Blockade of CD40-CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren's syndrome.

OBJECTIVE: To examine the role of CD40-CD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (NOD/ShiLtJ) model of Sjögren's syndrome (SS). METHODS: We assessed leucocyte infiltration in salivary glands (SGs) from NOD/ShiLtJ mice by immunohistochemistry and examined transcriptomics data of SG tissue from these animals for evidence of a CD40 pathway gene signature. Additionally, we dosed MR1 (anti-CD154 antibody) in NOD mice after the onset of SS-like disease and examined the effects of MR1 treatment on sialadenitis, autoantibody production, SG leucocyte infiltration, gene expression downstream of CD40 and acquaporin 5 (AQP5) expression. RESULTS: We could detect evidence of CD40 expression and pathway activation in SG tissue from NOD mice. Additionally, therapeutic treatment with MR1 suppressed CD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in SGs but had minimal effects on AQP5 expression in NOD/ShiLtJ SGs. CONCLUSION: CD40-CD154 interactions play an important role in key pathological processes in a mouse model of SS, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.

Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo.

The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

Half-life extended biotherapeutics.

INTRODUCTION: Many of the biotherapeutics approved or under development suffer from a short half-life necessitating frequent applications in order to maintain a therapeutic concentration over an extended period of time. The implementation of half-life extension strategies allows the generation of long-lasting therapeutics with improved pharmacokinetic and pharmacodynamic properties. AREAS COVERED: This review gives an overview of the different half-life extension strategies developed over the past years and their application to generate next-generation biotherapeutics. It focuses on srategies already used in approved drugs and drugs that are in clinical development. These strategies include those aimed at increasing the hydrodynamic radius of the biotherapeutic and strategies which further implement recycling by the neonatal Fc receptor (FcRn). EXPERT OPINION: Half-life extension strategies have become an integral part of development for many biotherapeutics. A diverse set of these strategies is available for the fine-tuning of half-life and adaption to the intended treatment modality and disease. Currently, half-life extension is dominated by strategies utilizing albumin binding or fusion, fusion to an immunoglobulin Fc region and PEGylation. However, a variety of alternative strategies, such as fusion of flexible polypeptide chains as PEG mimetic substitute, have reached advanced stages and offer further alternatives for half-life extension.

Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells.

Murine CMV (MCMV) infection induces effector CD8(+) T cells that continue to increase in frequency after acute infection ("inflation") and are stably maintained at a high frequency, with up to 20% of the CD8(+) T-cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8(+) T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8(+) T cells. Treatment of MCMV-infected mice with MHC-Class I-saporin tetramers led to partial (80-90%) depletion of epitope-specific CD8(+) T cells-rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8(+) T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the "inflated" CD8(+) T cell responses during persistent infection.

Immunization with recombinant HLA classes I and II, HIV-1 gp140, and SIV p27 elicits protection against heterologous SHIV infection in rhesus macaques.

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID(50)) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.

Soluble donor-like MHC class I proteins induce CD4(+)CD25(+)CD8(-)FoxP3(+) cells with potential to ameliorate graft chronic injury.

Conventional immunosuppressive therapies failed to prevent allograft chronic rejection. New approaches to modulate recipient immune response are needed. Donor-like MHC class I soluble proteins demonstrated therapeutic potential to suppress chronic rejection. The present study was designed to clarify the ability of MHC class I soluble proteins to induce T regulatory cells with true regulatory potential in a fully allogeneic rat cardiac transplant model. Donor-like MHC class I proteins upregulate small population of splenic CD8(-) negative CD4(+)CD25(+)FoxP3(+) positive cells. CD4(+) splenocytes after MHC therapy suppress lymphocyte proliferation against donor antigens in vitro. ACI recipients of WF hearts treated with CD4(+) cells, induced with donor-like MHC class I proteins (CD4-MHC), demonstrated stable survival of the transplanted organ (MST >120 days; n=17). Histology revealed that grafts of recipients treated with CD4-MHC had 23.6% vessels affected 100 days postgrafting. On the contrary, hearts obtained from long-term surviving hosts treated with CD4(+) cells induced with high-dose CsA (CD4-CsA) had 50-70% of affected vessels. CD4-MHC class I treated transplants were mostly CD3(-) negative, had low level of mast and FoxP3(+) cell infiltration compared to CD4-CsA treated hearts. Intragraft CD4(+) cells were close to mast cells in morphology. The same graft tissues had similar number of CD4(+) positive cells and mast cells suggesting existence of CD4(+) positive mast cells. On the other hand, a negligible number of FoxP3(+) positive cells in the grafts after CD4-MHC treatment supports the idea of CD4(+) positive FoxP3(+) negative mast cells population. We demonstrate that donor-like MHC class I protein therapy induces population of CD4(+)CD25(+)CD8(-)FoxP3(+) cells with potential to ameliorate development of transplant vascular disease and evoke CD4(+) positive FoxP3 negative mast cells in the secondary hosts.

Downregulation of RhoA and changes in T cell cytoskeleton correlate with the abrogation of allograft rejection.

Proper actin cytoskeleton architecture and dynamics are indispensable for events in the immunological response such as T cell migration, redistribution of T cell receptors, and interaction with antigen presenting cells. Thus, T cell activation, downstream signaling events and effector functions are all actin-dependent. Actin cytoskeleton architecture and dynamics are regulated by proteins belonging to the superfamily of small GTP-binding proteins, such as RhoA GTPase. We previously showed that the administration of an MHC class I allochimeric molecule [alpha1h1/u]-RT1.Aa, which contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) sequences, to the ACI recipient of heterotopic WF heart resulted in the restriction of the TCR repertoire, inhibition of T cell infiltration into the heterotopic cardiac allografts, abrogation of acute and chronic rejection, and induction of indefinite survival of the allograft. Here we show that the allochimeric molecule treatment caused downregulation of RhoA GTPase in T cells. This resulted in dramatic changes in the distribution of actin and the actin-binding protein, Hip55, in these cells, which in turn, inhibited T cell infiltration into the graft. This indicates that the immunosuppressive activity of the allochimeric molecule is achieved via downregulation of the RhoA pathway and disruption of the proper organization of T cell actin cytoskeleton to inhibit T cell functions such as motility and/or TCR signaling events.

Development of a novel Hsp70-based DNA vaccine as a multifunctional antigen delivery system.

DNA vaccination is a simple and effective method to induce immune responses against a variety of tumors as well as infectious diseases. Vaccination with major histocompatibility complex (MHC) class I tumor peptide has been carried out to induce an antigen-specific and tumor-reactive cytotoxic T lymphocytes (CTLs) response in vivo. In this study, we describe a novel DNA vaccine based on heat shock protein 70 (Hsp70), which can chaperon antigenic peptides and initiate innate and adaptive immune responses, to induce a more effective immune response. Ovalbumin (OVA) MHC class I epitope peptide (OVA(257-264): SIINFEKL) was selected as a model antigen and polyhistidine was used to facilitate the cytosolic delivery of the antigen-Hsp70 after endocytic uptake. A novel plasmid DNA vector encoding polyhistidine, Hsp70 and OVA(257-264) (pHis-Hsp70-pep) was designed. When mice were immunized with pHis-Hsp70-pep by intradermal injection in combination with electroporation, strong antigen-specific CTL responses were generated. pHis-Hsp70-pep also showed a significant protective effect against tumor challenge with an OVA-expression EL4 tumor line. These results indicate that the Hsp70-based DNA vaccine is useful as a multifunctional antigen delivery system to induce the antigen-specific immune response.

sHLA-I contaminating molecules as novel mechanism of ex vivo/in vitro transcriptional and posttranscriptional modulation of transforming growth factor-beta in CD8+ T lymphocytes and neutrophils after intravenous immunoglobulin treatment.

BACKGROUND: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders. Among others' data, an in vitro increase of intracellular TGF-beta expression when culturing CD4+ T lymphocytes in the presence of IVIG has been reported. As IVIG infusion involves administration of soluble contaminants likewise all hemoderivative preparations, we hypothesized that, besides several other immunomodulatory proposed mechanisms, the clinical effects of IVIG therapy might be, at least partly, due to contaminating soluble HLA Class I (sHLA-I) molecules capable to exert pleiotropic immunomodulatory effects among which TGF-beta(1) modulation. STUDY DESIGN AND METHODS: Ex vivo and in vitro transcriptional and posttranscriptional modulation of TGF-beta(1) in CD8+ T lymphocytes and neutrophils after IVIG infusion was analyzed. RESULTS: Ex vivo analysis of cells drawn from 10 enrolled IVIG recipients pointed out a significant increase of TGF-beta(1) mRNA and intracellular TGF-beta(1) molecules in both leukotypes. In vitro comparable results were obtained incubating CD8+ T lymphocytes and neutrophils from healthy donors with IVIG. The immunodepletion of sHLA-I and/or soluble Fas ligand (sFasL) abolished TGF-beta(1) modulation in both leukotypes. Coculture with human immunoglobulin (Ig)M monoclonal antibody or chimeric IgG (MabThera, Roche), whose manufacturing excludes "contamination," did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA-I molecules enhanced TGF-beta(1) mRNA in both white blood cells to levels comparable to those obtained with IVIG incubation. CONCLUSION: On the whole, these data lead us to speculate that the ability of IVIG administration to modulate TGF-beta(1) might be related to the immunomodulatory activities of sHLA-I and sFasL molecules on activated CD8+ T lymphocytes and neutrophils.

Enhanced generation of cytotoxic T lymphocytes by increased cytosolic delivery of MHC class I epitope fused to mouse heat shock protein 70 via polyhistidine conjugation.

Heat shock protein 70 (Hsp70)-associated antigens in a soluble form have been shown to elicit strong antigen-specific cytotoxic T lymphocyte (CTL) responses following immunization without any adjuvants. In order to improve the potential of Hsp70, we genetically designed a novel Hsp70-based antigen delivery system, in which the model MHC class I epitope of ovalbumin (OVA) (SIINFEKL; OVA257-264) was fused to mouse Hsp70. To facilitate the cytosolic delivery of the peptide following Hsp receptor-mediated endocytosis, polyhistidine of 25 or 50 residues was further fused to the fusion protein. Each fusion protein was then expressed in E. coli and purified. When added to DC2.4 cells, a mouse dendritic cell line, the fusion protein containing polyhistidine of 25 residues was efficiently taken up by the cells and efficiently distributed to the cytosol. The fusion protein also exhibited a significantly improved efficacy of MHC class I-restricted presentation of antigen. Vaccination of mice with the polyhistidine fusion protein generated strong antigen-specific CTL responses and antitumor activity. These findings suggest that polyhistidine fusion is a useful strategy to increase the potential of Hsp-based vaccination.

Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo.

Optimal immunity to Mycobacterium tuberculosis (Mtb) infection requires CD8(+) T cells, and several current Mtb vaccine candidates are being engineered to elicit enhanced CD8(+) T cell responses. However, the function of these T cells and the mechanism by which they provide protection is still unknown. We have previously shown that CD8(+) T cells specific for the mycobacterial Ags CFP10 and TB10.4 accumulate in the lungs of mice following Mtb infection and have cytolytic activity in vivo. In this study, we determine which cytolytic pathways are used by these CD8(+) T cells during Mtb infection. We find that Mtb-specific CD8(+) T cells lacking perforin have reduced cytolytic capacity in vivo. In the absence of perforin, the residual cytolytic activity is CD95 and TNFR dependent. This is particularly true in Mtb-infected lung tissue where disruption of both perforin and CD95 eliminates target cell lysis. Moreover, adoptive transfer of immune CD8(+) T cells isolated from wild-type, but not perforin-deficient mice, protect recipient mice from Mtb infection. We conclude that CD8(+) T cells elicited following Mtb infection use several cytolytic pathways in a hierarchical and compensatory manner dominated by perforin-mediated cytolysis. Finally, although several cytolytic pathways are available, adoptively transferred Mtb-specific CD8(+) T cells require perforin-mediated cytolysis to protect animals from infection. These data show that CD8(+) T cell-mediated protection during Mtb infection requires more than the secretion of IFN-gamma and specifically defines the CD8(+) cytolytic mechanisms utilized and required in vivo.

Low-avidity CD8lo T cells induced by incomplete antigen stimulation in vivo regulate naive higher avidity CD8hi T cell responses to the same antigen.

We have previously reported that multiple injections of soluble MHC class I tetramers assembled with wild-type HY peptide induces unresponsiveness to male skin grafts in naive female C57BL/6 (B6) mice. Induction of unresponsiveness is dependent on a population of unresponsive allospecific CD8(lo )T cells. Reduced expression of CD8 acts to limit a T cell response to HY peptide by limiting the avidity window of effective signal transduction. We and others have demonstrated that CD8(lo) T cells are an alternative stable phenotype of CD8alphabeta(+) T cells in vitro and in vivo after antigen stimulation. We show here that CD8(lo) T cells can suppress naive CD8(+) T cell responses to HY antigen in vitro and male skin graft rejection in vivo after adoptive transfer into female recipients. These novel regulatory T cells express surface TGF-beta1 and secrete T cytotoxic 2 cytokines after antigen-specific stimulation. Anti-TGF-beta antibody and latency-associated peptide inhibit the suppressive effects in vitro. We also show that HY-specific memory CD8(+) T cells overcome regulation by CD8(lo) T cells. These data define a novel peripheral regulatory CD8(+ )T cell population that arises after repeated antigen encounter in vivo. These cells have implications in the maintenance of tolerance and memory.

Delivery of an erythropoietin-Fc fusion protein by inhalation in humans through an immunoglobulin transport pathway.

A novel drug delivery platform has been developed that utilizes a naturally occurring receptor known as the neonatal Fc receptor (FcRn). The receptor is specific for the Fc fragment of IgG and is expressed in epithelial cells where it functions to transport immunoglobulins across these cell barriers. It has been shown that FcRn is expressed in both the upper and central airways in non-human primates as well as in humans. Pulmonary delivery of an erythropoietin- Fc fusion molecule (EpoFc) was previously demonstrated in non-human primates using this FcRn pathway. We have now conducted a phase I clinical study to test whether the FcRn pathway functioned similarly in man using human erythropoietin (Epo) fused to the Fc portion of human IgG1. The design was a three leg, non-randomized study conducted in healthy male volunteers with rising doses (3, 10, and 30 microg/kg) of the fusion protein targeted to the central lung regions. Using a target range of 10-30% vital capacity and 15 breaths per minute, approximately 70% of the lung-deposited dose of aerosolized EpoFc was delivered safely and effectively to the central lung regions. We showed dose-dependent concentrations of the fusion protein in the serum and an increase in circulating reticulocytes was evident in the highest dose group, thus demonstrating that large therapeutic molecules can be delivered to humans via the lung, with retention of biological activity, using the FcRn-mediated transport pathway.

Primary adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and liver enzyme elevation.

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.

Induction of impaired antitumor immunity by fusion of MHC class II-deficient dendritic cells with tumor cells.

To dissect the role of Ag presentation through MHC class I and/or II pathways by dendritic cell (DC)-tumor fusion cells, we have created various types of DC-tumor fusion cells by alternating fusion cell partners. Fusions of MC38/MUC1 carcinoma cells with DC from wild-type (WT-DC), MHC class I knockout (IKO-DC), class II knockout (IIKO-DC), or class I and II knockout (I/IIKO-DC) mice created WTDC-fusion cells (FC), IKO-FC, IIKO-FC, and I/IIKO-FC, respectively. MHC class II- and MUC1-positive fusion cells were constructed by fusion of B16/MUC1 melanoma cells with IKO-DC (IKO/B16-FC). Immunization of MUC1 transgenic mice with 5 x 10(5) WTDC-FC, IKO-FC, IIKO-FC, or I/IIKO-FC provided 100, 91.7, 61.5, and 15.4% protection, respectively, against tumor challenge with MC38/MUC1 cells. In contrast, all mice immunized with irradiated MC38/MUC1 tumor cells or WT-DC developed tumors. One group of mice was immunized with 5 x 10(5) IKO/B16-FC and then challenged with B16/Ia(+)/MUC1 on one flank and MC38/MUC1 on the other flank. Immunization of these mice with IKO/B16-FC resulted in 100 and 78.6% protection against B16/Ia(+)/MUC1 and MC38/MUC1 tumor challenge, respectively. The antitumor immunity induced by immunization with IKO/B16-FC was able to inhibit the growth of MHC class II-negative tumor. In addition, in vivo results correlated with the induction of Ag-specific CTL. Collectively, the data indicate that MHC class II Ag presentation targeting activation of CD4 T cells is indispensable for antitumor immunity.

Increased adjuvant activity of minimal CD8 T cell peptides incorporated into lipid-core-peptides.

A problem facing the use of subunit peptide and protein vaccines is their inability to stimulate protective immune responses. Many different approaches have been utilized to overcome this inefficient immune activation. The approach we have taken is to modify the vaccine antigen so that it now has adjuvant properties. To do this, multiple copies of minimal CD8 T cell epitopes were attached to a poly lysine lipid core. These constructs are known as lipid-core-peptides (LCP). The research presented here examines the adjuvant activity of LCP. Using mouse models, we were able to show that LCP were indeed able to activate antigen-presenting cells in vitro and to activate cytotoxic T-cell responses in vivo. More importantly, LCP were able to stimulate the development of a protective antitumour immune response.

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor.

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.

Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections.

Control of hepatitis C virus (HCV) infection could be influenced by the timing and magnitude of CD4+ T cell responses against individual epitopes. We characterized CD4+ T cells targeting seven Pan troglodytes (Patr) class II-restricted epitopes during primary and secondary HCV infections of a chimpanzee. All Patr-DR-restricted HCV epitopes bound multiple human HLA-DR molecules, indicating the potential for overlap in epitopes targeted by both species. Some human MHC class II molecules efficiently stimulated IL-2 production by chimpanzee virus-specific T cell clones. Moreover, one conserved epitope designated NS3(1248) (GYKVLVLNPSV) overlapped a helper epitope that is presented by multiple HLA-DR molecules in humans who spontaneously resolved HCV infection. Resolution of primary infection in the chimpanzee was associated with an initial wave of CD4+ T cells targeting a limited set of dominant epitopes including NS3(1248.) A second wave of low-frequency CD4+ T cells targeting other subdominant epitopes appeared in blood several weeks later after virus replication was mostly contained. During a second infection 7 years later, CD4+ T cells against all epitopes appeared in blood sooner and at higher frequencies but the pattern of dominance was conserved. In summary, primary HCV infection in this individual was characterized by T cell populations targeting two groups of MHC class II-restricted epitopes that differed in frequency and kinetics of appearance in blood. The hierarchial nature of the CD4+ T cell response, if broadly applicable to other HCV-infected chimpanzees and humans, could be a factor governing the outcome of HCV infection.