T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers.

BACKGROUND: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. METHODS: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis. RESULTS: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected. CONCLUSIONS: The human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Two sides of the same coin: Protective versus pathogenic CD4+ resident memory T cells.

The ability of the adaptive immune system to form memory is key to providing protection against secondary infections. Resident memory T cells (TRM) are specialized T cell populations that reside within tissue sites where they await reencounter with their cognate antigen. TRM are distinct from circulating memory cells, including central and effector memory T cells, both functionally and transcriptionally. Since the discovery of TRM, most research has focused on CD8+ TRM, despite that CD4+ TRM are also abundant in most tissues. In the past few years, more evidence has emerged that CD4+ TRM can contribute both protective and pathogenic roles in disease. A complexity inherent to the CD4+ TRM field is the ability of CD4+ T cells to polarize into a multitude of distinct subsets and recognize not only viruses and intracellular bacteria but also extracellular bacteria, fungi, and parasites. In this review, we outline the key features of CD4+ TRM in health and disease, including their contributions to protection against SARS-CoV-2 and potential contributions to immunopathology associated with COVID-19.

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

GITR shapes humoral immunity by controlling the balance between follicular T helper cells and regulatory T follicular cells.

Follicular helper CD4+ T-cells (Tfh) control humoral immunity by driving affinity maturation and isotype-switching of activated B-cells. Tfh localize within B-cell follicles and, upon encounter with cognate antigen, drive B-cell selection in germinal centers (GCs) as GC-Tfh. Tfh functionality is controlled by Foxp3-expressing Tfh, which are known as regulatory T follicular cells (Tfr). Thus far, it remains unclear which factors determine the balance between these functionally opposing follicular T-cell subsets. Here, we demonstrate in human and mouse that Tfh and GC-Tfh, as well as their regulatory counterparts, express glucocorticoid-induced TNF receptor related protein (GITR) on their surface. This costimulatory molecule not only helps to identify follicular T-cell subsets, but also increases the ratio of Tfh vs. Tfr, both within and outside the GC. Correspondingly, GITR triggering increases the number of IL-21 producing CD4+ T-cells, which also produce more IFN-γ and IL-10. The latter are known switch factors for IgG2c and IgG1, respectively, which corresponds to a concomitant increase in IgG2c and IgG1 production upon GITR-mediated costimulation. These results demonstrate that GITR can skew the functional balance between Tfh and Tfr, which offers new therapeutic possibilities in steering humoral immunity.

Functional Heterogeneity of CD4+ Tumor-Infiltrating Lymphocytes With a Resident Memory Phenotype in NSCLC.

Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

Blimp-1 induces and Hobit maintains the cytotoxic mediator granzyme B in CD8 T cells.

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA+ effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm.

The TNF Receptor Superfamily-NF-κB Axis Is Critical to Maintain Effector Regulatory T Cells in Lymphoid and Non-lymphoid Tissues.

After exiting the thymus, Foxp3+ regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt+ Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans.

The Transcription Factor Hobit Identifies Human Cytotoxic CD4+ T Cells.

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4+ T cells that conforms to the phenotype of cytotoxic CD8+ T cells has received increased recognition. These cytotoxic CD4+ T cells display constitutive expression of granzyme B and perforin at the protein level and mediate HLA class II-dependent killing of target cells. In humans, this cytotoxic profile is found within the human cytomegalovirus (hCMV)-specific, but not within the influenza- or Epstein-Barr virus-specific CD4+ T cell populations, suggesting that, in particular, hCMV infection induces the formation of cytotoxic CD4+ T cells. We have previously described that the transcription factor Homolog of Blimp-1 in T cells (Hobit) is specifically upregulated in CD45RA+ effector CD8+ T cells that arise after hCMV infection. Here, we describe the expression pattern of Hobit in human CD4+ T cells. We found Hobit expression in cytotoxic CD4+ T cells and accumulation of Hobit+ CD4+ T cells after primary hCMV infection. The Hobit+ CD4+ T cells displayed highly overlapping characteristics with Hobit+ CD8+ T cells, including the expression of cytotoxic molecules, T-bet, and CX3CR1. Interestingly, γδ+ T cells that arise after hCMV infection also upregulate Hobit expression and display a similar effector phenotype as cytotoxic CD4+ and CD8+ T cells. These findings suggest a shared differentiation pathway in CD4+, CD8+, and γδ+ T cells that may involve Hobit-driven acquisition of long-lived cytotoxic effector function.

Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells.

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

Dried Saliva Spots: A Robust Method for Detecting Streptococcus pneumoniae Carriage by PCR.

The earliest studies in the late 19th century on Streptococcus pneumoniae (S. pneumoniae) carriage used saliva as the primary specimen. However, interest in saliva declined after the sensitive mouse inoculation method was replaced by conventional culture, which made isolation of pneumococci from the highly polymicrobial oral cavity virtually impossible. Here, we tested the feasibility of using dried saliva spots (DSS) for studies on pneumococcal carriage. Saliva samples from children and pneumococcus-spiked saliva samples from healthy adults were applied to paper, dried, and stored, with and without desiccant, at temperatures ranging from -20 to 37 °C for up to 35 days. DNA extracted from DSS was tested with quantitative-PCR (qPCR) specifically for S. pneumoniae. When processed immediately after drying, the quantity of pneumococcal DNA detected in spiked DSS from adults matched the levels in freshly spiked raw saliva. Furthermore, pneumococcal DNA was stable in DSS stored with desiccant for up to one month over a broad range of temperatures. There were no differences in the results when spiking saliva with varied pneumococcal strains. The collection of saliva can be a particularly useful in surveillance studies conducted in remote settings, as it does not require trained personnel, and DSS are resilient to various transportation conditions.

Carriage of Streptococcus pneumoniae in aged adults with influenza-like-illness.

Incidence of pneumococcal disease is disproportionally high in infants and elderly. Nasopharyngeal colonisation by Streptococcus pneumoniae is considered a prerequisite for disease but unlike in children, carriage in elderly is rarely detected. Here, we tested for S. pneumoniae in nasopharyngeal and saliva samples collected from community-dwelling elderly with influenza-like-illness (ILI). Trans-nasal nasopharyngeal, trans-oral nasopharyngeal and saliva samples (n = 270 per sample type) were collected during winter/spring 2011/2012 from 135 persons aged 60-89 at onset of ILI and 7-9 weeks later following recovery. After samples were tested for pneumococci by conventional culture, all plate growth was collected. DNA extracted from plate harvests was tested by quantitative-PCRs (qPCR) specific for S. pneumoniae and serotypes included in the 13-valent pneumococcal conjugated vaccine (PCV13). Pneumococci were cultured from 14 of 135 (10%) elderly with none of the sampled niches showing superiority in carriage detection. With 76/270 (28%) saliva, 31/270 (11%) trans-oral and 13/270 (5%) trans-nasal samples positive by qPCR, saliva was superior to nasopharyngeal swabs (p<0.001) in qPCR-based carriage detection. Overall, from all methods used in the study, 65 of 135 (48%) elderly carried pneumococci at least once and 26 (19%) at both study time points. The difference between carriage prevalence at ILI (n = 49 or 36%) versus recovery (n = 42 or 31%) was not significant (p = 0.38). At least 23 of 91 (25%) carriage events in 19 of 65 (29%) carriers were associated with PCV13-serotypes. We detected a large reservoir of pneumococci in saliva of elderly, with PCV13-serotype distribution closely resembling the contemporary carriage of serotypes reported in the Netherlands for PCV-vaccinated infants.