Madeline J. Lee

Background: ¹⁷⁷Lu-PSMA-617 radioligand treatment represents a major advancement in the management of metastatic castration-resistant prostate cancer (mCRPC), but key questions remain about tumor cell dynamics and evolution in patients who have received this drug. Here, we used a circulating tumor cell (CTC) capture assay to assess the prognostic value of CTC subsets defined by PSMA and EpCAM, and their dynamics, during ¹⁷⁷Lu-PSMA-617 treatment in a cohort of mCRPC patients. Methods: Using a deep learning-based holographic imaging platform combined with in-flow protein marker analysis, we prospectively analyzed serial CTC samples from patients receiving ¹⁷⁷Lu-PSMA-617 therapy at pre-treatment and on-treatment time points (n = 39 of 100 enrolled patients). PSMA and EpCAM expression on CTCs was determined by immunofluorescence staining. Proportions and absolute counts of CTC subsets delineated by PSMA and EpCAM expression were assessed in PSA50 responders (n = 19) and non-responders (n = 20). Within-patient changes in CTC subsets were compared using Wilcoxon signed-rank tests with Benjamini–Hochberg correction. Associations of CTC subset abundance and dynamics with overall survival were assessed using landmark Kaplan–Meier estimates (time zero at on-treatment sample collection) with log-rank tests and univariable Cox proportional hazards models. Results: PSMA and EpCAM expression defined three CTC subsets: PSMA+/EpCAM+, PSMA+/EpCAM−, and PSMA−/EpCAM+. The mean proportion of PSMA−/EpCAM+ CTCs increased following ¹⁷⁷Lu-PSMA-617 in both PSA50 responders (6.2% to 18.7%; p = 0.041) and non-responders (7.9% to 15.4%; p = 0.023). This corresponded to a decrease in the proportion of PSMA+/EpCAM− CTCs, whereas the proportion of PSMA+/EpCAM+ CTCs did not decline. PSMA+ CTCs remained detectable in on-treatment samples from 37/39 patients, including 17/19 PSA50 responders. Pre-treatment counts of >5 CTCs, relative to ≤5 CTCs, were associated with shorter overall survival in the PSMA+/EpCAM+ subset (HR 3.66, 95%CI 1.35–9.94, p = 0.011), with a similar but nonsignificant trend in the PSMA+/EpCAM− subset (HR 7.00, 95%CI 0.94–51.9, p = 0.057), whereas on-treatment counts were not significantly associated with survival. Finally, on-treatment declines in PSMA+/EpCAM+ (HR 0.852, p = 0.731) and PSMA+/EpCAM− (HR 0.66, p = 0.305) CTC subsets were not significantly associated with overall survival relative to sustained high levels of these cells. Conclusions: In mCRPC patients receiving ¹⁷⁷Lu-PSMA-617, PSMA+ CTCs frequently persist during treatment, and the pre-treatment abundance of PSMA+ CTC subsets may have prognostic value. These exploratory findings warrant validation in larger cohorts.

Cytokines dimerize two receptor chains to activate Janus kinases and signal transducer and activator of transcription (STAT) transcription factors that regulate immune cells, but they have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs. Trikines coactivated phosphorylation of STAT5 (pSTAT5) and pSTAT3 signatures distinct from natural cytokines by assembling trimeric combinations of interleukin-2 (IL-2), IL-10, and IL-21 receptors. In preclinical models, an IL-2–based Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without observable toxicity. An IL-10–based Trikine induced immune infiltration into poorly immunogenic tumors, showing efficacy in preclinical models of small cell lung cancer and pancreatic cancer. Trikines obviate the need for cell engineering to customize STAT signatures and may hold potential for immunotherapy.

Natural killer (NK) cells are innate lymphocytes known for their ability to kill infected or malignant cells, but they have an overlooked role in regulating antibody responses. In mice, NK cells can kill T follicular helper (T _FH ) cells, decreasing somatic hypermutation and antibody titers. Although human NK cell activation correlates with poor vaccine response, the mechanisms of NK cell regulation of adaptive immunity in humans are poorly understood. Here we found that, in ancestral severe acute respiratory syndrome coronavirus 2 infection, individuals with the broadest neutralization profile had fewer NK cells that expressed inhibitory and immaturity markers, whereas NK cells from narrow neutralizers were highly activated and expressed interferon-stimulated genes. ISG-mediated activation in NK cells from healthy donors increased cytotoxicity toward induced T _FH -like cells via NKG2D and NKp30. This work reveals that NK cell activation and dysregulated inflammation play a role in poor antibody response to severe acute respiratory syndrome coronavirus 2 and opens exciting avenues for designing improved vaccines and adjuvants to target emerging pathogens.

Timely initiation of antiretroviral therapy (ART) remains a major challenge in the effort to treat children living with HIV (“CLH”) and little is known regarding the dynamics of immune normalization following ART in CLH with varying times to and durations of ART. Here, we leveraged two cohorts of virally-suppressed CLH from Nairobi, Kenya to examine differences in the peripheral immune systems between two cohorts of age-matched children (to control for immune changes with age): one group which initiated ART during early HIV infection and had been on ART for 5-6 years at evaluation (early, long-term treated; “ELT” cohort), and one group which initiated ART later and had been on ART for approximately 9 months at evaluation (delayed, short-term treated; “DST” cohort). We profiled PBMC and purified NK cells from these two cohorts by mass cytometry time-of-flight (CyTOF). Although both groups of CLH had undetectable viral RNA load at evaluation, there were marked differences in both immune composition and immune phenotype between the ELT cohort and the DST cohort. DST donors had reduced CD4 T cell percentages, decreased naive to effector memory T cell ratios, and markedly higher expression of stress-induced markers. Conversely, ELT donors had higher naive to effector memory T cell ratios, low expression of stress-induced markers, and increased expression of markers associated with an effective antiviral response and resolution of inflammation. Collectively, our results demonstrate key differences in the immune systems of virally-suppressed CLH with different ages at ART initiation and durations of treatment and provide further rationale for emphasizing early onset of ART. AUTHOR SUMMARY Many children living with HIV lack access to both antiviral treatments and testing for HIV infection and are therefore unable to initiate treatment in a timely manner. When children do begin treatment, their immune systems take time to recover from the uncontrolled HIV infection. In this study, we examine how the immune systems of children living with HIV normalize after treatment onset by looking at two groups of children whose HIV is well-controlled by treatment and who therefore don’t have virus replicating in their blood. One group started treatment within the first year of life and has been on treatment for 5-6 years, while the other began treatment after the first year and has been treated for around 9 months. Although both of these groups are virally-suppressed, we found significant differences in their immune profiles, with the children who had delayed and short-term treatment showing signs of inflammation and immune dysfunction. Collectively, our study helps us understand how variation in the timing and duration of ART treatment impacts the immune system in children with viral suppression and therefore provides clinicians with additional knowledge that can inform the care of children living with HIV, improving their health and quality of life.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-β, that underlie this dysregulation. However, the role of cell–cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell–cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16− NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-β. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I interferon and TGFβ, that underlie this dysregulation. However, the role of cell-cell interactions in mediating changes in NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell co-culture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro co-cultures in which NK cells from healthy donors were incubated with monocytes from COVID-19+ or healthy donors. Co-culture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a transwell setting, we found that only CD56 ^bright CD16 ^- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from transwell co-cultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines as well as TGFβ. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19. BACKGROUND: Natural killer (NK) cells are innate lymphocytes that are critical antiviral effectors. Because of their role in controlling acute viral infections, multiple studies have evaluated the role of NK cells in SARS-CoV-2 infection. Such studies revealed that NK cell phenotype and function are significantly altered by severe COVID-19; the peripheral NK cells of severe COVID-19 patients are highly activated and proliferative(1–5), with increased expression of cytotoxic molecules, Ki-67, and several surface markers of activation(3, 5–8). However, these NK cells also have dysfunctional cytotoxic responses to both tumor target cells(1, 2, 9, 10) and SARS-CoV-2-infected target cells(9, 10). Given that peripheral NK cells are thought to migrate to the lung during COVID-19(11–13), these results suggest that the NK cells of severe COVID-19 patients may be incapable of mounting a successful antiviral response to SARS-CoV-2 infection. Although the unique phenotype and dysfunctionality of NK cells in severe COVID-19 has been well-characterized, the processes underlying these phenomena have not. Only one study has conducted in vitro mechanistic experiments to identify a possible cause of NK cell dysfunction: Witkowski et al. identified serum-derived TGFβ as a suppressor of NK cell functionality in severe COVID-19 patients(9). However, this study did not identify the source of serum TGFβ. Additionally, given the high degree of complexity within the immune system, there are likely other causes of NK cell dysfunction in COVID-19 that have thus far remain unexplored. One such mechanism may be the myriad of interactions between NK cells and other peripheral immune cells. NK cells are known to interact with CD4 and CD8 T cells, dendritic cells, neutrophils, and macrophages/monocytes(14), which can prime NK cell cytotoxicity or induce tolerance. Previous work by our lab suggested the potential for NK cell-monocyte crosstalk in severe COVID-19 through the expression of ligands for NK cell activating receptors on the monocytes of these patients(3). Crosstalk between NK cells and monocytes plays a role in regulating the NK cell response to other infections, including HIV-1(15, 16), mouse(17) and human cytomegalovirus(18), and malaria(19) through mechanisms including secretion of NK cell-regulating cytokines by monocytes. In this study, we used a combination of computational and in vitro methods to dissect the interactions between NK cells and monocytes in severe COVID-19. We utilized primary NK cells and monocytes from a large cohort of COVID-19 patients to demonstrate that co-culture of healthy NK cells with monocytes from severe COVID-19 donors can partially recapitulate the activated phenotype observed in the NK cells from COVID-19 patients. We then interrogated the mechanisms by which this activation occurs by performing NK cell-monocyte co-cultures in a transwell setting and using O-link to analyze the cytokines present in this system. Collectively, our work identifies monocytes as a driver of NK cell activation in severe COVID-19 and reveals interactions between NK cells and monocytes that may underlie this process.

Natural killer (NK) cells are critical effectors of antiviral immunity. Researchers have therefore sought to characterize the NK cell response to coronavirus disease 2019 (COVID-19) and the virus that causes it, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The NK cells of patients with severe COVID-19 undergo extensive phenotypic and functional changes. For example, the NK cells from critically ill patients with COVID-19 are highly activated and exhausted, with poor cytotoxic function and cytokine production upon stimulation. The NK cell response to SARS-CoV-2 is also modulated by changes induced in virally infected cells, including the ability of a viral peptide to bind HLA-E, preventing NK cells from receiving inhibitory signals, and the downregulation of major histocompatibility complex class I and ligands for the activating receptor NKG2D. These changes have important implications for the ability of infected cells to escape NK cell killing. The implications of these findings for antibody-dependent NK cell activity in COVID-19 are also reviewed. Despite these advances in the understanding of the NK cell response to SARS-CoV-2, there remain critical gaps in our current understanding and a wealth of avenues for future research on this topic.

The twenty-first century has seen the emergence of many epidemic and pandemic viruses, with the most recent being the SARS-CoV-2-driven COVID-19 pandemic. As obligate intracellular parasites, viruses rely on host cells to replicate and produce progeny, resulting in complex virus and host dynamics during an infection. Single-cell RNA sequencing (scRNA-seq), by enabling broad and simultaneous profiling of both host and virus transcripts, represents a powerful technology to unravel the delicate balance between host and virus. In this review, we summarize technological and methodological advances in scRNA-seq and their applications to antiviral immunity. We highlight key scRNA-seq applications that have enabled the understanding of viral genomic and host response heterogeneity, differential responses of infected versus bystander cells, and intercellular communication networks. We expect further development of scRNA-seq technologies and analytical methods, combined with measurements of additional multi-omic modalities and increased availability of publicly accessible scRNA-seq datasets, to enable a better understanding of viral pathogenesis and enhance the development of antiviral therapeutics strategies.