Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer.

BACKGROUND: Major histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. METHODS: We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single-cell neighborhoods from mIF images followed by multivariate discriminant analysis. RESULTS: Spatial quantitation of tumor cell MHC-I expression revealed intratumoral and intertumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single-cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. CONCLUSIONS: Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Coassociation of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent colocalization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer.

Background: MHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. Methods: We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Results: Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Conclusions: Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

The role of wildfire, prescribed fire, and mountain pine beetle infestations on the population dynamics of black-backed woodpeckers in the black hills, South Dakota.

Wildfire and mountain pine beetle infestations are naturally occurring disturbances in western North American forests. Black-backed woodpeckers (Picoides arcticus) are emblematic of the role these disturbances play in creating wildlife habitat, since they are strongly associated with recently-killed forests. However, management practices aimed at reducing the economic impact of natural disturbances can result in habitat loss for this species. Although black-backed woodpeckers occupy habitats created by wildfire, prescribed fire, and mountain pine beetle infestations, the relative value of these habitats remains unknown. We studied habitat-specific adult and juvenile survival probabilities and reproductive rates between April 2008 and August 2012 in the Black Hills, South Dakota. We estimated habitat-specific adult and juvenile survival probability with Bayesian multi-state models and habitat-specific reproductive success with Bayesian nest survival models. We calculated asymptotic population growth rates from estimated demographic rates with matrix projection models. Adult and juvenile survival and nest success were highest in habitat created by summer wildfire, intermediate in MPB infestations, and lowest in habitat created by fall prescribed fire. Mean posterior distributions of population growth rates indicated growing populations in habitat created by summer wildfire and declining populations in fall prescribed fire and mountain pine beetle infestations. Our finding that population growth rates were positive only in habitat created by summer wildfire underscores the need to maintain early post-wildfire habitat across the landscape. The lower growth rates in fall prescribed fire and MPB infestations may be attributed to differences in predator communities and food resources relative to summer wildfire.

A re-evaluation of a case-control model with contaminated controls for resource selection studies.

1. A common sampling design in resource selection studies involves measuring resource attributes at sample units used by an animal and at sample units considered available for use. Few models can estimate the absolute probability of using a sample unit from such data, but such approaches are generally preferred over statistical methods that estimate a relative probability of use. 2. The case-control model that allows for contaminated controls, proposed by Lancaster & Imbens (1996) and Lele (2009), can estimate the absolute probability of using a sample unit from use-availability data. However, numerous misconceptions have likely prevented the widespread application of this model to resource selection studies. We address common misconceptions regarding the case-control model with contaminated controls and demonstrate its ability to estimate the absolute probability of use, prevalence and parameters associated with categorical covariates from use-availability data. 3. We fit the case-control model with contaminated controls to simulated data with varying prevalence (defined as the average probability of use across all sample units) and sample sizes (n1 = 500 used and na = 500 available samples; n1 = 1000 used and na = 1000 available samples). We then applied this model to estimate the probability Ozark hellbenders (Cryptobranchus alleganiensis bishopi) would use a location within a stream as a function of covariates. 4. The case-control model with contaminated controls provided unbiased estimates of all parameters at N = 2000 sample size simulation scenarios, particularly at low prevalence. However, this model produced increasingly variable maximum likelihood estimates of parameters as prevalence increased, particularly at N = 1000 sample size scenarios. We thus recommend at least 500-1000 used samples when fitting the case-control model with contaminated controls to use-availability data. Our application to hellbender data revealed selection for locations with coarse substrate that are close to potential sources of cover. 5. This study unites a disparate literature, addresses and clarifies many commonly held misconceptions and demonstrates that the case-control model with contaminated controls is a viable alternative for estimating the absolute probability of use from use-availability data.