The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7–9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.
<p>Supplementary Figure 5: STAT1, pSTAT1Y701, and PD-L1 have similar baseline expression and impact on survival between tissue types</p>
<div>AbstractPurpose:<p>IFN signaling in the tumor microenvironment is a critical determinant of both response and resistance of cancer to immune checkpoint inhibitors (ICI). We hypothesized that distinct patterns of IFN signaling in melanoma are associated with clinical response or resistance to ICIs.</p>Experimental Design:<p>Two tissue microarrays containing samples from 97 patients with metastatic melanoma who received nivolumab, pembrolizumab, or a combination of ipilimumab and nivolumab at Yale New Haven Hospital between 2011 and 2017 were randomized into discovery and validation cohorts. Samples were stained and visualized using multiplexed immunofluorescence microscopy for STAT1, STAT1 phosphorylated at Y701 (pSTAT1<sup>Y701</sup>), and PD-L1, and signals were quantified using the automated quantitative analysis method of quantitative immunofluorescence. Treatment response was assessed using RECIST, and overall survival was analyzed. For <i>in vitro</i> studies, human melanoma cell lines were stimulated with IFNγ and IFNβ, and Western blotting was performed.</p>Results:<p>Pretreatment STAT1 levels were higher in responders to ICIs [complete response/partial response/stable disease (SD) for > 6 months] than in nonresponders (SD < 6 months/progressive disease). Higher pretreatment STAT1 levels were associated with improved survival after ICIs in both the discovery and validation cohorts. Western blot analysis of human melanoma cell lines stimulated with IFN demonstrated distinct patterns of upregulation of STAT1 compared with pSTAT1<sup>Y701</sup> and PD-L1. When combining STAT1 and PD-L1 markers, patients with STAT1<sup>high</sup>PD-L1<sup>low</sup> tumors had improved survival compared with those with STAT1<sup>low</sup>PD-L1<sup>high</sup> tumors.</p>Conclusions:<p>STAT1 may better predict melanoma response to ICIs than current strategies, and combined STAT1 and PD-L1 biomarkers may provide insight into IFN-responsive versus IFN-resistant states.</p></div>
9522 Background: Interferon (IFN) signaling in tumors is critical for both response and resistance to immune checkpoint inhibitors (ICI). While PD-L1 is an approved biomarker for ICI response in some cancers, most have no approved biomarkers for ICI response. Further, there are no clinical strategies to predict IFN sensitive vs resistant states. We hypothesized that distinct patterns of IFN signaling in melanoma, including STAT1 (precursor to IFN signaling), phosphorylated STAT1 (pSTAT1, marker of active IFN signaling), and PD-L1 (canonical inhibitory output), are associated with response or resistance to ICI. Methods: Samples from 2 tissue microarrays of 97 patients total with metastatic melanoma who received ICI from 2011-2017 were randomized into discovery [D] or validation [V] cohorts. Multiplexed immunofluorescent microscopy was used to assess STAT1, pSTAT1, and PD-L1. 54 tumors were assessed for CD3. Signals were quantified via AQUA (Automated Quantitative Analysis). High vs low signals were defined around the discovery cohort median AQUA scores. Treatment response was assessed using RECIST, and 3-year survival was analyzed. For in vitro studies, 4 human melanoma cell lines were stimulated with IFNγ/β, and Western blots were performed for STAT1, pSTAT1, and PD-L1. Results: Median follow up time was 20.2 mos (range 0.9-95.4 mos). Higher overall histospot, tumor, and nuclear STAT1 were in responders (CR/PR/SD > 6 mos) compared to non-responders (SD < 6 mos/PD, histospot p = 0.029 [D] and 0.040 [V]) and were associated with improved survival (histospot HR 0.25 [95% CI 0.08-0.76] p = 0.015 [D]; HR 0.32 [95% CI 0.12-0.84] p = 0.021 [V]). PD-L1 and pSTAT1 did not validate as associated with ICI response/survival. While many tumors had concordant high/low STAT1, pSTAT1, and PD-L1, many also had a discordant STAT1 or PD-L1 predominance. To assess the basis of this discordance, we performed IFN stimulation and Western blots of melanoma cell lines. IFNβ upregulated STAT1 greater than PD-L1 in both acute and chronic settings (p < 0.001 and < 0.001), while chronic IFNγ upregulated PD-L1 compared to the acute setting (p = 0.039) but not STAT1 (p = 0.105). Therefore, we hypothesized patients with discordant STAT1 and PD-L1 expression may have differential responses to ICI. In the overall cohort, STAT1highPD-L1high tumors (IFN engaged) were associated with improved survival compared to STAT1lowPD-L1low tumors (IFN low, HR 0.28 [95% CI 0.11-0.70] p = 0.007). Interestingly, STAT1highPD-L1low tumors were associated with improved survival compared to STAT1lowPD-L1high tumors (HR 0.28 [95% CI 0.08-0.94] p = 0.04). Accordingly, STAT1highPD-L1high and STAT1highPD-L1low tumors had greater CD3 AQUA scores compared to STAT1lowPD-L1low tumors (p < 0.001 and p = 0.018 respectively). Conclusions: Combined STAT1 and PD-L1 expression patterns may better predict melanoma response to ICI and provide insight into IFN sensitive versus resistant states.
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