Despite widespread flu vaccination, influenza remains a major cause of morbidity and mortality in the elderly. The early immune response, including CD4+ helper T cells, and CD8+ CTLs, plays a critical role in efficient control of virus and initiation of adaptive immunity. However, the correlates of protection in the elderly are not well understood. Here, we sought to investigate early cellular responses to re-exposure with seasonal flu vaccine antigens in a cohort of pre-vaccinated young and old rhesus macaques (human age equivalent of 30–45 yrs, and 55–85 yrs). In vitro T cell responses were examined following overnight stimulation of PBMCs with flu vaccine antigens by flow cytometry for Th1-type and Th17-type cytokine responses, and cytolytic functions. Notably, older macaques showed significantly impaired IL-17 production and CD107a upregulation in response to vaccine antigens, along with greater HLA-DR +/PD-1 +CD8 T cells. Further, they produced significantly higher TNF-α than young macaques. These results suggest that circulating flu vaccine-specific T cells in older macaques are deficient in mucosal barrier-protective and cytotolytic functions and possess a dominant proinflammatory function. These findings suggest that re-exposure to flu vaccine antigens in older individuals likely induces a proinflammatory response that is impaired in cytolytic capacity and the ability to repair lung mucosal damage during influenza infection, thereby resulting in more severe disease outcomes. Further studies on in vivo modulation of proinflammatory T cells while enhancing IL-17 functions during vaccination could support the development of more effective influenza vaccine strategies for older adults.

Previous studies have indicated that the loss of CD161-expressing CD4(+) Th17 cells is linked to the progression of chronic HIV. These cells are significantly depleted in peripheral blood and gut mucosa of HIV-infected individuals, contributing to inflammation and disruption of the gut barrier. However, the impact of HIV infection on CD161-expressing CD8(+) T cells remain unclear. Here, we examined the functions of peripheral blood and mucosal CD161(+)CD8(+) T cells in the macaque model of HIV infection. In contrast to the significant loss of CD161(+)CD4(+) T cells, CD161(+)CD8(+) T cell frequencies were maintained in blood and gut during chronic SIV infection. Furthermore, gut CD161(+)CD8(+) T cells displayed greater IL-17 production and maintained Th1-type and cytolytic functions, contrary to impaired IL-17 and granzyme-B production in CD161(+)CD4(+) T cells of SIV-infected macaques. These results suggest that augmented Th17-type effector functions of CD161(+)CD8(+) T cells during SIV infection is a likely mechanism to compensate for the sustained loss of gut mucosal Th17 cells. Targeting the cytokine and cytolytic effector functions of CD161(+)CD8(+) T cells in the preclinical setting of chronic SIV infection with antiretroviral therapy has implications in the restoration of gut barrier disruption in persons with HIV infection.

BackgroundAlthough BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity.MethodsHere, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL).ResultsPrior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection.ConclusionsOverall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.

Although most SARS-CoV-2 infections are mild, some patients develop systemic inflammation and progress to acute respiratory distress syndrome (ARDS). However, the cellular mechanisms underlying this spectrum of disease remain unclear. γδT cells are T lymphocyte subsets that have key roles in systemic and mucosal immune responses during infection and inflammation. Here we show that peripheral γδT cells are rapidly activated following aerosol or intra-tracheal/intra-nasal (IT/IN) SARS-CoV-2 infection in nonhuman primates. Our results demonstrate a rapid expansion of Vδ1 γδT cells at day1 that correlate significantly with lung viral loads during the first week of infection. Furthermore, increase in levels of CCR6 and Granzyme B expression in Vδ1 T cells during viral clearance imply a role in innate-like epithelial barrier-protective and cytotoxic functions. Importantly, the early activation and mobilization of circulating HLA-DR + CXCR3 + γδT cells along with significant correlations of Vδ1 T cells with IL-1Ra and SCF levels in bronchoalveolar lavage suggest a novel role for Vδ1 T cells in regulating lung inflammation during aerosol SARS-CoV-2 infection. A deeper understanding of the immunoregulatory functions of MHC-unrestricted Vδ1 T cells in lungs during early SARS-CoV-2 infection is particularly important in the wake of emerging new variants with increased transmissibility and immune evasion potential.

Infection with SARS-CoV-2, the novel coronavirus responsible for the current COVID-19 pandemic, results in a wide range of pathologies from asymptomatic/mild outcome to fulminant pneumonia and progression to acute respiratory distress syndrome (ARDS). The cellular mechanisms underlying this spectrum of disease remain unknown. We investigated the dynamic changes in frequency and activation status of peripheral innate lymphocyte subsets in two different species of nonhuman primates. Cohorts of Rhesus macaques (n=8) and African Green monkeys (n=8) were infected with SARS-CoV-2 either by small particle or mucosal (IN/IT) route. Flow cytometric analysis of PBMCs revealed a rapid and significant increase in circulating γδ T cell frequencies and activation at d1 postexposure across all animals inoculated by aerosol, which was correlative with peak viral loads in lung lavage and Vδ2 T cell frequencies. Further, elevated GranzymeB expression in both Vδ1 and Vδ2 subsets of γδ T cells was suggestive of increased cytotoxic function in early infection. In contrast, mucosally-infected animals showed little change in circulating γδ T cell frequencies from d1 through d7 post-infection, nor were there remarkable differences in early MAIT and NK cell frequencies. Collectively, these data suggest γδ T cells may be early responders to deep lung SARS-CoV-2 infection and the kinetics and quality of immune response are dependent upon route of exposure despite resulting in similar peak viral loads from infection. Further understanding of γδ T cell functions within the pulmonary compartment during SARS-CoV-2-driven acute inflammation may reveal tractable therapeutic approaches to further mitigate ARDS-like responses in COVID-19 patients.

Gut dysbiosis is a common feature associated with the chronic inflammation of HIV infection. Toward understanding the interplay of chronic treated HIV infection, dysbiosis, and systemic inflammation, we investigated longitudinal fecal microbiome changes and plasma inflammatory markers in the nonhuman primate model. Following simian immunodeficiency virus (SIV) infection in rhesus macaques, significant changes were observed in several members of the phylum Firmicutes along with an increase in Bacteroidetes. Viral suppression with antiretroviral therapy (ART) resulted in an early but partial recovery of compositional changes and butyrate producing genes in the gut microbiome. Over the course of chronic SIV infection and long-term ART, however, the specific loss of Faecalibacterium prausnitzii and Treponema succinifaciens significantly correlated with an increase in plasma inflammatory cytokines including IL-6, G-CSF, I-TAC, and MIG. Further, the loss of T. succinifaciens correlated with an increase in circulating biomarkers of gut epithelial barrier damage (IFABP) and microbial translocation (LBP and sCD14). As F. prausnitzii and T. succinifaciens are major short-chain fatty acid producing bacteria, their sustained loss during chronic SV-ART may contribute to gut inflammation and metabolic alterations despite effective long-term control of viremia. A better understanding of the correlations between the anti-inflammatory bacterial community and healthy gut barrier functions in the setting of long-term ART may have a major impact on the clinical management of inflammatory comorbidities in HIV-infected individuals.

HIV-associated inflammation has been implicated in the premature aging and increased risk of age-associated comorbidities in cART-treated individuals. However, the immune mechanisms underlying the chronic inflammatory state of cART-suppressed HIV infection remain unclear. Here, we investigated the role of γδT cells, a group of innate IL-17 producing T lymphocytes, in the development of systemic inflammation and leaky gut phenotype during cART-suppressed SIV infection of macaques. Plasma levels of inflammatory mediators, intestinal epithelial barrier disruption (IEBD) and microbial translocation (MT) biomarkers, and Th1/Th17-type cytokine functions were longitudinally assessed in blood and gut mucosa of SIV-infected, cART-suppressed macaques. Among the various gut mucosal IL-17/IL-22-producing T lymphocyte subsets including Th17, γδT, CD161+ CD8+ T, and MAIT cells, a specific decline in the Vδ2 subset of γδT cells and impaired IL-17/IL-22 production in γδT cells significantly correlated with the subsequent increase in plasma IEBD/MT markers (IFABP, LPS-binding protein, and sCD14) and pro-inflammatory cytokines (IL-6, IL-1β, IP10, etc.) despite continued viral suppression during long-term cART. Further, the plasma inflammatory cytokine signature during long-term cART was distinct from acute SIV infection and resembled the inflammatory cytokine profile of uninfected aging (inflammaging) macaques. Overall, our data suggest that during cART-suppressed chronic SIV infection, dysregulation of IL-17/IL-22 cytokine effector functions and decline of Vδ2 γδT cell subsets may contribute to gut epithelial barrier disruption and development of a distinct plasma inflammatory signature characteristic of inflammaging. Our results advance the current understanding of the impact of chronic HIV/SIV infection on γδT cell functions and demonstrate that in the setting of long-term cART, the loss of epithelial barrier-protective functions of Vδ2 T cells and ensuing IEBD/MT occurs before the hallmark expansion of Vδ1 subsets and skewed Vδ2/Vδ1 ratio. Thus, our work suggests that novel therapeutic approaches toward restoring IL-17/IL-22 cytokine functions of intestinal Vδ2 T cells may be beneficial in preserving gut epithelial barrier function and reducing chronic inflammation in HIV-infected individuals.

Unfortunately, the original version of this article was published with error in the materials and methods section.

The development of chronic inflammation, called inflammaging, contributes to the pathogenesis of age-related diseases. Although it is known that both B and T lymphocyte compartments of the adaptive immune system deteriorate with advancing age, the impact of aging on immune functions of Th17-type CD161-expressing innate immune cells and their role in inflammaging remain incompletely understood. Here, utilizing the nonhuman primate model of rhesus macaques, we report that a dysregulated Th17-type effector function of CD161+ immune cells is associated with leaky gut and inflammatory phenotype of aging. Higher plasma levels of inflammatory cytokines IL-6, TNF-α, IL-1β, GM-CSF, IL-12, and Eotaxin correlated with elevated markers of gut permeability including LPS-binding protein (LBP), intestinal fatty acid binding protein (I-FABP), and sCD14 in aging macaques. Further, older macaques displayed significantly lower frequencies of circulating Th17-type immune cells comprised of CD161+ T cell subsets, NK cells, and innate lymphoid cells. Corresponding with the increased markers of gut permeability, production of the type-17 cytokines IL-17 and IL-22 was impaired in CD161+ T cell subsets and NK cells, along with a skewing towards IFN-γ cytokine production. These findings suggest that reduced frequencies of CD161+ immune cells along with a specific loss in Th17-type effector functions contribute to impaired gut barrier integrity and systemic inflammation in aging macaques. Modulating type-17 immune cell functions via cytokine therapy or dietary interventions towards reducing chronic inflammation in inflammaging individuals may have the potential to prevent or delay age-related chronic diseases and improve immune responses in the elderly population.

Incidence of newly-acquired HIV infections in persons over age 50 is increasing, from 13% in 2006 to 21% in 2013. Advanced age corresponds with lower 12-month survival and ART is contraindicated with the simultaneous use of many chronic disease medications. To address the need for age-specific interventions, we are studying aged rhesus macaques (RM) infected with SIV. In a pilot study, four young (6.27–7.37 years) and six older (17.71–23.82 years) RM were injected i.v. with SIVmac239. The older RM exhibited lower survival times compared to younger macaques (112.5 ± 76.8 days v. 353 ± 74.9 days, respectively). In young adult RM with SIV, we reported that increasing monocyte turnover (via BrdU uptake) as well as declining CD4+ cell counts, predicted disease progression. Among the aged RM, we observed that 3 animals developed increased or high monocyte turnover (HMT) after the first month of infection (35.2% ± 2.36) while the other 3 maintained steady state or low monocyte turnover (LMT) (5.74% ± 4.92) similar to that in the young animals (9.97% ± 2.34). Older RM with LMT survived longer (151 ± 85.6 days) than those with HMT (74 ± 54.5 days). The young adult and aged RM with LMT exhibited a dramatic decline of CD4+ T cells during peak viral load (−46% and −41%) 2 wks post infection compared to only a 22% decrease in aged RM with HMT. Also, the numbers of naïve T cells (CD4 and CD8) were lower in the HMT aged animals than in the LMT and young adults, at baseline and 2 wks post infection. These results suggest that there may be an additional or alternative preferential host cell target of SIV early in infection of older susceptible RM that we speculate to be macrophages. Work is continuing to examine the role of macrophages in pathogenesis of HIV infection in the elderly.

Infection with HIV-1 results in CD4+ T cell depletion and the subsequent loss of immune function results in AIDS. Although HAART lowers plasma viremia, it requires life-long drug therapy. However, elite controllers are able to exert control over viral replication in the absence of cART therapy by maintaining polyfunctional HIV-specific T cells with high avidity TCR binding that target CTL epitopes where escape mutants have reduced fitness. To mimic increase immunosurveillance of elite controllers and prevent viral replication in a non-controlled setting, we will transduce T cells with CD4-CAR vectors to bolster and redirect the “designer T cells” (dTc) towards an HIV-specific target. Chimeric antigen receptors (CARs) are fusion proteins of an extracellular antigenbinding domain linked to intracellular T cell signaling domains. In CD4-CARs, the CD4 extracellular domain binds directly to diverse HIV envelopes and redirects the CTL activity. Env/CD4 binding is independent of both the MHC restriction and Nef down regulation. As in elite controllers, CD4-CARs targets an essential viral function where mutations would lower viral fitness and make viral escape difficult to achieve. The first-generation CD4-CAR vector, with only the T-cell receptor zeta (TCRζ) intracellular domain, killed Env+ cells in vitro but failed to control viremia in clinical trials. Recent studies with anti-tumor CARs show that additional co-stimulatory signals (CD28 and 4-1BB) improved CTL function in vitro, in murine models, and showed objective tumor regression in clinical trials. We hypothesize that genetically modified T cells designed to increase CTL killing of HIV+ cells, to persist in vivo, and to prevent viral infection will provide durable viral suppression in vivo and eliminate the need for life-long drug therapy as modeled by elite controllers. To determine what signaling domains would be necessary to mimic this immune protection, we developed a series of CD4-CAR vectors with various intracellular signaling domains. To protect dTc from infection, we co-transduced cells with the membrane-associated C46 (maC46), a fusion inhibitor tethered to dTc, that binds HIV and blocks viral replication thereby providing a strong selective advantage to transduced cells. Therefore, we stimulated PBMCs with αCD3αCD28 and co-transduced T cells with the series of CD4-CAR and maC46 vectors. To evaluate the functional activity of the dTc, we measured 1) HIV Env-specific cytotoxicity, 2) cytokine production using IFN-γ ELIspot, and 3) CAR-specific proliferation of dTc. CD4-CAR T cells demonstrated Env+ specific killing, increased frequency of IFN-γ spot-forming cells, and CAR-specific proliferation. Our long-term goal, to adoptively transfer dTc with increased CTL function (cytokine production, proliferative capacity, viral suppression). The control of viremia in the absence of HAART would revolutionize HIV treatments for patients with AIDS.

Background To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. Methods We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors. CD4-CAR-transduced T cells were added to Env(+) 293T cells at E: T of 1:1. Killing of target cells was measured as reduced impedance. Results We observed gene expression in 60-70% of rhesus CD3(+) CD8(+) T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR-transduced populations specifically killed Env(+) cells. Conclusions In these studies, we showed that designer T cells were redirected to kill Env(+) cells. Control of viremia without HAART would revolutionize treatment for HIV patients.