Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRβ sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (>1%) TCRβ clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRβ clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRβ clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRβ sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy

Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGFβ/PD-L1 blockade-treated squamous cell carcinoma.

Transforming growth factor beta (TGFβ) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFβ and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFβ inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFβ and PD-L1 to treat mouse SCCs and found TGFβ inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFβ and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC

Fibroblast Growth Factor 8 Deficiency Compromises the Functional Response of the Serotonergic System to Stress

<div><p>Functionally heterogeneous populations of serotonergic neurons, located within the dorsal raphe nucleus (DR), play a role in stress-related behaviors and neuropsychiatric illnesses such as anxiety and depression. Abnormal development of these neurons may permanently alter their structure and connections, making the organism more susceptible to anxiety-related disorders. A factor that critically regulates the development of serotonergic neurons is fibroblast growth factor 8 (Fgf8). In this study, we used acute restraint stress followed by behavioral testing to examine whether Fgf8 signaling during development is important for establishing functional stress- and anxiety-related DR neurocircuits in adulthood. Wild-type and heterozygous male mice globally hypomorphic for <i>Fgf8</i> were exposed to acute restraint stress and then tested for anxiety-like behavior on the elevated plus-maze. Further, we measured c-Fos immunostaining as a marker of serotonergic neuronal activation and tissue 5-hydroxyindoleacetic acid concentrations as a marker of serotonin functional output. Results showed that Fgf8 hypomorphs exhibited 1) an exaggerated response of DR anxiety-promoting circuits and 2) a blunted response of a DR panic-inhibiting circuit to stress, effects that together were associated with increased baseline anxiety-like behavior. Overall, our results provide a neural substrate upon which <i>Fgf8</i> deficiency could affect stress response and support the hypothesis that developmental disruptions of serotonergic neurons affect their postnatal functional integrity.</p></div

Serotonin metabolism is dysregulated in Fgf8-deficient mice.

<p>The 5-HIAA tissue content in anxiety-related and panic-related DR circuits of WT and Fgf8-deficient (HET) mice following non-stress (NS) or restraint stress (S) conditions. Stress increased 5-HIAA content in select anxiety-related projection sites of the DRD (BLA, PrL and IL) as well as in the panic-inhibiting DRVL/VLPAG and DLPAG projection. In WT mice, restraint stress increased 5-HIAA content in the panic-inhibiting DRVL/VLPAG and DLPAG circuit but not in anxiety-related DR projection sites (BLA, PrL, and IL). In Fgf8 HET mice, converse effects were observed. In the DLPAG, Fgf8 HET mice also had lower 5-HIAA content following stress compared to WT mice. N = 4−9/group. Data are presented as mean ± SEM, p<0.05, ^avs. WT NS, ^bvs. WT S, ^cvs. HET NS, ^&main effect of stress, ^#genotype x stress interaction. See abbreviations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101420#pone-0101420-t001" target="_blank">Table 1</a>.</p

Representative photomicrographs illustrating c-Fos-ir and Tph-ir neurons in the DR at −4.72 mm bregma.

<p>WT (A, B) and Fgf8-deficient (C, D) mice exposed to non-stress (A, C) or restraint stress (B, D) conditions. Black boxes with lower case letters, e-l, correspond to higher magnification insets found in the corners of panels A–D. Arrows indicate c-Fos-ir nuclei (blue/black nuclear staining); white arrowheads indicate Tph-ir neurons (brown cytoplasmic staining); black arrowheads indicate c-Fos/Tph-ir double-immunostained neurons. Scale bar represents 100 µm for A–D and 40 µm for insets e–l.</p

5-HIAA concentrations (pg/µg protein) across brain regions in WT and Fgf8 HET mice following non-stress or stress conditions.

<p>Data are presented as mean ± SEM, ^&main effect of stress, n.s. = not significant. See abbreviations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101420#pone-0101420-t001" target="_blank">Table 1</a>.</p

Fgf8-deficient mice have elevated anxiety-like behavior.

<p>Anxiety-like behavior was tested using the elevated plus-maze following non-stress (NS) or restraint stress (S) in WT and Fgf8-deficient (HET) mice. Restraint stress significantly increased anxiety-like behavior in WT mice as measured by a lower percentage of time spent in the open arms. Non-stress Fgf8 HET mice spent significantly less time in the open arms and more time in the closed arms than WT non-stress mice. No differences were found for the percent time spent in the center area. N = 12−16/group. Data are presented as mean ± SEM, ^ap<0.01 vs. WT NS, ^cp<0.05 vs. HET NS, *main effect of genotype, ^#genotype x stress interaction.</p

Details of tissue sample collection for HPLC-ED analysis of 5-HIAA and 5-HT concentrations.

<p>Details of tissue sample collection for HPLC-ED analysis of 5-HIAA and 5-HT concentrations.</p

Serotonergic neuronal activation is dysregulated in Fgf8-deficient mice.

<p>The percentage of DR c-Fos/Tph-ir neurons at five rostrocaudal levels in WT and Fgf8-deficient (HET) mice following non-stress (NS) or stress (S) conditions. DR subregions are organized by row, and anatomical levels are organized in columns from rostral (left) to caudal (right). Distance from bregma (in mm) is shown above each column. Restraint stress increased serotonergic neuronal activation (% c-Fos/Tph-ir) in the anxiety-related DRD and panic-inhibiting DRVL/VLPAG of WT mice compared to non-stress WT mice. In contrast, activation of serotonergic neurons was similar between the non-stress and stress conditions in Fgf8 HET mice. Non-stress Fgf8 HET mice had increased basal activation of the anxiety-related DRD at −4.72 mm bregma compared to non-stress WT mice and blunted stress-induced activation of the panic-inhibiting DRVL/VLPAG at −4.54 mm bregma compared to WT mice. N = 5−6/group. Data are presented as mean ± SEM, p<0.05, ^avs. WT NS, ^bvs. WT S, ^&main effect of stress, ^#genotype x stress interaction. See abbreviations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101420#pone-0101420-t001" target="_blank">Table 1</a>.</p