Dual Role of PI-3 Kinase Gamma in Susceptibility of C57BL/6 Mice to Leishmania mexicana

Denman Undergraduate Research Forum (1st Place, Biological Sciences, 2005) (3rd Place, Biological Sciences, 2006)Biological Sciences Research Colloquium Award Winner 2006Phosphotidylinositol-3 kinase gamma (PI3K-y), a class IB PI3K that is activated by heterotrimeric G-protein coupled receptors, plays a role in the homing of leukocytes to infected tissues. We analyzed the role of PI3K-y in the development of cutaneous Leishmania mexicana infection in PI3K-y -/- C57BL/6 mice, which model the kinetics of human infection due to the development of chronic non-healing lesions. Leishmania species are obligate intramacrophage parasites. The difference between the thickness of the infected ear and the control ear was used as the measure of infection progression. Following L. mexicana infection, PI3K-y -/- developed smaller lesions containing fewer parasites as compared to wild type mice. Production of TH1 (IL-12 and IFN-g) and TH2 (IL-4 and IL-10) cytokines were also lower in PI3K-y -/- mice, indicating that increased resistance of PI3K-y -/- mice to L. mexicana was not due to enhancement of a TH1-like response. Flow cytometric analysis was performed on cells derived from infected ears using appropriate antibodies to detect macrophages and T-cell subpopulations. Lesions from PI3K-y -/- mice contained approximately half the number of macrophages observed in wild type lesions (1.3X10^5 for PI3K-y -/- versus 4.0X10^5 for wild types). To test the role of PI3K-y in the activation of the oxidative burst, bone marrow-derived macrophages were infected with an excess of Leishmania mexicana promastigotes, and then stimulated with media, IFN-y, IFN-y and LPS. Macrophage-specific cytokines IL-6, IL-10, IL-12, and TNF-a from supernatants were then analyzed. Production of all cytokines were similar at 12 hours for all three stimulation types, but at 24 hours there was a significant increase in the levels of all cytokines in PI3K-y -/- mice but not in WT mice. PI3K-y -/- had fewer infected cells and fewer parasites per cell at 12 hours than WT cells, but at 24 hours an even greater difference was seen with PI3K-y -/- cells showing a 38% infection rate versus 75% and 2.4 parasites per cell versus 3.4. The difference in infection size in vivo can most likely be attributed to a shortage of host cells at the site of infection and an increased ability of infected cells to kill engulfed parasites.A three-year embargo was granted for this item

Variants Near CCK Receptors are Associated With Electrophysiological Responses to Pre-pulse Startle Stimuli in a Mexican American Cohort

Neurophysiological measurements of the response to prepulse and startle stimuli have been suggested to represent an important endophenotype for both substance dependence and other select psychiatric disorders. We have previously shown, in young adult Mexican Americans (MA), that presentation of a short delay acoustic prepulse, prior to the startle stimuli can elicit a late negative component at about 400 msec (N4S), in the event-related potential (ERP), recorded from frontal cortical areas. In the present study we investigated whether genetic factors associated with this endophenotype could be identified. The study included 420 (age 18 – 30 years) MA men (n=170) and women (n=250). DNA was genotyped using an Affymetrix Axiom Exome1A chip. An association analysis revealed that the CCKAR and CCKBR (cholecystokinin A and B receptor) genes each had a nearby variant that showed suggestive significance with the amplitude of the N4S component to prepulse stimuli. The neurotransmitter cholecystokinin (CCK), along with its receptors, CCKAR and CCKBR, have been previously associated with psychiatric disorders, suggesting that variants near these genes may play a role in the prepulse/startle response in this cohort

The influence of the germline HSD3B1 adrenal-permissive variant (c.1100 C) on somatic alteration landscape, transcriptome, and immune-cell infiltration in prostate cancer

215 Background: A missense-encoding germline polymorphism in the HSD3B1 gene has been shown to result in adrenal-permissive (c.1100 C, p.367 Thr) or adrenal-restrictive (c.1100 A, p.367 Asn) phenotypes with respect to androgen synthesis from adrenal precursors, and the adrenal-permissive variant is associated with inferior outcomes to hormonal therapies. However, the tumoral characteristics (somatic alteration landscape, transcriptome, immune-cell composition) of prostate cancers that arise in the context of these germline genotypes are unknown. Methods: We utilized the Caris Life Sciences (Phoenix, AZ) database to study the relationship between germline HSD3B1 c.1100 genotypes and somatic tumoral characteristics in 5,421 prostate cancer biopsies. Germline HSD3B1 status was inferred using variant allele frequencies (VAFs) derived from tumor DNA sequencing: c.1100C VAF of 0% defined the AA genotype, VAF of 40-60% defined the AC genotype, and VAF of 100% defined the CC genotype. Each HSD3B1 genotype was interrogated for its associated mutational landscape (by exome sequencing) and tumor transcriptome (by RNA-seq); immune-cell subsets were explored using the quanTIseq deconvolution algorithm. Results: The prevalence of the HSD3B1 c.1100 AA genotype (restrictive–homozygous) was 54.4%, AC (heterozygous) was 34.2%, and CC (permissive–homozygous) was 11.4%. Liver/lung metastases had the highest prevalence of the CC genotype (14.2%), while lymph node metastases had the lowest (10.9%). Among the AR-associated genes, compared to the AA genotype, the CC genotype had greater frequency of TMPRSS2-ERG fusions (35.5% vs 29.7%, P<0.01), AR-V7 (17.2% vs 14.7%, P=0.17), and mRNA expression of AR (median 124 vs 115 TPM, P=0.01), HOXB13 (37 vs 34 TPM, P=0.08) and KLK2 (490 vs 448 TPM, P=0.12); there were no associations with SPOP or FOXA1 mutations or expression. Outside of AR pathways, the MAPK activation signature was higher in the CC relative to the AA genotype (0.27 vs -0.04 MPAS, P=0.01). When examining immune-cell subsets and immunotherapy targets, the CC genotype unexpectedly showed higher CD276 ( B7-H3) expression (30 vs 27 TPM, P<0.01), higher dendritic cells ( P=0.02) and T-regs ( P=0.03), and lower neutrophils ( P=0.10) compared to the AA genotype. Conclusions: The homozygous adrenal-permissive HSD3B1 variant (c.1100 CC) is associated with distinct tumoral features characterized by elevated AR signaling and MAPK activation. It is also associated with a unique immune-cell regulatory landscape, with higher B7-H3 expression, increased intratumoral dendritic cells and decreased immunosuppressive neutrophils. This distinct molecular landscape of HSD3B1 c.1100 CC–associated prostate cancers warrants special therapeutic considerations, including possibly using B7-H3–targeted therapies

NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo

We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKETM) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting

Divergent immune microenvironments in two tumor nodules from a patient with mismatch repair-deficient prostate cancer

Abstract Patients with prostate cancer (PC) generally do not respond favorably to immune checkpoint inhibitors, which may be due to a low abundance of tumor-infiltrating lymphocytes even when mutational load is high. Here, we identified a patient who presented with high-grade primary prostate cancer with two adjacent tumor nodules. While both nodules were mismatch repair-deficient (MMRd), exhibited pathogenic MSH2 and MSH6 alterations, had a high tumor mutational burden (TMB), and demonstrated high microsatellite instability (MSI), they had markedly distinct immune phenotypes. The first displayed a dense infiltrate of lymphocytes (“hot nodule”), while the second displayed significantly fewer infiltrating lymphocytes (“cold nodule”). Whole-exome DNA analysis found that both nodules shared many identical mutations, indicating that they were derived from a single clone. However, the cold nodule appeared to be sub-clonal relative to the hot nodule, suggesting divergent evolution of the cold nodule from the hot nodule. Whole-transcriptome RNA analysis found that the cold nodule demonstrated lower expression of genes related to antigen presentation (HLA) and, paradoxically, classical tumor immune tolerance markers such as PD-L1 (CD274) and CTLA-4. Immune cell deconvolution suggested that the hot nodule was enriched not only in CD8+ and CD4 + T lymphocytes, but also in M1 macrophages, activated NK cells, and γδ T cells compared to the cold nodule. This case highlights that MMRd/TMB-high PC can evolve to minimize an anti-tumor immune response, and nominates downregulation of antigen presentation machinery (HLA loss) as a potential mechanism of adaptive immune evasion in PC