, 13 of which were found to be polymorphic. The levels of allelic variation at these loci provided paternity exclusion probabilities of 0.852 when neither parent was known, and 0.981 when one parent was known. In addition, our analysis revealed that, although genotypes can be rapidly scored using fluorescence-based fragment analysis, the presence of complex or multiple short tandem repeat (STR) motifs at a microsatellite locus could generate similar fragment patterns from alleles that have different nucleotide sequences and perhaps different evolutionary origins. Even so, this collection of microsatellite loci is suitable for parentage analyses and will allow us to test various hypotheses about the relationship between social behavior and kinship in wild owl monkey populations. Furthermore, given the limited number of platyrrhine-specific microsatellite loci available in the literature, this STR panel represents a valuable tool for population studies of other cebines and callitrichines.Fil: Babb, Paul L.. University of Pennsylvania; Estados UnidosFil: McIntosh, Annick M.. Haverford College; Estados UnidosFil: Fernandez Duque, Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; Argentina. University of Pennsylvania; Estados UnidosFil: Di Fiore, Anthony. University of Texas at Austin; Estados UnidosFil: Schurr, Theodore G.. University of Pennsylvania; Estados Unido

An Optimized Microsatellite Genotyping Strategy for Assessing Genetic Identity and Kinship in Azara&apos;s Owl Monkeys (Aotus azarai)

Abstract In this study, we characterize a panel of 20 microsatellite markers that reproducibly amplify in Azara&apos;s owl monkeys (Aotus azarai) for use in genetic profiling analyses. A total of 128 individuals from our study site in Formosa, Argentina, were genotyped for 20 markers, 13 of which were found to be polymorphic. The levels of allelic variation at these loci provided paternity exclusion probabilities of 0.852 when neither parent was known, and 0.981 when one parent was known. In addition, our analysis revealed that, although genotypes can be rapidly scored using fluorescence-based fragment analysis, the presence of complex or multiple short tandem repeat (STR) motifs at a microsatellite locus could generate similar fragment patterns from alleles that have different nucleotide sequences and perhaps different evolutionary origins. Even so, this collection of microsatellite loci is suitable for parentage analyses and will allow us to test various hypotheses about the relationship between social behavior and kinship in wild owl monkey populations. Furthermore, given the limited number of platyrrhine-specific microsatellite loci available in the literature, this STR panel represents a valuable tool for population studies of other cebines and callitrichines

A schematic of the <i>APOE</i> gene.

<p>Structure and nucleotide position numbers follow Fullerton <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Fullerton1" target="_blank">[27]</a> and Ensembl (ENSG00000130203). The location of primers used in this study are given above (forward primers) and below (reverse primers) the labeled exons. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760.s002" target="_blank">Table S1</a> for primer and PCR-cycling information. An intronic SNP differentiating the two chimpanzee populations is highlighted in orange (position 2098*). SNP locations in red (3071 and 3073) represent putative <i>APOE</i> non-synonymous changes based on the chimpanzee genome assembly (Pan_troglodytes-2.1.4). Positions in blue (3205, 3937 and 4075) correspond to the amino acids (61, 112 and 158, respectively) that define the three human <i>APOE</i> alleles (E2, E3, E4). Position 4219^† (in green) represents the single, synonymous difference between the <i>P. t. verus</i> sequences generated in this study and that of Fullerton <i>et al.</i> (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Fullerton1" target="_blank">[27]</a>. *corresponds to Ensembl coordinates 19∶45411002 for the human genome and 19∶50097633 for the chimpanzee genome. <b>^†</b>corresponds to Ensembl coordinates 19∶45412223 for the human genome.</p

Variation at key <i>APOE</i> functional sites in <i>Homo</i> and <i>Pan</i>.

*<p>Gene nucleotide positions following notation of Fullerton et al. 2000 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Fullerton1" target="_blank">[27]</a>, and as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone-0047760-g001" target="_blank">Figure 1</a>.</p>**<p>Based on two reads, one each from two fossil specimens: Vi33.25 and Vi33.26.</p

Lineage-specific mutations mapped onto a schematic of the APOE protein (A) and primate phylogeny (B).

<p>Protein structure is modeled after Bu 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Bu1" target="_blank">[65]</a>, and tree topology represents known evolutionary relationships based on genome-wide data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Scally1" target="_blank">[46]</a>. Human mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-McLean1" target="_blank">[66]</a> at key residues 61, 112 and 158 are in red. Including residue 61, the human APOE protein has four fixed, <i>Homo</i>-specific, non-synonymous mutations, all of which seem to be shared with the Denisovan hominin (inferred from reads mapped to the human genome at <a href="http://www.genome.ucsc.edu" target="_blank">http://www.genome.ucsc.edu</a>). The chimpanzee APOE protein is monomorphic within and between subspecies, and is identical to the bonobo APOE protein. Mutation R15H (dotted arrow) is shared by gorillas, chimpanzees and bonobos likely as a result of incomplete lineage sorting rather than independent evolution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047760#pone.0047760-Scally1" target="_blank">[46]</a>.</p