Biology subjects

Prlic, M.

Publications and source records attributed to Prlic, M..

15 recordsLinked to original sources

bioRxiv2026

Human tissue-resident CD8 T cells contribute to trophoblast homeostasis in health and during acute inflammation

Previous studies have highlighted that some T cell subsets in tissues can provide signals to support tissue cell homeostasis and differentiation. If and how T cell-tissue cell signaling is altered in healthy compared to inflamed tissues is poorly understood. Here, we address if communication between human T cells and tissue cells changes from steady state to an acutely inflamed state in the human placenta. We used single cell analysis strategies to examine invasive cytotrophoblasts (iCTBs) and immune cells isolated from third trimester healthy and acutely inflamed human placentas. We performed cell communication analysis to predict cell-cell communication networks, and found evidence that iCTBs provided signals to support the recruitment of T cells, as well as the formation of tissue-resident memory CD8 T cells (Trm). In exchange, Trm provide signals to support iCTB homeostasis. During acute inflammation, iCTBs and macrophages underwent profound transcriptional changes, while most T cell subsets only underwent limited transcriptional changes. This was not due to T cell exhaustion or tolerance, as T cells were functionally intact. Cell communication analysis and validation at the protein level provide evidence that T cells can maintain their homeostatic support to iCTBs during acute inflammation.

immunology↗

bioRxiv2026

Menstruation is associated with cyclical granulysin peaks in vaginal secretions despite stable expression by cervicovaginal immune cells

ProblemThe anti-microbial protein granulysin is present in vaginal secretions during the follicular phase of the menstrual cycle but nearly disappears during the luteal phase. The reason for this change is unknown. Method of studyParticipants (n = 23) with regular menstrual cycles collected daily vaginal swabs for granulysin ELISAs. Endocervical cytobrushes, ectocervical biopsies, vaginal biopsies, and PBMC were collected across the cycle to enumerate granulysin-expressing cells by flow cytometry. Cycle phase was determined by daily urinary luteinizing hormone testing and confirmed by serum progesterone levels. ResultsGranulysin levels in secretions were up to 10,000 times higher during menstruation than during the luteal phase (menstruation, median 3,924 pg/mL [IQR 400-17,280]; luteal, median and IQR undetectable [<7.81 pg/mL]). In the endocervical canal, granulysin-expressing cells were much more abundant during menstruation than during the mid-follicular or mid-luteal phases. In contrast, the number of granulysin-expressing cells in the ectocervix and vagina remained stable during the cycle. The most abundant granulysin-expressing cell types in the mucosa were CD8 T cells and NK cells. In a minority of participants, granulysin was consistently detected in luteal-phase swabs; this phenomenon was associated with parity. ConclusionsGranulysin in vaginal secretions is associated with menstruation and concomitant with a spike in granulysin-expressing cells in the endocervical canal. This result explains the much higher granulysin levels in secretions during the follicular than the luteal phase. In contrast, immune cells from ectocervical and vaginal biopsies express granulysin independently of the menstrual cycle, indicating their continuous ability to respond to microbial infection.

immunology↗

bioRxiv2026

Human lung γδ T cells maintain functionality during inflammatory lung disease

{gamma}{delta} T cells provide mucosal defense against infection while also contributing to tissue repair. However, data regarding the effect of the human lung environment on {gamma}{delta} T cell functionality remains limited. To address whether lung inflammation impacts {gamma}{delta} T cell functionality, we analyzed lung and matched hilar lymph node (LN) tissue from deceased donors and patients with interstitial lung disease (ILD). We performed high-parameter spectral flow cytometry to examine the expression pattern of phenotypic biomarkers and assess ex vivo function. We identified lung-specific enrichment of {gamma}{delta} T cells with an effector memory phenotype relative to matched regional LN. We then used an ex vivo stimulation approach to interrogate the capacity to protect against infection (granzyme B [GzmB], interferon-{gamma} [IFN{gamma}] and tumor necrosis factor [TNF]) and promote epithelial cell proliferation (amphiregulin [AREG]). We found that {gamma}{delta} T cells in lung and LN from deceased donors had similar functional properties. While {gamma}{delta} T cell populations from ILD lungs largely maintained cytokine production capacity, expression was diminished relative to LN counterparts. Importantly, lung {gamma}{delta} T cells maintained polyfunctional GzmB, IFN{gamma} and TNF expression across cohorts. Overall, we report human lung {gamma}{delta} T cells are regionally distinct with conserved functionality in a fibrotic environment.

immunology↗

bioRxiv2026

Tissue-specific adaptation of human T cells is preserved during tissue inflammation

T cells play an essential role in protecting tissues against pathogens and regulating tissue homeostasis. Previous studies highlight that T cells display tissue-specific phenotypic and functional properties, suggesting that T cells adapt to their local environment. Whether inflammation disrupts tissue-specific T cell adaptation remains poorly understood. To address this open question, we examined the T cell compartment - including conventional CD4 and CD8 T cells, regulatory T cells, {gamma}{delta} T cells, and MAIT cells - from healthy and inflamed human mucosal tissues. Using high-parameter spectral flow cytometry, we examined phenotype ex vivo and the functional capacity following stimulation, utilizing conventional gating and unsupervised clustering analysis approaches. Overall, we analyzed 65 tissue samples including mild, moderate, and severely inflamed oral gingiva, healthy and inflamed lung, along with healthy and inflamed tissue from the decidual-placental interface. Across these mucosal barrier tissues, we find that tissue location plays a dominant role in shaping the composition, phenotype, and functional capacity of the T cell compartment. Importantly, these tissue-specific adaptations were largely maintained during states of tissue inflammation. This included the ability to exert tissue repair functions, which was preserved across T cell subsets, even in severely inflamed tissues.

immunology↗

bioRxiv2026

Regulatory T cells restrain IL-15-mediated cytotoxic and bystander T cell activity in mucosal tissue without compromising antigen-driven memory

ABSTRACT/SUMMARYMany pathogenic human infections enter the host via a mucosal surface. These nonlymphoid tissues are abundantly populated by polyclonal memory CD8 T cells that persist following infections for protection upon repeat exposure. Memory T cells can be triggered via T cell receptor recognition of their cognate antigen upon re-infection to exert effector functions, including cytotoxicity and cytokine production, and assist in pathogen elimination. Alternatively, some T cells are bystander activated by cytokines without an antigenic signal. This layered approach boosts efacient pathogen clearance but also poses a threat to host tissues if this response is not properly controlled. Here, we investigate the regulatory mechanisms modulating the tissue memory CD8 T cell response upon recall, leveraging viral rechallenge mouse models to distinguish antigen-driven versus cytokine-activated memory tissue CD8 T cell immunity. We and that regulatory T cells (Treg) participate in restricting cytotoxic and bystander activity without compromising the antigen-driven protective memory CD8 T cell response in mucosal T cells. Critically, Treg provide extrinsic regulation of tissue CD8 T cell cytotoxicity in part through restriction of available IL-2 and IL-15 trans-presentation. Our andings help deane the extrinsic environmental and cellular cues in mucosal tissues that direct tissue-memory CD8 T cells.

immunology↗

bioRxiv2026

CaptureBody - an anti-CD45 x anti-IgG bispecific antibody enables accurate unmixing for spectral flow cytometry

Accurate spectral unmixing is a critical step for flow cytometry data analysis and requires a single stain control for every fluorescent parameter used in an experiment. Currently, compensation particles are often used for making single stain controls when a target protein is of low abundance or a cell type is of low frequency. However, compensation particles introduce incongruencies in emission spectra compared to cells resulting in spectral unmixing or compensation errors. To enable the use of cells regardless of the abundance of target proteins or immune cell type, we generated a bispecific antibody that links a human anti-CD45 and mouse anti-IgG variable region. We refer to this new bispecific tool as CaptureBody (CB) and highlight the benefits of its final nanobody-based design. We provide all sequences and methods necessary for the in-house expression of a CaptureBody to disseminate their use for spectral flow cytometry experiments.

immunology↗

bioRxiv2025

Mucosal tissue NK cells tune their function between optimal anti-pathogen activity and tissue protection

Preserving barrier integrity while mounting effective immunity is essential at mucosal surfaces. In examining the immune cells that mediate both inflammation and tissue homeostasis, we uncovered a dual role for natural killer (NK) cells in barrier immunity. While NK cells are known to control viral infections, here we identify a previously unrecognized reparative function for NK cells in mucosal tissues. Using single-cell RNA sequencing and high-dimensional flow cytometry, we reveal a distinct population of human mucosal NK cells marked by tissue residency, immunoregulatory profiles, and limited cytotoxic potential at homeostasis, yet highly responsive to inflammatory cues. In a mouse model of acute HSV-2 infection, NK cells are required for limiting infection-associated tissue damage. Mucosal NK cells express the epithelial growth factor amphiregulin (Areg), and their depletion leads to increased tissue barrier damage despite preserved viral clearance, suggesting a novel role in tissue protection. Mechanistically, we demonstrate that the barrier-derived cytokines IL-18 and IL-33 induce Areg expression by both human and mouse NK cells, linking local inflammatory cues to reparative NK cell programming that is able to potentiate wound healing. Together, our findings reveal a context-dependent, dual function of mucosal NK cells in immune defense and mucosal tissue protection, expanding current models of NK cell biology.

immunology↗

bioRxiv2023

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in tissue biopsies and other human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

immunology↗

bioRxiv2023

A novel non-invasive method to sample immune cells in the lower female genital tract

T cells in the human female genital tract (FGT)2 are key mediators of susceptibility to and protection from infection, including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT, but current sampling methods require a healthcare provider and are expensive, limiting the ability to study these populations longitudinally. To address these challenges, we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are non-invasive, self-applied, and low-cost. To demonstrate reproducibility, we sampled the cervicovaginal fluid (CVF)3 of healthy, reproductive-aged individuals using menstrual discs over three sequential days. CVF was processed for cervicovaginal cells, and high parameter flow cytometry was used to characterize immune populations. We identified large numbers of live, CD45+ leukocytes, as well as distinct populations of T cells and B cells. Within the T cell compartment, activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches, including identification of both tissue resident and migratory populations. In addition, the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies.

immunology↗

bioRxiv2023

TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner

Transforming growth factor {beta} (TGF-{beta}) directly acts on naive, effector and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-{beta} signaling. TGF-{beta} availability is altered by infections and cancer, however the dose-dependent effects of TGF-{beta} on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-{beta} signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-{beta} could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or pro-inflammatory signals. In contrast, even high doses of TGF-{beta} had a comparatively modest effect on IFN-{gamma} expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-{beta} signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-{beta} signals is of importance. We found that exposure to TGF-{beta} prior to as well as after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-{beta} altered [~]10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-{beta}-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-{beta} is not simply suppressing CD8 Tmem, but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.

immunology↗

bioRxiv2021

Convergent clonal selection of donor- and recipient-derived CMV-specific T cells in hematopoietic stem cell transplant patients

Competition between antigen-specific T cells for peptide:MHC (p:MHC) complexes shapes the ensuing T cell response. Mouse model studies provided compelling evidence that competition is a highly effective mechanism controlling the activation of naive T cells. However, assessing the effect of T cell competition in the context of a human infection requires defined pathogen kinetics and trackable naive and memory T cell populations of defined specificity. A unique cohort of non-myeloablative hematopoietic stem cell transplant (nmHSCT) patients allowed us to assess T cell competition in response to CMV reactivation, which was documented with detailed virology data. In our cohort, HSCT donors and recipients were CMV-seronegative and -positive, respectively, thus providing genetically distinct memory and naive T cell populations. We used single-cell transcriptomics to track donor versus recipient-derived T cell clones over the course of 90 days. We found that donor-derived T cell clones proliferated and expanded substantially following CMV-reactivation. However, for immunodominant CMV epitopes, recipient-derived memory T cells remained the overall dominant population. This dominance was maintained despite more robust clonal expansion of donor-derived T cells in response to CMV reactivation. Interestingly, the donor-derived T cells that were recruited into these immunodominant memory populations shared strikingly similar TCR properties with the recipient-derived memory T cells. This selective recruitment of identical and nearly identical clones from the naive into the immunodominant memory T cell pool suggests that competition does not interfere with rejuvenating a memory T cell population, but results in selection of convergent clones to the memory T cell pool. SignificanceAn existing memory T cell population specific for a single epitope is sufficient to effectively curtail responses to any new antigens if the original epitope is present in a vaccination regimen or heterologous infections. We asked if T cell competition precludes recruitment of any new, naive T cells to an existing memory T cell pool in context of CMV-specific T cell responses in a cohort of transplant patients. Our data indicate that competition does not prevent recruitment of naive T cells into the memory T cell pool, but selects for T cells with nearly or fully congruent T cell receptor specificities. We discuss the implications of rejuvenating a memory T cell pool while preserving the T cell receptor repertoire.

immunology↗

bioRxiv2021

Inflammatory signals are sufficient to elicit TOX expression in mouse and human CD8 T cells

T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which induces a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in specific pathogen-free laboratory mice typically do not express TOX, functional human Tmem show heterogeneous TOX expression levels. Whether TCR-independent mechanisms can alter TOX expression in human and murine Tmem has not been defined. We report that human and mouse Tmem increase TOX expression following stimulation with inflammatory cytokines IL-12, IL-15, and IL-18. TOX and PD-1 expression patterns often appear to be directly correlated, however, we found that TOX is not necessary for cytokine-driven expression of PD-1. Together, these observations highlight that inflammation is sufficient to alter TOX and PD-1 expression and that the signals regulating TOX expression appear well conserved in human and murine Tmem.

immunology↗

bioRxiv2019

Progressive differentiation of memory CD8 T cells in the cervicovaginal tissue

Tissue-resident memory CD8 T cells (CD8 TRM) are critical for maintaining barrier immunity. CD8 TRM have been mainly studied in the skin and gut with recent studies suggesting that the signals that control tissue-residence and phenotype are highly tissue-dependent. We examined the T cell compartment in healthy human cervicovaginal tissue (CVT) and found that most CD8 T cells were granzyme B+ and TCF-1-. To address if this phenotype is driven by CVT tissue-residence, we used a mouse model to control for environmental factors. Using localized and systemic infection models, we found that CD8 TRM in the mouse CVT gradually acquired a granzyme B+, TCF-1- phenotype as seen in human CVT. In contrast to CD8 TRM in the gut, these CD8 TRM were not stably maintained regardless of the initial infection route, which led to reductions in local immunity. Our data show that residence in the CVT is sufficient to progressively shape the size and function of its CD8 TRM compartment. SummaryThe tissue-resident memory (TRM) CD8 T cell compartment in human and mouse cervicovaginal tissue (CVT) is remarkably similar. The CVT TRM compartment is maintained autonomously and does not reach phenoypical or numerical equilibrium. The numerical decline leads to impaired viral control in a secondary challenge.

immunology↗

bioRxiv2019

A targeted multi-omic analysis approach measures protein expression and low abundance transcripts on the single cell level

High throughput single-cell RNA sequencing (sc-RNAseq) has become a frequently used tool to assess immune cell function and heterogeneity. Recently, the combined measurement of RNA and protein expression by sequencing was developed, which is commonly known as CITE-Seq. Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcript, but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets.\n\nHere, we describe a novel targeted transcriptomics approach that combines analysis of over 400 genes with simultaneous measurement of over 40 proteins on more than 25,000 cells. This targeted approach requires only about 1/10 of the read depth compared to a whole transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic transcript-protein datasets, we adapted One-SENSE for intuitive visualization of the relationship of proteins and transcripts on a single-cell level.

immunology↗

bioRxiv2019

Elimination of HSV-2 infected cells is mediated predominantly by paracrine effects of tissue-resident T cell derived cytokines

The mechanisms underlying rapid elimination of herpes simplex virus-2 (HSV-2) in the human genital tract despite low tissue-resident CD8+ T-cell density (TRM) are unknown. We analyzed shedding episodes during chronic HSV-2 infection: viral clearance always occurred within 24 hours of detection even if viral load exceeded 107 HSV DNA copies; surges in granzyme B and interferon-{gamma} occurred within the early hours after reactivation. We next developed a mathematical model of an HSV-2 genital ulcer to integrate mechanistic observations of TRM in situ proliferation, trafficking, cytolytic effects and cytokine alarm signaling from murine studies with viral kinetics, histopathology and lesion size data from humans. A sufficiently high density of HSV-2 specific TRM predicted rapid contact-mediated elimination of infected cells. At lower TRM densities, TRM must initiate a rapidly diffusing, polyfunctional cytokine response in order to eliminate of a majority of infected cells and eradicate briskly spreading HSV-2 infection.\n\nOne Sentence SummaryControl of herpes simplex virus-2 is primarily mediated by rapidly diffusing cytokines secreted by tissue-resident T cells.

immunology↗