Search

Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics., Graphical abstract, Highlights • Severe COVID-19 associates with higher antibody production and neutralization titers • Neutralization potency of anti-RBD antibodies predicts disease severity and survival • Immunomodulatory COVID-19-directed therapies modulate antibody responses • COVID-19 sera neutralize D614 and G614 variants, but not pre-emergent WIV1-CoV, Garcia-Beltran et al. show that the development of more potent neutralizing antibodies during SARS-CoV-2 infection predicts COVID-19 survival. Protective antibody responses exhibit potent neutralization against the currently circulating SARS-CoV-2 D614G spike variant but lack significant activity against pre-emergent WIV1-CoV spike, suggesting that convalescent patients are likely to remain susceptible to future pandemics.

CD4+ T lymphocytes are the principal target of human immunodeficiency virus (HIV), but infected macrophages also contribute to viral pathogenesis. The killing of infected cells by CD8+ cytotoxic T lymphocytes (CTLs) leads to control of viral replication. Here we found that the killing of macrophages by CTLs was impaired relative to the killing of CD4+ T cells by CTLs, and this resulted in inefficient suppression of HIV. The killing of macrophages depended on caspase-3 and granzyme B, whereas the rapid killing of CD4+ T cells was caspase independent and did not require granzyme B. Moreover, the impaired killing of macrophages was associated with prolonged effector cell-target cell contact time and higher expression of interferon-γ by CTLs, which induced macrophage production of pro-inflammatory chemokines that recruited monocytes and T cells. Similar results were obtained when macrophages presented other viral antigens, suggestive of a general mechanism for macrophage persistence as antigen-presenting cells that enhance inflammation and adaptive immunity. Inefficient killing of macrophages by CTLs might contribute to chronic inflammation, a hallmark of chronic disease caused by HIV.

Summary Natural killer (NK) cells are innate cytolytic effectors that target HIV-infected CD4+ T cells. In conjunction with antibodies recognizing the HIV envelope, NK cells also eliminate HIV-infected targets through antibody-dependent cellular cytotoxicity (ADCC). However, how these NK cell functions impact infected macrophages is less understood. We show that HIV-infected macrophages resist NK cell-mediated killing. Compared with HIV-infected CD4+ T cells, initial innate NK cell interactions with HIV-infected macrophages skew the response toward cytokine production, rather than release of cytolytic contents, causing inefficient elimination of infected macrophages. Studies with chimeric antigen receptor (CAR) T cells demonstrate that the viral envelope is equally accessible on CD4+ T cells and macrophages. Nonetheless, ADCC against macrophages is muted compared with ADCC against CD4+ T cells. Thus, HIV-infected macrophages employ mechanisms to evade immediate cytolytic NK cell function while preserving inflammatory cytokine responses. These findings emphasize the importance of eliminating infected macrophages for HIV cure efforts.

Chronic HIV infection results in a loss of HIV-specific CD8 + T cell effector function, termed “exhaustion,” which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8 + T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8 + T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions. IMPORTANCE Despite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.

CD8+ CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain–containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8+ T cells, and antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8+ T cells. Tim-3 was found within CD8+ T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3hiCD8+ T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3–mediated immune dysfunction.

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)–expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3–expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3–expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1–infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)–specific CD8+ T cells obtained from HIV-1–infected human subjects responded to HIV-1–infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)–specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)–specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)–targeted HIV-1 vaccines and immunotherapeutics.

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8+ and Th1 CD4+ T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3+ cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4+ T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3+ primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.

Co-infection of HCV with HIV has been associated with more rapid progression of HCV-related disease. HCV-specific T-cell immune responses, which are essential for disease control, are attenuated in co-infection with HIV. T-cell exhaustion has recently been implicated in the deficient control of chronic viral infections. In the current study, we investigated the role of programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) expression in T-cell exhaustion during HCV/HIV co-infection. We show that in HCV/HIV co-infection, both total and HCV-specific T cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared with HCV mono-infection. Co-expression of these two markers on HCV-specific CD8(+) T cells positively correlated with a clinical parameter of liver disease progression. HCV-specific CD8(+) T cells showed greater frequencies of Tim-3/PD-1 co-expression than HIV-specific CD8(+) T cells, which may indicate a greater degree of exhaustion in the former. Blocking Tim-3 or PD-1 pathways restored both HIV- and HCV-specific CD8(+) T-cell expansion in the blood of co-infected individuals. These data demonstrate that co-expression of Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, especially in the setting of HIV co-infection.

Although CD4+ T cells represent the major reservoir of persistent HIV and SIV infection, accumulating evidence suggests that macrophages also contribute. However, investigations of the role of macrophages are often underrepresented at HIV pathogenesis and cure meetings. This was the impetus for a scientific workshop dedicated to this area of study, held in Cambridge, MA in January 2017. The workshop brought together experts in the fields of HIV/SIV immunology and virology, macrophage biology and immunology, and animal models of HIV/SIV infection to discuss the role of macrophages as a physiologically relevant viral reservoir, and the implications of macrophage infection for HIV pathogenesis and strategies for cure. While still controversial, there is an emerging theory that infected macrophages likely persist in the setting of combination antiretroviral therapy. These macrophages could then drive persistent inflammation and contribute to the viral reservoir, which indicates the importance of addressing macrophages as well as CD4+ T cells with future therapeutic strategies.

The T cell Ig- and mucin domain–containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8+ T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8+ T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3+CD8+ T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3+PD-1+CD8+ T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8+ T cells in all tissues examined. Tim-3+CD8+ T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3−CD8+ T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8+ T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR–driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8+ T cell responses.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers1–5. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition6–10. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Background Regulatory B cells (Breg), the B cells producing interleukin 10 (IL-10), have been identified in mice and humans. Mouse Breg can suppress innate and T cell responses and are implicated in pathogenesis of some autoimmune diseases and immune evasion of some pathogens. However, the role of Breg in humans is less clear. Methods PBMC and gut biopsy samples were obtained from healthy donors and HIV infected individuals. Flow cytometry and Luminex were used to quantify cytokine production. Flow cytometry were used to analyze Breg’s phenotype. Results Breg were elevated in both peripheral blood and gut tissue of untreated HIV-1 infected individuals and the elevation correlated with viral load in early HIV-1 infection. Breg from HIV-1 infected individuals were CD19 + TIM-1 + .Antiretroviral therapy could reduce elevated Breg frequency. Treatment of B cells from healthy donors with microbial translocation products could differentiate them toward a Breg phenotype. Ex vivo Bregs from HIV-1 infected individuals suppressed cytokine production /degranulation of HIV-1 specific T cells that was in part IL-10 dependent. Conclusion

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8(+) T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3(+) CD8(+) T cells to make perforin and 2) the direct ability of Tim-3(+) CD8(+) T cells to kill autologous HIV infected CD4(+) target cells. Surprisingly, Tim-3(+) CD8(+) T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8(+) T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4(+) T cells and d) their ability to suppress HIV infection of CD4(+) T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8(+) T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8(+) T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.

A rare subset of IL-10-producing B cells, named regulatory B cells (Bregs), suppresses adaptive immune responses and inflammation in mice. In this study, we examined the role of IL-10-producing B cells in HIV-1 infection. Compared to uninfected controls, IL-10-producing B cell frequencies were elevated in both blood and sigmoid colon during the early and chronic phase of untreated HIV-1 infection. Ex vivo IL-10-producing B cell frequency in early HIV-1 infection directly correlated with viral load. IL-10-producing B cells from HIV-1 infected individuals were enriched in CD19(+)TIM-1(+) B cells and were enriched for specificity to trimeric HIV-1 envelope protein. Anti-retroviral therapy was associated with reduced IL-10-producing B cell frequencies. Treatment of B cells from healthy donors with microbial metabolites and Toll-like receptor (TLR) agonists could induce an IL-10 producing phenotype, suggesting that the elevated bacterial translocation characteristic of HIV-1 infection may promote IL-10-producing B cell development. Similar to regulatory B cells found in mice, IL-10-producing B cells from HIV-1-infected individuals suppressed HIV-1-specific T cell responses in vitro, and this suppression is IL-10-dependent. Also, ex vivo IL-10-producing B cell frequency inversely correlated with contemporaneous ex vivo HIV-1-specific T cell responses. Our findings show that IL-10-producing B cells are induced early in HIV-1 infection, can be HIV-1 specific, and are able to inhibit effective anti-HIV-1 T cell responses. HIV-1 may dysregulate B cells toward Bregs as an immune evasion strategy.