Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection.

Here, we show that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, and control chronic viral challenges. The cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2-dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2-producing effector subset with superior protective powers. In contrast, the non-IL-2-producing effector cells respond to IL-2 signals and gain effector traits at the expense of memory formation. Despite having distinct properties during the effector phase, IL-2-producing and nonproducing CD8 T cells appear to converge transcriptionally as memory matures to form populations with equal recall abilities. Therefore, the potential to produce IL-2 during the effector, but not memory stage, is a consequential feature that dictates the protective capabilities of the response.

Predicting the Probability of Chlamydia Reinfection in African American Women Using Immunologic and Genetic Determinants in a Bayesian Model.

BACKGROUND: African Americans have the highest rates of Chlamydia trachomatis (CT) infection in the United States and also high reinfection rates. The primary objective of this study was to develop a Bayesian model to predict the probability of CT reinfection in African American women using immunogenetic data. METHODS: We analyzed data from a cohort of CT-infected African American women enrolled at the time they returned to a clinic in Birmingham, AL, for the treatment of a positive routine CT test result. We modeled the probability of CT reinfection within 6 months after treatment using logistic regression in a Bayesian framework. Predictors of interest were presence or absence of an HLA-DQB1*06 allele and CT-specific CD4+ IFN-γ response, both of which we had previously reported were independently associated with CT reinfection risk. RESULTS: Among 99 participants evaluated, the probability of reinfection for those with a CT-specific CD4+ IFN-γ response and no HLA-DQB1*06 alleles was 14.1% (95% credible interval [CI], 3.0%-45.0%), whereas the probability of reinfection for those without a CT-specific CD4+ IFN-γ response and at least one HLA-DQB1*06 allele was 61.5% (95% CI, 23.1%-89.7%). CONCLUSIONS: Our model demonstrated that presence or absence of an HLA-DQB1*06 allele and CT-specific CD4+ IFN-γ response can have an impact on the predictive probability of CT reinfection in African American women.

Stimulated peripheral blood mononuclear cells from chlamydia-infected women release predominantly Th1-polarizing cytokines.

Chlamydia trachomatis infection (chlamydia) is the most prevalent sexually transmitted bacterial infection and causes significant reproductive morbidity in women. Little is known about how immunity to chlamydia develops in women, though animal models of chlamydia indicate that T-helper type 1 (Th1) responses are important for chlamydia clearance and protective immunity, whereas T-helper type 2 (Th2) responses are associated with persisting infection. In chlamydia-infected women, whether the predominant immune response is Th1- or Th2-polarizing remains controversial. To determine the cytokine profiles elicited by peripheral blood mononuclear cells (PBMCs) from chlamydia-infected women, we stimulated PBMCs with C. trachomatis elementary bodies and recombinant C. trachomatis Pgp3 and measured supernatant levels of select cytokines spanning Th1- and Th2-polarizing responses. We found that stimulated PBMCs from chlamydia-infected women secreted cytokines that indicate strong Th1-polarizing responses, especially interferon-gamma, whereas Th2-polarizing cytokines were expressed at significantly lower levels. In chlamydia-infected women, the predominant cytokine responses elicited on stimulation of PBMCs with C. trachomatis antigens were Th1-polarizing, with interferon-gamma as the predominant cytokine.

An Adaptive Chlamydia trachomatis-Specific IFN-γ-Producing CD4+ T Cell Response Is Associated With Protection Against Chlamydia Reinfection in Women.

Background: Adaptive immune responses that mediate protection against Chlamydia trachomatis (CT) remain poorly defined in humans. Animal chlamydia models have demonstrated that CD4+ Th1 cytokine responses mediate protective immunity against reinfection. To better understand protective immunity to CT in humans, we investigated whether select CT-specific CD4+ Th1 and CD8+ T cell cytokine responses were associated with protection against CT reinfection in women. Methods: Peripheral blood mononuclear cells were collected from 135 CT-infected women at treatment and follow-up visits and stimulated with CT antigens. CD4+ and CD8+ T-cells expressing IFN-γ, TNF-α, and/or IL-2 were assessed using intracellular cytokine staining and cytokine responses were compared between visits and between women with vs. without CT reinfection at follow-up. Results: A CD4+TNF-α response was detected in the majority (77%) of study participants at the treatment visit, but a lower proportion had this response at follow-up (62%). CD4+ IFN-γ and CD4+ IL-2 responses occurred less frequently at the treatment visit (32 and 18%, respectively), but increased at follow-up (51 and 41%, respectively). CD8+ IFN-γ and CD8+ TNF-α responses were detected more often at follow-up (59% for both responses) compared to the treatment visit (30% for both responses). At follow-up, a CD4+IFN-γ response was detected more often in women without vs. with reinfection (60 vs. 33%, P = 0.005). Conclusions: Our findings suggest that a CT-specific CD4+ IFN-γ response is associated with protective immunity against CT reinfection and is thus an important component of adaptive immunity to CT in women.

Performance of Chlamydia trachomatis OmcB Enzyme-Linked Immunosorbent Assay in Serodiagnosis of Chlamydia trachomatis Infection in Women.

Chlamydia trachomatis serological assays with improved sensitivity over commercially available assays are needed to evaluate the burden of C. trachomatis infection and the effectiveness of prevention efforts. We evaluated the performance of a C. trachomatis outer membrane complex protein B (OmcB) enzyme-linked immunosorbent assay (ELISA) in the detection of anti-C. trachomatis antibody responses in C. trachomatis-infected women. OmcB ELISA was less sensitive than our C. trachomatis elementary body (EB) ELISA, but it was highly specific. The magnitude of the antibody response was higher in African-Americans and those with prior C. trachomatis infection. Unlike EB ELISA, the IgG1 response to C. trachomatis OmcB was short-lived and was not maintained by repeat C. trachomatis infection.

T cell phenotypes in women with Chlamydia trachomatis infection and influence of treatment on phenotype distributions.

T cell phenotypes involved in the immune response to Chlamydia trachomatis (CT) have not been fully elucidated in humans. We evaluated differences in T cell phenotypes between CT-infected women and CT-seronegative controls and investigated changes in T cell phenotype distributions after CT treatment and their association with reinfection. We found a higher expression of T cell activation markers (CD38+HLA-DR+), T helper type 1 (Th1)- and Th2-associated effector phenotypes (CXCR3+CCR5+ and CCR4+, respectively), and T cell homing marker (CCR7) for both CD4+ and CD8+ T cells in CT-infected women. At follow-up after treatment of infected women, there were a lower proportion of CD4+ and CD8+ T cells expressing these markers. These findings suggest a dynamic interplay of CD4+ and CD8+ T cells in CT infection, and once the infection is treated, these cell markers return to basal expression levels. In women without reinfection, a significantly higher proportion of CD8+ T cells co-expressing CXCR3 with CCR5 or CCR4 at follow-up was detected compared to women with reinfection, suggesting they might play some role in adaptive immunity. Our study elucidated changes in T cell phenotypes during CT infection and after treatment, broadening our understanding of adaptive immune mechanisms in human CT infections.

Distinct peripheral vs mucosal T-cell phenotypes in chlamydia-infected women.

PROBLEM: Differences in circulating (peripheral) and mucosal T-cell phenotypes in chlamydia-infected women remain largely unknown. METHOD OF STUDY: Thirteen paired mononuclear cell specimens from blood and cervicovaginal lavages collected from chlamydia-infected women were stained and analyzed using ten-color cell surface flow cytometry for T-cell distribution, activation status, homing, and T helper (Th)-associated chemokine receptors (CKRs). RESULTS: A higher proportion of genital mucosal T-cells were activated (CD38+ HLA-DR+ ) and expressed CCR5 and Th1-associated CKR CXCR3+ CCR5+ compared to peripheral T-cells, but a lower proportion of mucosal T-cells expressed homing CKR CCR7, Th-2 associated CKR CCR4, and CXCR3+ CCR4+ for both T-cell subsets. CONCLUSION: T-cell phenotypes differed in the peripheral vs genital mucosa compartments in chlamydia-infected women. As chlamydia infects mucosal epithelial cells, the finding of a higher frequency of activated T-cells and Th-1 phenotypes in the mucosa likely reflects an adaptive immune response to infection.

Immunoglobulin-Based Investigation of Spontaneous Resolution of Chlamydia trachomatis Infection.

Chlamydia trachomatis elementary body enzyme-linked immunosorbent assay (ELISA) was used to investigate serum anti-CT immunoglobulin G1 (IgG1; long-lived response) and immunoglobulin G3 (IgG3; short-lived response indicating more recent infection) from treatment (enrollment) and 6-month follow-up visits in 77 women previously classified as having spontaneous resolution of chlamydia. Of these women, 71.4% were IgG1+IgG3+, consistent with more recent chlamydia resolution. 15.6% were IgG3- at both visits, suggesting absence of recent chlamydia. Using elementary body ELISA, we demonstrated approximately 1 in 6 women classified as having spontaneous resolution of chlamydia might have been exposed to C. trachomatis but not infected. Further, we classified their possible infection stage.

The Predominant CD4+ Th1 Cytokine Elicited to Chlamydia trachomatis Infection in Women Is Tumor Necrosis Factor Alpha and Not Interferon Gamma.

Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection and can cause significant reproductive morbidity in women. There is insufficient knowledge of C. trachomatis-specific immune responses in humans, which could be important in guiding vaccine development efforts. In contrast, murine models have clearly demonstrated the essential role of T helper type 1 (Th1) cells, especially interferon gamma (IFN-γ)-producing CD4+ T cells, in protective immunity to chlamydia. To determine the frequency and magnitude of Th1 cytokine responses elicited to C. trachomatis infection in humans, we stimulated peripheral blood mononuclear cells from 90 chlamydia-infected women with C. trachomatis elementary bodies, Pgp3, and major outer membrane protein and measured IFN-γ-, tumor necrosis factor alpha (TNF-α)-, and interleukin-2 (IL-2)-producing CD4+ and CD8+ T-cell responses using intracellular cytokine staining. The majority of chlamydia-infected women elicited CD4+ TNF-α responses, with frequency and magnitude varying significantly depending on the C. trachomatis antigen used. CD4+ IFN-γ and IL-2 responses occurred infrequently, as did production of any of the three cytokines by CD8+ T cells. About one-third of TNF-α-producing CD4+ T cells coproduced IFN-γ or IL-2. In summary, the predominant Th1 cytokine response elicited to C. trachomatis infection in women was a CD4+ TNF-α response, not CD4+ IFN-γ, and a subset of the CD4+ TNF-α-positive cells produced a second Th1 cytokine.

A Context-Dependent Role for IL-21 in Modulating the Differentiation, Distribution, and Abundance of Effector and Memory CD8 T Cell Subsets.

The activation of naive CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory, effector memory (TEM), and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express IL-21R, we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4ß7 on CD8 T cells primed in vitro and on circulating CD8 T cells in the mixed bone marrow chimeras. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection; nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in lymphopenic environments. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses.

UNLABELLED: T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8(+) T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8(+) T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8(+) T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8(+) T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8(+) T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. IMPORTANCE: Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8(+) T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.