Hyperprogressive disease (HPD) is an adverse outcome of immunotherapy consisting of an acceleration of tumor growth associated with prompt clinical deterioration. The definitions based on radiological evaluation present important technical limitations. No biomarkers have been identified yet. In this study, 70 metastatic NSCLC patients treated with anti-PD-1/PD-L1 immunotherapy after progression to platinum-based therapy were prospectively studied. Samples from peripheral blood were obtained before the first (baseline) and second cycles of treatment. Peripheral blood mononuclear cells (PBMCs) were isolated and differentiation stages of CD4 lymphocytes quantified by flow cytometry and correlated with HPD as identified with radiological criteria. A strong expansion of highly differentiated CD28− CD4 T lymphocytes (CD4 THD) between the first and second cycle of therapy was observed in HPD patients. After normalizing, the proportion of posttreatment/pretreatment CD4 THD was significantly higher in HPD when compared with the rest of patients (median 1.525 vs. 0.990; p = 0.0007), and also when stratifying by HPD, non-HPD progressors, and responders (1.525, 1.000 and 0.9700 respectively; p = 0.0025). A cut-off value of 1.3 identified HPD with 82% specificity and 70% sensitivity. An increase of CD28− CD4 T lymphocytes ≥ 1.3 (CD4 THD burst) was significantly associated with HPD (p = 0.008). The tumor growth ratio (TGR) was significantly higher in patients with expansion of CD4 THD burst compared to the rest of patients (median 2.67 vs. 0.86, p = 0.0049), and also when considering only progressors (median 2.67 vs. 1.03, p = 0.0126). A strong expansion of CD28− CD4 lymphocytes in peripheral blood within the first cycle of therapy is an early differential feature of HPD in NSCLC treated with immune-checkpoint inhibitors. The monitoring of T cell dynamics allows the early detection of this adverse outcome in clinical practice and complements radiological evaluation.
SUMMARY: HIV-1 primarily infects T lymphocytes and uses these motile cells as migratory vehicles for effective dissemination in the host. Paradoxically, the virus at the same time disrupts multiple cellular processes underlying lymphocyte motility, seemingly counterproductive to rapid systemic infection. Here we show by intravital microscopy in humanized mice that perturbation of the actin-cytoskeleton via the lentiviral protein Nef, and not changes to chemokine receptor expression or function, is the dominant cause of dysregulated infected T cell motility in lymphoid tissue by preventing stable cellular polarization required for fast migration. Accordingly, disrupting the Nef hydrophobic patch that facilitates actin-cytoskeletal perturbation initially accelerates systemic viral dissemination after female genital transmission. However, the same feature of Nef was subsequently critical for viral persistence in immune-competent hosts. Therefore, a highly conserved activity of lentiviral Nef proteins has dual effects and imposes both fitness costs and benefits on the virus at different stages of infection.
BackgroundAlthough discovery research has identified the importance of dozens of pro- and anti-inflammatory immune mediators in the pathogenesis, maintenance, exacerbation and resolution of inflammatory diseases, most human cohort studies have incorporated few or no immunological intermediate phenotypes in their analyses. Significant hindrances have been (1) the limited panel of biomarkers known to be readily detected in healthy human populations and (2) the stability, hence utility, of such biomarkers to repeated analysis.MethodsThe frequency and stability of 14 plasma biomarkers linked to in vivo immune regulation of allergic and autoimmune inflammatory disorders was determined in 140 healthy pediatric and adult participants. The impact of initial and multiple subsequent freeze/thaw cycles on pro-inflammatory (CCL2, CXCL10, IL-18, TNFα, IL-6), anti-inflammatory (IL-10, sTNF-RII, IL-1Ra), acute phase proteins (CRP, PTX3) and other biomarkers (sST2, IL-1RAcP) was subsequently quantified.ResultsMultiple biomarkers capable of providing an innate immune signature of inflammation were readily detected directly ex vivo in healthy individuals. These biomarker levels were unaffected when comparing paired data sets from freshly obtained, never frozen plasma or serum and matched aliquots despite extensive freeze/thaw cycles. Neither age nor sex affected stability. Similarly, no quantitative differences were found following repetitive analysis of inflammatory biomarkers in culture samples obtained following in vitro stimulation with TLR and RLR ligands.ConclusionsA broad panel of in vivo and ex vivo cytokine, chemokine and acute phase protein biomarkers that have been linked to human chronic inflammatory disorders are readily detected in vivo and remain stable for analysis despite multiple freeze thaw cycles. These data provide the foundation and confidence for large scale analyses of panels of inflammatory biomarkers to provide better understanding of immunological mechanisms underlying health versus disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1154-3) contains supplementary material, which is available to authorized users.
The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.
Macrophages are susceptible to HIV infection and play an important role in viral dissemination through cell-cell contacts with T cells. However, our current understanding of macrophage-to-T cell HIV transmission is derived from studies that do not consider the robust migration and cell-cell interaction dynamics between these cells. Here, we performed live-cell imaging studies in 3-dimensional (3D) collagen that allowed CD4+ T cells to migrate and to locate and engage HIV-infected macrophages, modeling the dynamic aspects of the in situ environment in which these contacts frequently occur. We show that HIV+ macrophages form stable contacts with CD4+ T cells that are facilitated by both gp120-CD4 and LFA-1–ICAM-1 interactions and that prolonged contacts are a prerequisite for efficient viral spread. LFA-1–ICAM-1 adhesive contacts function to restrain highly motile T cells, since their blockade substantially destabilized macrophage-T cell contacts, resulting in abnormal tethering events that reduced cell-cell viral spread. HIV-infected macrophages displayed strikingly elongated podosomal extensions that were dependent on Nef expression but were dispensable for stable cell-cell contact formation. Finally, we observed persistent T cell infection in dynamic monocyte-derived macrophage (MDM)-T cell cocultures in the presence of single high antiretroviral drug concentrations but achieved complete inhibition with combination therapy. Together, our data implicate macrophages as drivers of T cell infection by altering physiological MDM-T cell contact dynamics to access and restrain large numbers of susceptible, motile T cells within lymphoid tissues. IMPORTANCE Once HIV enters the lymphoid organs, exponential viral replication in T cells ensues. Given the densely packed nature of these tissues, where infected and uninfected cells are in nearly constant contact with one another, efficient HIV spread is thought to occur through cell-cell contacts in vivo. However, this has not been formally demonstrated. In this study, we performed live-cell imaging studies within a 3-dimensional space to recapitulate the dynamic aspects of the lymphoid microenvironment and asked whether HIV can alter the morphology, migration capacity, and cell-cell contact behaviors between macrophages and T cells. We show that HIV-infected macrophages can engage T cells in stable contacts through binding of virus- and host-derived adhesive molecules and that stable macrophage-T cell contacts were required for high viral spread. Thus, HIV alters physiological macrophage-T cell interactions in order to access and restrain large numbers of susceptible, motile T cells, thereby playing an important role in HIV progression.
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