Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8(+) T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.     

The adjuvant effects of Mycobacterium tuberculosis heat shock protein 70 result from the rapid and prolonged activation of antigen-specific CD8+ T cells in vivo

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8(+) T cells in vivo, we tracked Ag-specific CD8(+) T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8(+) T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8(+) T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8(+) T cells.     

Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses

In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8(+) T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8(+) T cells, IFN-gamma, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.     

Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory

Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.     

CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4+ and CD8+ T cells independently of IL-12

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4(+) and CD8(+) T cells. Normally, sensitization for CHS responses induces hapten-specific CD4(+) T cells producing type 2 cytokines and CD8(+) T cells producing IFN-gamma. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4(+) and CD8(+) T cells producing IFN-gamma in the skin draining lymph nodes. Identical results were observed using IL-12(-/-) mice, indicating that CD40 ligation promotes CHS responses and development of IFN-gamma-producing CD4(+) and CD8(+) T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-gamma-producing cells.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells

CD4(+)CD25(+) regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4(+)CD25(-) T cells and are potent suppressors of CD4(+)CD25(-) T cell activation in vitro. We demonstrate that CD4(+)CD25(+) T cells also suppress both proliferation and IFN-gamma production by CD8(+) T cells induced either by polyclonal or Ag-specific stimuli. CD4(+)CD25(+) T cells inhibit the activation of CD8(+) responders by inhibiting both IL-2 production and up-regulation of IL-2Ralpha-chain (CD25) expression. Suppression is mediated via a T-T interaction as activated CD4(+)CD25(+) T cells suppress the responses of TCR-transgenic CD8(+) T cells stimulated with soluble peptide-MHC class I tetramers in the complete absence of APC. These results broaden the immunoregulatory role played by CD4(+)CD25(+) T cells in the prevention of autoimmune diseases, but also raise the possibility that they may hinder the induction of effector CD8(+) T cells to tumor or foreign Ags.     

Partial activation of neonatal CD11c+ dendritic cells and induction of adult-like CD8+ cytotoxic T cell responses by synthetic microspheres

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8(+) cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8(+) T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-gamma-secreting CD8(+) splenocytes. Neonatal CD8(+) T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c(+) dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c(+) DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4(+) T cell help. The effective induction of neonatal CD8(+) T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c(+) DC may mature sufficiently to stimulate naive CD8(+) neonatal T cells, even in the absence of strong maturation signals.     

Antigen-specific transfer of functional programmed death ligand 1 from human APCs onto CD8+ T cells via trogocytosis

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8(+) T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8(+) T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell-cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8(+) T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1-expressing CD8(+) T cells. In summary, our data demonstrate that human CD8(+) T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1-expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8(+) T cells could have a regulatory role in immune responses.     

Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.     

Fas ligand costimulates the in vivo proliferation of CD8+ T cells

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8(+) T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL(+) CD8(+) T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL(-)counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8(+) T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8(+) T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.     

Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.     

Granzyme B expression by CD8+ T cells is required for the development of experimental cerebral malaria

Parasite burden predicts disease severity in malaria and risk of death in cerebral malaria patients. In murine experimental cerebral malaria (ECM), parasite burden and CD8(+) T cells promote disease by mechanisms that are not fully understood. We found that the majority of brain-recruited CD8(+) T cells expressed granzyme B (GzmB). Furthermore, gzmB(-/-) mice harbored reduced parasite numbers in the brain as a consequence of enhanced antiparasitic CD4(+) T cell responses and were protected from ECM. We showed in these ECM-resistant mice that adoptively transferred, Ag-specific CD8(+) T cells migrated to the brain, but did not induce ECM until a critical Ag threshold was reached. ECM induction was exquisitely dependent on Ag-specific CD8(+) T cell-derived perforin and GzmB, but not IFN-γ. In wild-type mice, full activation of brain-recruited CD8(+) T cells also depended on a critical number of parasites in this tissue, which in turn, was sustained by these tissue-recruited cells. Thus, an interdependent relationship between parasite burden and CD8(+) T cells dictates the onset of perforin/GzmB-mediated ECM.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

Stimulation by soluble CD70 promotes strong primary and secondary CD8+ cytotoxic T cell responses in vivo

Identification of the signals required for optimal differentiation of naive CD8(+) T cells into effector and memory cells is critical for the design of effective vaccines. In this study we demonstrate that CD27 stimulation by soluble CD70 considerably enhances the magnitude and quality of the CD8(+) T cell response. Stimulation with soluble CD70 in the presence of Ag significantly enhanced the proliferation of CD8(+) T cells and their ability to produce IL-2 and IFN-gamma in vitro. Administration of Ag and soluble CD70 resulted in a massive (>300-fold) expansion of Ag-specific CD8(+) T cells in vivo, which was due to the enhanced proliferation and survival of activated T cells. In mice that received Ag and soluble CD70, CD8(+) T cells developed into effectors with direct ex vivo cytotoxicity. Furthermore, unlike peptide immunization, which resulted in a diminished response after rechallenge, CD27 stimulation during the primary challenge evoked a strong secondary response upon rechallenge with the antigenic peptide. Thus, in addition to increasing the frequency of primed Ag-specific T cells, CD27 signaling during the primary response instills a program of differentiation that allows CD8(+) T cells to overcome a state of unresponsiveness. Taken together these results demonstrate that soluble CD70 has potent in vivo adjuvant effects for CD8(+) T cell responses.     

The size and phenotype of virus-specific T cell populations is determined by repetitive antigenic stimulation and environmental cytokines

Based on the expression of the TNFR SFP CD27, two Ag-primed CD8(+) T cell subsets can be discerned in the circulation of healthy individuals: CD27(+) T cells that produce a variety of cytokines but do not display immediate cytolytic activity; and cytotoxic CD27(-) T cells, which secrete only IFN-gamma and TNF-alpha. The mechanism that controls the generation of these different phenotypes is unknown. We show that CMV reactivation not only increases the number of virus-specific T cells but also induces their transition from a CD27(+) to a CD27(-) phenotype. In support of a relation between pool size and phenotype in a cohort of latently infected individuals, the number of Ag-specific CD27(-) CD8(+) T cells was found to be linearly related to the total number of CMV-specific CD8(+) T cells. In vitro studies revealed that the acquisition of the CD27(-) phenotype on CMV-specific T cells depended on the interaction of CD27 with its cellular ligand, CD70. Expression of CD70 was proportional to the amount of antigenic stimulation and blocked by the CD4(+) T cell-derived cytokine IL-21. Thus, induction of CD70, which may vary in distinct viral infections, appears to be a key factor in determining the size and phenotype of the CMV-specific T cell population in latently infected individuals.     

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.     

CD27/CFSE-based ex vivo selection of highly suppressive alloantigen-specific human regulatory T cells

Naturally occurring CD4(+)CD25(+) regulatory T cells (Treg) are crucial in immunoregulation and have great therapeutic potential for immunotherapy in the prevention of transplant rejection, allergy, and autoimmune diseases. The efficacy of Treg-based immunotherapy critically depends on the Ag specificity of the regulatory T cells. Moreover, the use of Ag-specific Treg as opposed to polyclonal expanded Treg will reduce the total number of Treg necessary for therapy. Hence, it is crucial to develop ex vivo selection procedures that allow selection and expansion of highly potent, Ag-specific Treg. In this study we describe an ex vivo CFSE cell sorter-based isolation method for human alloantigen-specific Treg. To this end, freshly isolated CD4(+)CD25(+) Treg were labeled with CFSE and stimulated with (target) alloantigen and IL-2 plus IL-15 in short-term cultures. The alloantigen-reactive dividing Treg were characterized by low CFSE content and could be subdivided by virtue of CD27 expression. CD27/CFSE cell sorter-based selection of CD27(+) and CD27(-) cells resulted in two highly suppressive Ag-specific Treg subsets. Each subset suppressed naive and Ag-experienced memory T cells, and importantly, CD27(+) Treg also suppressed ongoing T cell responses. Summarizing, the described procedure enables induction, expansion, and especially selection of highly suppressive, Ag-specific Treg subsets, which are crucial in Ag-specific, Treg-based immunotherapy.     

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.