The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane

Lack of myeloid cell infiltration as an acquired resistance strategy to immunotherapy

Background Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments.Methods We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients.Results Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-β signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8+ T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients.Conclusion An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.</p

IFNγ production of tumor-specific CD8 T cells.

<p>Frequencies and IFNγ levels of transferred pmel-1 T cells were analysed in the blood at the peak of the response on day 21. MFI indicates intensity of IFNγ staining per pmel-1 population (geo-means). Two-way student t-test was used to calculate statistical significance.</p

Macrophage reduction slows down melanoma outgrowth.

<p>Kaplan-Meier survival curves from control and PLX3397-treated mice are shown of 26 mice per group, compiled from three independent experiments. Mice were sacrificed when tumors reached 1000 mm³. Statistical analyses was performed with log-rank test.</p

Systemic inhibition of CSF-1R decreases B16F10-induced accumulation of blood monocyte subsets.

<p>(A) Myeloid populations in the blood were monitored by flow cytometry following B16F10 tumor growth. Percentages of CD11b⁺ myeloid cells out of total white blood cells (WBC) and frequencies of CD11b⁺F4/80⁺CSF-1R⁺ monocytes or CD11b⁺Gr-1^high granulocytic cells within the CD11b⁺ population are depicted of control- and PLX3397-treated mice. Data are compiled from two different experiments with ten mice per group. Linear regression analysis indicated a significant difference in frequencies between control- and PLX3397-treatment measurements for all three graphs and a significant slope difference of CD11b⁺F4/80⁺CSF-1R⁺ cells (<i>p</i> = 0.04). (B) Frequencies of CD11b⁺F4/80⁺ monocytes in the blood as a proportion of total white blood cells at day 13 of B16F10 tumor growth. (C) Flow cytometry plots of blood CD11b⁺F4/80⁺ cells at day 13 of B16F10 tumor growth. (D) Frequencies of Ly6C^- cells within this gate are plotted for eight mice per group. Means and standard error of the mean of five mice per group are depicted from one out of two similar experiments.</p

Decreased frequencies of tumor infiltrating macrophages.

<p>(A) B16F10 tumors were resected from mice at a size of 200 mm³, embedded in paraffin and stained for F4/80, as a marker for macrophages. Photos are representative for the distinct tumor areas and for more than ten analysed tumors. (B) Three tumors with comparable sizes were resected, dissociated to single cells and analysed by flow cytometry. Frequencies of myeloid subsets (from all CD11b⁺) are depicted. Frequencies of CD45⁺ infiltrating cells are depicted for CD8 T cells. Means and standard error of the mean are shown.</p

CSF-1R inhibition enhances CD8-mediated immunotherapy of melanoma.

<p>(A) Tumor growth curves of individual mice are shown for the four different treatment groups. Immunotherapy is designated as ‘pept vacc’ and contains transfer of pmel-1 CD8 T cells and peptide vaccination. Numbers in the panels indicate the group size and the number of mice that are still alive at day 50. This experiment was repeated once with comparable outcome and indicated survival rates are compiled from both. (B) Average tumor sizes and standard error of the mean per group from experiment shown in panel (A). Statistical analysis was performed with two-way ANOVA after Bonferroni correction.</p

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccine

A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.Pattern Recognition and Bioinformatic