Biology subjects

Brown, M. G.

Publications and source records attributed to Brown, M. G..

3 recordsLinked to original sources

bioRxiv2024

Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation

Early embryos often have unique chromatin states prior to zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 reductive nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. Previous work found that histone H3 chromatin incorporation decreases while its variant H3.3 increases leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription and heterochromatin, suggesting a link between H3.3 and ZGA. Here, we test what factors regulate H3.3 incorporation at ZGA. We find that H3 nuclear availability falls more rapidly than H3.3 leading up to ZGA. We generate H3/H3.3 chimeric proteins at the endogenous H3.3A locus and observe that chaperone binding, but not gene structure, regulates H3.3 behavior. We identify the N/C ratio as a major determinant of H3.3 incorporation. To isolate how the N/C ratio regulates H3.3 incorporation we test the roles of genomic content, zygotic transcription, and cell cycle state. We determine that cell cycle regulation, but not H3 availability or transcription, controls H3.3 incorporation. Overall, we propose that local N/C ratios control histone variant usage via cell cycle state during ZGA.

cell biology↗

bioRxiv2024

Spatial analysis reveals combinative role for natural killer and CD8 T cells in antitumor immunity despite profound MHC class I loss in non-small cell lung cancer

BackgroundMHC 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. MethodsWe 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 IFN{gamma}-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis ResultsSpatial 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 over-all 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<1x10-3). 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 IFN{gamma}+/- NK cells and T cells. We discovered that both IFN{gamma}+ NK and CD8 T cells were more frequently associated with other IFN{gamma}+ lymphocytes in comparison to IFN{gamma}- NK cells and CD8 T cells (p<1x10-30). Moreover, IFN{gamma}+ lymphocytes were most often found clustered near MHC-I+ tumor cells. ConclusionsTumor-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 IFN{gamma}. Frequent co-localization of IFN{gamma}+ NK cells with other IFN{gamma}+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated. What is already known on this topicMHC-I loss occurs frequently in NSCLC and corresponds with waning immunity in the tumor microenvironment (TME). NK cells recognize "missing-self" targets and could be leveraged to target NSCLC tumors with MHC-I loss. While NK cell presence at tumor margins has been documented in NSCLC, they were shown to lose function in this environment. What this study addsWe developed spatial analysis pipelines leveraging the local heterogeneity of the TME at single cell resolution to test whether NK cells and T cells together contribute antitumoral immunity in NSCLC. We discovered that a high density of tumor-infiltrating NK cells corresponded with DFS, and this association was increased in patients with high coincident CD8 T cells, especially those in central tumor. Intriguingly, both cell types were found clustered together in MHC-I-bearing tumors, especially when both expressed IFN{gamma}, suggesting coordinated lymphocyte activities may enhance immune control of NSCLC. How this study might affect research, practice, or policyThis study provides a rationale for developing novel immunotherapies that simultaneously increase NK and T cell anti-tumoral immunity. Associations linking NK cells with patient survival and increased immune effector activity in NSCLC, even in MHC-I-deficient tumors, further highlights the need to devise and deploy NK cell activating strategies which may be highly efficacious in CD8 T cell refractory NSCLC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/581048v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1e383c0org.highwire.dtl.DTLVardef@1dee268org.highwire.dtl.DTLVardef@1e21bd4org.highwire.dtl.DTLVardef@1910a9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Boosting Live Malaria Vaccine with Cytomegalovirus Vector Can Prolong Immunity through Innate and Adaptive Mechanisms

Vaccines to persistent parasite infections have been challenging, and current iterations lack long-term protection. Cytomegalovirus (CMV) chronic vaccine vectors drive protection against SIV, tuberculosis and liver-stage malaria correlated with antigen-specific CD8 T cells with a Tem phenotype. This phenotype is likely driven by a combination of antigen-specific and innate adjuvanting effects of the vector, though these mechanisms are less well understood. Sterilizing immunity from live Plasmodium chabaudi vaccination lasts less than 200 days. While P. chabaudi-specific antibody levels remain stable after vaccination, the decay of parasite-specific T cells correlates with loss of challenge protection. Therefore, we enlisted murine CMV as a booster strategy to prolong T cell responses against malaria. To study induced T cell responses, we included P. chabaudi MSP-1 epitope B5 (MCMV-B5). We found that MCMV vector alone significantly protected against a challenge P. chabaudi infection 40-60 days later, and that MCMV-B5 was able to make B5-specific Teff, in addition to previously-reported Tem, that survive to the challenge timepoint. Used as a booster, MCMV-B5 prolonged protection from heterologous infection beyond day 200, and increased B5 TCR Tg T cell numbers, including both a highly-differentiated Tem phenotype and Teff, both previously reported to protect. B5 epitope expression was responsible for maintenance of Th1 and Tfh B5 T cells. In addition, the MCMV vector had adjuvant properties, contributing non-specifically through prolonged stimulation of IFN-{gamma}. In vivo neutralization of IFN-{gamma}, but not IL-12 and IL-18, late in the course of MCMV, led to loss of the adjuvant effect. Mechanistically, sustained IFN-{gamma} from MCMV increased CD8+ dendritic cell numbers, and led to increased IL-12 production upon Plasmodium challenge. In addition, neutralization of IFN-{gamma} before challenge reduced the polyclonal Teff response to challenge. Our findings suggest that, as protective epitopes are defined, an MCMV vectored booster can prolong protection through the innate effects of IFN-{gamma}. Authors summaryMalaria represents a challenging target for vaccination. This is in part because of the requirement for CD4 T cell immunity in addition to the standard B cell responses current vaccines induce. However, human malaria vaccine approaches thus far have limited longevity of protection due to decay of T cell responses. This includes the most advanced malaria vaccine, a virus-like particle expressing one recombinant liver-stage antigen (RTS,S), and liver-stage parasites attenuated by radiation (PfSPZ), as well as live vaccination with drug-treatment. Our work seeks to prolong this protection using MCMV, a promising vaccine vector known to promote CD8 T cell responses. We observed that boosting the live malaria vaccine with MCMV including a Plasmodium antigen led to longer protection from P. chabaudi parasitemia, and can be used to promote maintenance of antigen-specific CD4 T cells. In investigating the mechanisms of the MCMV booster, we found that the cytokine IFN-{gamma} is required for prolonged protection and enhances priming of the innate immune system for prolonged protection from malaria. Our research informs both the quest for a longer-lived malaria vaccine and that to understand mechanisms of protection from persistent infection.

immunology↗