Research Highlights

Pseudomonas aeruginosa Infection Induces Intragraft Lymphotoxicity that Triggers Lung Transplant Antibody-mediated Rejection Liao F, Zhou D, Cano M, et al. Sci Transl Med. 2025;17(784):eadp1349. Advances in surgical techniques and immunosuppression strategies have improved early survival after lung transplantation, yet long-term outcomes remain poor, with the shortest median survival of all solid organ transplants.1 Reduced survival is largely attributable to chronic lung allograft dysfunction (CLAD), the leading cause of death beyond the first year and affecting more than half of all recipients within 5 y.2 CLAD is associated with both T-cell and antibody-mediated rejection (AMR).3 AMR has proven particularly challenging due to the lack of standardized diagnostic criteria, variability in histological findings, and inconsistent associations between donor-specific antibodies (DSAs) and allograft dysfunction, in addition to a dearth of validated biomarkers.4,5 Infectious complications are also a driver of poor outcomes after lung transplantation. Pseudomonas aeruginosa in particular has been associated with the development and progression of CLAD and is considered a significant risk factor for both bronchiolitis obliterans syndrome and restrictive allograft syndrome.6 The unique exposure of the lung allograft to the external environment combined with chronic immunosuppression and impaired mucociliary clearance creates an environment that supports Pseudomonas infection and persistence promoting chronic airway inflammation, direct epithelial injury, and alloimmune responses.7 Liao et al8 (Figure 1) explored the role of Pseudomonas in lung transplant recipients with a technically challenging, fully major histocompatibility complex–mismatched (BALB/c into C57BL/6), orthotopic (single) lung transplant model using dual costimulatory blockade. After allograft acceptance at 1 mo, a P aeruginosa isolate (PA103) was instilled intratracheally, inducing significant histopathological changes (alveolar injury) and C4d deposition that resembled AMR. Despite the bacterial clearance, IgM DSAs appeared with subsequent isotype switching (to IgG). In contrast, heat-inactivated isolates generated minimal injury and DSA production.FIGURE 1.: Understanding the development of AMR in lung allograft rejection based on infection, isotype switch capacity, germinal B cells, Treg, and Myd88 signaling. Created with Biorender. AID, activation-induced cytidine deaminase; AMR, antibody-mediated rejection; DSA, donor-specific antibody; GC, germinal center; Treg, regulatory T cell; Tx, transplant; µs, secretory chain.To determine the contribution of B cells, the authors used AID–/–μs–/– mice that lacked activation-induced cytidine deaminase (required for antibody diversity by regulating class-switch recombination) and immunoglobulin secretory chain expression but maintained IgM receptor expression and the ability to present antigen. The lack of injury and antibody response in recipients indicated the necessary role of B cells and antibody production in response to pathogen-associated molecular patterns. An interesting phenomenon of this model is that tolerant lung allografts remain so when retransplanted into a nonimmunosuppressed recipient of the same genotype (isogenic recipient). Retransplantation into B6 recipients that expressed the tomato red (TdRed) reporter protein under the control of the β-actin promoter, followed P aeruginosa infection, recruited CD43–CD5– B cells (indicative of B-2 lineage). This was associated with higher frequencies of germinal center (GC) B cells (TdRed+ B220+) that coexpressed CD95, CXCR5, and BCL6, which were not reflected in peripheral lymphoid organs. To further understand the role of GC B cells, the team generated mice lacking Bcl6 (the necessary transcription factor) that also reported expression of Blimp-1 (Prdm1EYFP), which drives the formation of plasma cell. Pseudomonas aeruginosa–infected Cd19Cre/+Bcl6fl/flPrdmlEYFP lung transplant recipients developed few GC B cells compared with controls. IgM DSA generation was preserved (but IgG generation was compromised), with relative protection against rejection and fewer CD273+CD80+CD73lo intragraft memory B cells. Plasma cells (CD138+B220loIAbloCD38lo with intracellular immunoglobulins) were detectable in all recipients, regardless of GC B-cell capability or whether P aeruginosa was instilled. A lack of CD4+FoxP3+ regulatory T cells also promotes rejection, and 18 h after P aeruginosa infection, the graft showed a significant reduction in numbers. There were further reductions in CD4+ T cells and B cells (terminal deoxynucleotidyl transferase dUTP nick end labeling positive—a nonspecific marker of cell death), which were reconstituted after the resolution of infection. CD8+ T cells were relatively spared. The use of an isogenic mutant lacking the multitoxin component type 3 secretion system induced only moderate DSA titers and failed to initiate allograft rejection, with limited lymphocyte death. Targeted depletion of graft-resident Foxp3+CD4+ T cells was accomplished by transplanting accepted allografts from a Foxp3DTR recipient into a wild-type recipient and administering diphtheria toxin (denoted FoxP3Δ). Instillation of saline generated characteristic DSA titers (IgG2c>IgG1 and IgG>IgM), albeit less than titers seen after P aeruginosa infection in wild-type mice, possibly due to the persistence of intragraft IgM+CD23+CD93+CD21+IgDlo/− B cells. This was also associated with elevated T-bet (expressed by circulating B cells) and its transcriptional target CXCR3 in GC and total B cells, with concomitant elevations in serum expression of interferon-γ and B cell–activating factor. Higher T-bet+ and CXCR3+ B cells and lower FoxP3+ CD4+ T cells were also seen in AMR human lung biopsies. Finally, the team investigated the contribution of the pleiotropic adaptor protein Myd88 (which also controls T-bet), transplanting allografts into Cd19cre/+Myd88fl/fl (B cells deficient in Myd88; or controls) and infecting with P aeruginosa. Cd19cre/+Myd88fl/fl recipients had substantially fewer pre-GC B cells with lower CXCR3 expression, defective IgG2c production (and modest class switching bias toward IgG1), reduced intragraft IgG2c+CD273+CD80+ memory B-cell abundance, and protection against rejection. This study highlights potential PAMP-related contributions to pathogenesis and tailored treatment options (CXCR3-directed therapies), as well as the immunologic risk associated with superimposed infections, including rejection9 and erosion of tolerance.10 Early Transcriptional Effects of Inflammatory Cytokines Reveal Highly Redundant Cytokine Networks Lee JL, Yang L, Kotzin JJ, et al. J Exp Med. 2025;222(4):e20241207 Inflammatory cytokines are essential and multifunctional signaling molecules secreted by a broad range of leukocytes in response to external or internal stimulus triggers.1 Cytokines are crucial to immune defense by facilitating communication between immune cells and stromal tissue components,2 and their secretion triggers a further cascade of events that promote inflammatory responses that ultimately lead to the initiation of cell differentiation, proliferation, and/or death.3 Key inflammatory cytokines include members of interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) families, and although their actions are mediated by different receptor groups (Figure 1),4 there is recognized functional overlap due to shared signaling mediators, for example, nuclear factor kappa B.5,6 Inflammatory cytokines regulate each other’s expression and release, forming interconnected signaling pathway networks. RNA sequencing, which enables a systematic analysis of the early transcriptional effects, was used by Lee et al7 to study 3 major inflammatory cytokines, IL-1β, IL-6, and TNF-α (after systemic administration into C57BL/6 mice), and their function across key immune cell types. This publication is from the collaborative Immunological Genome Project (https://www.immgen.org), which aims to better understand the murine immune system and provide the knowledge as a public resource. They have generated an overarching definition of inflammatory gene expression and regulatory networks of immune systems and how they work together to mediate the inflammatory response.FIGURE 1.: Redundancy in inflammatory cytokine signaling. There is crosstalk and overlap between the responses triggered by different cytokines. TNF-α, IL-1β, and IL-6 signaling cascades converge on NF-κB, whereas IL-6 and γc cytokine–signaling cascades share STAT3 for signal transduction. Created with Biorender. γc, gamma chain; CTLA4, cytotoxic T-lymphocyte-associated protein 4; FoxP3, forkhead box P3; IL, interleukin; NF-κB, nuclear factor kappa B; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor alpha.To characterize the transcriptional patterns of IL-1β, IL-6, and TNF-α across immune cell types, the authors first carried out an “ultra-low input” RNA sequencing for major cell types 2 h after exposure to IL-1β, IL-6, and TNF-α. The pattern of immune response to either TNF-α or IL-1β was robust, concordant, and predominately myeloid-centric (particularly neutrophils), and this subset displayed the most extensive transcriptional changes, with considerably less gene expression in B and T lymphocytes. Enrichment analysis revealed a representation of (and overlap with) inflammatory pathways. Relevant transcription factors driving inflammation included MYC, nuclear factor kappa B, the chromatin remodeler chromodomain helicase DNA-binding 1, and interferon regulatory factor 8. Interestingly, there was concurrent transcriptional downregulation, but this did not show any distinct pathway enrichment. The authors also found that TNF-α and IL-1β induced feed-forward loops, stimulating the production of their own transcripts, which enhanced and reflected the rapid innate immune response. This was prominent in myeloid clusters (and absent in lymphoid cells) with incomplete reciprocity in that IL-6 transcripts were induced by IL-1β and TNF-α (but not vice versa). In addition, there was no induction of receptor expression, demonstrating a separation between inflammatory and gamma chain cytokines. Using a Cleavage Under Targets and Release Using Nuclease chromatin profiling, there were marked changes induced by IL-1β for H3K4me3 (promoter), modest changes for other histone modifications, and an additional strong response for repressed genes. Assessment of temporal changes in inflammatory cytokines from 2 to 24 h showed a maximal response at 2–4 h, attenuation by 8 h, and diminution by 24 h. The caveat to this broad pattern was evidence of some transcripts that persisted or appeared later, which were enriched in cell cycle and RNA metabolism. Interestingly, the similarity in inflammatory cytokine production from leukocyte subsets seen in these data has been contradicted by arguments for cell specificity.5 Overlap has long been recognized because of receptor convergence or shared downstream signaling moieties. However, this appeared to vary based on the exogenous stimulus and cell type, as well as contributions from the analytical pipeline used to introduce variability. The same intersection can be demonstrated for gamma chain and inflammatory cytokine profiles, with contributions from common transcription factors (including MYC). This work makes us reconsider the interconnectivity of cytokine networks. Despite the disparate effects of TNF-α and IL-1β and the contrasting implications of pharmacological blockade, target transcripts are similar. There are some cell-specific differences, but perhaps the finer tuning is occurring with posttranslational modification, organ architecture that changes cytokine accessibility, exogenous insult, and/or organismal disparities. These data complement previous work identifying a cytokine hierarchy8 as well as shared molecular and cellular events occurring in vivo that influence infective and inflammatory outcomes. Lee et al7 provided a valuable compendium of the impact of the key inflammatory cytokines across all lineages of the mouse immune system, recognizing that the gross systemic dysregulation of immune processes in response to infectious can be harmful. The question that is not answered by this work is whether the response may fluctuate—temporally, spatially, by cell specificity, and in magnitude—with immunosuppression and how this impacts the allograft and the recipient.