Abstract The immune component of the tumor microenvironment (TME) is highly prognostic of patient outcome, further understanding of TME and the spatial organization of the immune cells within the TME is needed for better patient prognosis and treatment planning. Current brightfield immunohistochemistry techniques quantify immune cell counts and density but generally cannot assess the spatial relationship between tumor and immune cells due to the limited number of markers on a single slide. Here, we present a brightfield-based multiplexed immunohistochemistry workflow that combines multiple labels per staining round with iterative cycles of staining, destaining, and hyperspectral imaging to enable high-dimensional immune profiling within a single tissue section. This approach is directly compared with a sequential immunofluorescence technique applied to adjacent tissue sections to compare and evaluate its performance. By integrating serial imaging, sequential labeling, and image registration, we are able to spatially map the TME using standard brightfield microscopy.
Anti-DNA antibodies are a diverse set of antibodies that bind to structures on DNA that vary with clinical setting. In systemic lupus erythematosus (SLE), anti-DNA antibodies interact with the B-DNA backbone, binding DNA irrespective of species origin. In contrast, in the sera of otherwise healthy individuals (normal human subjects [NHSs]), anti-DNA antibodies bind to certain bacterial DNA. Furthermore, sera from patients with SLE and NHS controls can bind to the left-handed Z-DNA conformation. In the current studies, we have explored the antibody response to mycobacterial DNA from patients with tuberculosis (TB) and NHSs, focusing on DNA from Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis bacillus Calmette-Guérin (BCG), given their high GC content and predicted capacity to form Z-DNA. Using ELISAs, we demonstrated that plasmas from patients with TB and NHSs contain antibodies to mycobacterial DNA, with a greater response to Mtb DNA than BCG DNA. To elucidate these findings, we conducted an in silico analysis with Z-Hunt-III, synteny analysis, and an in-house R Shiny program, Z-GENIE, to identify Z-DNA-forming sequences (ZFSs) and genomic regions that may be antigenic. Our analysis suggested that regions of difference and single-nucleotide polymorphism-generated ZFSs may contribute to sequence differences, which may influence antigenicity. Additionally, we provide evidence that shared sequences between DNA of Mtb and nontuberculous mycobacteria could explain the observed pattern of antibody reactivity. Together, these findings suggest that anti-DNA antibodies from patients with TB and NHS controls can distinguish between the DNA of different mycobacterial species and could serve as serological markers for infection or exposure.
Medical schools in the United States have varying curricular designs and limited instructional time, which challenge cohesive and comprehensive immunology coverage necessary for future physicians. To address this challenge, the American Association of Immunologists formed a Medical Education Immunology Curriculum Task Force, to generate recommendations for U.S. medical schools in the field of immunology. In June 2024, faculty from all U.S. medical schools were invited to participate in an initial research survey designed to assess the current field of immunology content taught to medical students. Approximately 30% of the invited participants responded, with 25% of respondents from DO programs and 75% from MD programs, which reflects the overall distribution of these schools nationally. This article shares the core content identified by the participating faculty, along with various demographic and opinion-based metrics. The Task Force developed specific learning objectives (LOs) and associated immune-relevant clinical conditions for each core area of content. These were then vetted by the broader medical immunology education community by presentation at an immunology-focused professional meeting and through a second feedback survey. The final recommended content includes foundational topics and associated LOs, as well as a list of key associated clinical conditions aligned with each foundational topic. These LOs have the advantage of being inserted throughout integrated curriculum and/or could be used in stand-alone immunology course blocks in the medical school curriculum. The inclusion of these LOs in a medical curriculum will sufficiently educate future physicians regarding the impact of the immune system on overall health and illness.
TNF receptor-associated factor 1 (TRAF1) is a prosurvival signaling adaptor that contributes to NF-κB activation downstream of a subset of TNF receptor superfamily members. TRAF1 is overexpressed in many cancers of mature B cells, including chronic lymphocytic leukemia (CLL). Previous studies have established that TRAF1 S146 is a target of phosphorylation by the kinase PKN1 and that PKN1 is required to prevent cellular inhibitor of apoptosis protein (cIAP)-dependent degradation of TRAF1 in the CD40 signaling complex. The kinase inhibitor OTSSP167 inhibits PKN1 in the nanomolar range and its addition to primary CLL cells was previously shown to induce dose-dependent loss of TRAF1 and concomitant increases in activated caspase 3 and cell death. These studies identified PKN1 as a target for therapy of CLL. To identify more potent and specific PKN1 inhibitors for therapy of B-cell cancers, it is important to measure a direct target of PKN1, such as phospho-TRAF1. To this end, here we use overexpression of an S146A mutant of human TRAF1 in HEK293 cells to validate a recently generated TRAF1 phospho (p)S146-specific antibody and to confirm that this phosphorylation is lost upon treatment with OTSSP167. Using CRISPR/Cas9 knockout in RAJI cells, we also show that both PKN1 and the closely related family member PKN2 can phosphorylate TRAF1 S146. We further show that TRAF1 S146 is constitutively phosphorylated in OP9 cultured primary human CLL cells, including those with p53 mutations, and that this phosphorylation is sensitive to inhibition with OTSSP167. These findings provide support the development of more potent PKN1/2 inhibitors for CLL.
Progesterone exerts important immunomodulatory functions, yet the immune effects of structurally diverse synthetic progestins remain incompletely defined. Given their variable receptor-binding profiles and links to altered inflammatory outcomes, we examined how commonly used progestins regulate immune signaling in primary human cells. Peripheral blood mononuclear cells from healthy female donors were exposed to physiologically relevant concentrations of natural progesterone (P4) and representative synthetic progestins spanning multiple generations. Cytokine responses were quantified at both the protein and transcript levels. Baseline steroid receptor expression and age of donor was correlated with cytokine output. High-dimensional flow cytometry coupled with unsupervised clustering was used to define the cellular sources of inflammatory cytokine production. In parallel, naïve CD4+ T-cell differentiation was performed to determine effects on T helper (Th) cell polarization. Only P4 induced immunoregulatory cytokine IL-10, whereas all progestins, except first-generation norethindrone, induced markedly higher IL-6 production compared with media control. Gene expression analysis confirmed induction of multiple proinflammatory genes at physiologically relevant concentrations. High-dimensional flow cytometry identified CD14+ monocytes as the dominant cellular source of IL-6 following progestin exposure. In contrast, P4 selectively suppressed Th1 differentiation and promoted Th2 lineage commitment, whereas synthetic progestins failed to reproduce these immunoregulatory effects. Together, these findings demonstrate that synthetic progestins differentially regulate innate and adaptive immune responses compared with P4 and promote proinflammatory responses that may influence immune homeostasis.
