Chang-You Wu

Distinct lineages of T(H)1 cells have differential capacities for memory cell generation in vivo.

We studied here the long-term maintenance of distinct populations of T helper type 1 (TH1)-lineage cells in vivo and found that effector TH1 cells, defined by their secretion of interferon-γ (IFN-γ), are short-lived and do not efficiently develop into long-term memory TH1 cells. In contrast, a population of activated TH1-lineage cells that did not secrete IFN-γ after primary antigenic stimulation persisted for several months in vivo and developed the capacity to secrete IFN-γ upon subsequent stimulation. These data suggest that a linear differentiation pathway, as defined by the transition from IFN-γ–producing to resting memory cells, is relatively limited in vivo and support a revised model for TH1 memory differentiation.

Immunization with HIV-1 Gag Protein Conjugated to a TLR7/8 Agonist Results in the Generation of HIV-1 Gag-Specific Th1 and CD8+ T Cell Responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-α, and increased expression of CD80, CD86, and CD40 on CD11c+ dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8+ T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8+ T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8+ CD62low cells, ∼50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.

Vaccination with Heat-killed Leishmania Antigen or Recombinant Leishmanial Protein and CpG Oligodeoxynucleotides Induces Long-Term Memory CD4+and CD8+T Cell Responses and Protection Against Leishmania major Infection

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major–specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

Stat4 Is Expressed in Activated Peripheral Blood Monocytes, Dendritic Cells, and Macrophages at Sites of Th1-Mediated Inflammation

Stat4 is a key transcription factor involved in promoting cell-mediated immunity, whose expression in mature cells has been reported to be restricted to T and NK cells. We demonstrate here, however, that Stat4 expression is not restricted to lymphoid cells. In their basal state, monocytes do not express Stat4. Upon activation, however, IFN-γ- and LPS-treated monocytes and dendritic cells express high levels of Stat4. Monocyte-expressed Stat4 in humans is phosphorylated in response to IFN-α, but not IL-12. In contrast, the Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4 expression in activated monocytes, while having little effect on Stat6 expression. Moreover, macrophages in synovial tissue obtained from patients with rheumatoid arthritis express Stat4 in vivo, suggesting a potential role in a prototypical Th1-mediated human disease. IFN-α-induced Stat4 activation in human monocytes represents a previously unrecognized signaling pathway at sites of Th1 inflammation.

Requirements for the Maintenance of Th1 Immunity In Vivo Following DNA Vaccination: A Potential Immunoregulatory Role for CD8+ T Cells

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-γ-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-γ-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.

Th1 memory: implications for vaccine development.

Summary:  T‐helper 1 (Th1) cells play a critical role, via interferon‐γ (IFN‐γ) production, in mediating intracellular killing against a variety of infectious pathogens. Thus, understanding the regulation of Th1 responses could provide better insight into vaccine design for infections requiring Th1 immunity. The cellular and molecular mechanisms that control the induction of Th1 effector cells have been well characterized. More recently, there has been substantial progress in furthering our understanding of the factors that regulate the development of Th1 memory cells. It is clear that Th1 responses are functionally heterogeneous, as defined by their ability to produce IFN‐γ. Furthermore, this heterogeneity has profound implications for the capacity of distinct lineages of Th1 cells to develop into memory cells. This review emphasizes the mechanisms controlling the differentiation of naïve CD4+ T cells into effector and then memory cells in a progressive manner. It highlights the importance of IFN‐γ as a positive regulator for inducing Th1 responses but a negative regulator for sustaining Th1 effector cells. In conclusion, we discuss how this current understanding of Th1 differentiation will inform vaccine design and better define immune correlates of protection.

Prime-Boost Vaccination with HIV-1 Gag Protein and Cytosine Phosphate Guanosine Oligodeoxynucleotide, Followed by Adenovirus, Induces Sustained and Robust Humoral and Cellular Immune Responses

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.

Dendritic Cells Are Responsible for the Capacity of CpG Oligodeoxynucleotides to Act as an Adjuvant for Protective Vaccine Immunity Against Leishmania major in Mice

Vaccination with leishmanial Ag and CpG oligodeoxynucleotides (ODN) confers sustained cellular immunity and protection to infectious challenge up to 6 mo after immunization. To define the cellular mechanism by which CpG ODN mediate their adjuvant effects in vivo, the functional capacity of distinct dendritic cell (DC) subsets was assessed in the lymph nodes (LNs) of BALB/c mice, 36 h after immunization with the leishmanial antigen (LACK) and CpG ODN. After this immunization, there was a striking decrease in the frequency of the CD11c+B220+ plasmacytoid DCs with a proportionate increase in CD11c+CD8−B220− cells. CD11c+CD8+B220− cells were the most potent producers of interleukin (IL)-12 p70 and interferon (IFN)-γ, while plasmacytoid DCs were the only subset capable of secreting IFN-α. In terms of antigen presenting capacity, plasmacytoid DCs were far less efficient compared with the other DC subsets. To certify that DCs were responsible for effective vaccination, we isolated CD11c+ and CD11c− cells 36 h after immunization and used such cells to elicit protective immunity after adoptive transfer in naive, Leishmania major susceptible BALB/c mice. CD11c+ cells but not 10-fold higher numbers of CD11c− cells from such immunized mice mediated protection. Therefore, the combination of LACK antigen and CpG ODN adjuvant leads to the presence of CD11c+ DCs in the draining LN that are capable of vaccinating naive mice in the absence of further antigen or adjuvant.

Cytokine regulation of IL‐12 receptor β2 expression: differential effects on human T and NK cells

The biological activities of IL‐12 are mediated through a specific, high‐affinity receptor composed of IL‐12 receptor(R)β1 and IL‐12Rβ2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL‐12Rβ2 on CD4+ T cells in mouse and humans appears to be differentially regulated by IFN‐γ  and IFN‐α , respectively. Using an antibody specific for the human IL‐12Rβ2 subunit, the effect of IFN‐γ , IFN‐α , IL‐12 and IL‐2 on the regulation of IL‐12R expression and IL‐12 responsiveness of human T and NK cells was assessed. The presence of IFN‐α  or IFN‐γ  in cultures enhanced IL‐12Rβ2 expression of CD4+ and CD8+ T cells. The enhancing effect of IFN‐α  and IFN‐γ  was independent of endogenous IL‐12. Furthermore, the clearest effects of IFN‐α  and IFN‐γ  on IL‐12Rβ2 expression on T cells were seen by abrograting the inhibition induced by the presence of IL‐4 in cultures. In contrast to T cells, IFN‐α  and IFN‐γ  had little effect on regulating IL‐12Rβ2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL‐12Rβ2 expression by IFN‐α  and IFN‐γ  on human T and NK cells.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5′-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).