Prolonged expression of an anti-HIV-1 gp120 minibody to the female rhesus macaque lower genital tract by AAV gene transfer.

Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women; however, numerous pharmacokinetic limitations associated with coitally related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various green fluorescent protein (GFP)-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intravaginally, including p63+ epithelial stem cells. Moreover, intravaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79-day study period. These data provide proof of principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection.

Dendritic cell vaccination induces cross-reactive cytotoxic T lymphocytes specific for wild-type and natural variant human immunodeficiency virus type 1 epitopes in HLA-A*0201/Kb transgenic mice.

Dendritic cells (DC) are highly efficient at inducing primary T cell responses. Consequently, DC are being investigated for their potential to prevent and/or treat human immunodeficiency virus type 1 (HIV-1) infection. In the current study, we examined the capacity of DC to elicit CD8+ cytotoxic T lymphocyte (CTL) reactivity against an HLA-A*0201-restricted HIV-1 reverse transcriptase (pol) epitope (residues 476-484) and two naturally occurring variants. Previous work demonstrated that the wild-type pol epitope is recognized by CTLs from HIV-1-infected individuals, whereas the variant pol epitopes are not, despite binding to HLA-A*0201. In agreement with these observations, parenteral administration of wild-type pol peptide induced HLA-A*0201-restricted CTL activity in A2Kb transgenic mice. In contrast, similar treatment with the two variant pol peptides failed to stimulate CTL reactivity, and this lack of immunogenicity correlated with reduced peptide:HLA-A*0201 complex stability. However, CTL responses were induced in A2Kb transgenic mice upon adoptive transfer of syngeneic bone marrow DC pulsed with the variant pol peptides. Furthermore, DC pulsed with the wild-type pol peptide elicited CTLs that cross-reacted with the variant pol epitopes. These results demonstrate that DC effectively expand the T cell repertoire of a given epitope to include cross-reactive T cell clonotypes. Accordingly, DC vaccination may aid in immune recognition of HIV-1 escape variants by broadening the T cell response.

Dendritic cells from nonobese diabetic mice exhibit a defect in NF-kappa B regulation due to a hyperactive I kappa B kinase.

Insulin-dependent diabetes mellitus (IDDM) is characterized by the T cell-mediated destruction of insulin-producing beta cells. Accordingly, APCs, such as macrophage, have also been shown to be important in the disease process. However, the role(s) of dendritic cells (DCs) that exhibit potent APC function remains undefined in IDDM. Here we demonstrate that DCs derived from nonobese diabetic (NOD) mice, a model for IDDM, are more sensitive to various forms of stimulation compared with those from C57BL/6 and BALB/c mice, resulting in increased IL-12 secretion. This property is a consequence of hyperactivation of NF-kappaB, a transcription factor known to regulate IL-12 gene expression. Specifically, NOD DCs exhibit persistent hyperactivation of both IkappaB kinase and NF-kappaB in response to stimuli, in addition to selective degradation of IkappaBepsilon. Transfection of NOD DCs with a modified form of IkappaBalpha significantly reduced IL-12 secretion, suggesting that hyperactivation of NF-kappaB was in part responsible for increased IL-12 production. An enhanced capacity of NOD DCs to secrete IL-12 would be expected to contribute to the development of pathogenic Th1 (Tc1) cells during the diabetogenic response.

Crystal structure of an MHC class I presented glycopeptide that generates carbohydrate-specific CTL.

T cell receptor (TCR) recognition of nonpeptidic and modified peptide antigens has been recently uncovered but is still poorly understood. Immunization with an H-2Kb-restricted glycopeptide RGY8-6H-Gal2 generates a population of cytotoxic T cells that express both alpha/beta TCR, specific for glycopeptide, and gamma/delta TCR, specific for the disaccharide, even on glycolipids. The crystal structure of Kb/RGY8-6H-Gal2 now demonstrates that the peptide and H-2Kb structures are unaffected by the peptide glycosylation, but the central region of the putative TCR binding site is dominated by the extensive exposure of the tethered carbohydrate. These features of the Kb/RGY8-6H-Gal2 structure are consistent with the individual ligand binding preferences identified for the alpha/beta and gamma/delta TCRs and thus explain the generation of a carbohydrate-specific T cell response.

MHC class I presentation of live and heat-inactivated Sendai virus antigen in T2Kb cells depends on an intracellular compartment with endosomal characteristics.

T2Kb cells, which do not express TAP1/2 peptide transporters or the low molecular weight protein 2/7 (LMP2/7) proteasomal subunits, can still process and present both live and heat-inactivated Sendai virus (SV). As this operation may also reflect the existence of an alternative processing pathway in normal antigen-presenting cells (APC), the authors have characterized it using intracellular inhibitors and anti-Kb monoclonal antibodies (MoAbs). From the results with lipophilic amines (ammonium chloride, methylamine and chloroquine), cytoskeletal inhibitors (cytochalasin B and vinblastine), and an endoprotease inhibitor (phenylmethylsulfonyl fluoride, PMSF), the authors conclude that the processing of SV antigen in T2Kb cells has endosomal characteristics depending on cellular activities such as uptake, vesicular transport and intracellular-vesicular proteolysis. In addition, internalized 'empty' Kb molecules derived from the T2Kb cell surface appeared to be involved in the presentation of SV antigen, as demonstrated by a protocol using a combination of the Golgi inhibitor brefeldin A(BFA) and anti-Kb antibodies. The results thus indicate that T2Kb cells process SV antigen in an endosomal-like compartment which contain recycling 'empty' Kb molecules.

External glycopeptide binding to MHC class-I in relation to expression of TAP transporters, beta 2-microglobulin and to pH.

MHC class-I binding glycopeptides are easily visualized on the cell surface by carbohydrate specific monoclonal antibodies. By comparing the staining intensity between anti-carbohydrate and anti-MHC class-I specific monoclonal antibodies, an estimation of the fraction of peptide accessible 'empty' sites on the cell surface of MHC class-I molecules can be made. This system was used to analyze glycopeptide binding to MHC class-I molecules in relation to transporter associated with antigen processing (TAP) peptide transporters and beta 2-M expression, using gene targeted mice, and in relation to pH. Approximately 15, 40, and 95% 'empty' Db molecules were found on activated T cells from normal, beta 2-M-/- and TAP -/- mice, respectively. The ASN9-6h-Gal2 glycopeptide also bound to transfected 'empty' Db molecules on T1-Db, T2-Db and T3-Db cells with a preference for T2-Db cells, lacking TAP peptide transporters. The stability of glycopeptide binding to H-2Db is also highest on T2-Db cells. pH was found to influence binding either positively or negatively, using four different glycopeptides, binding either to Db or Kb. We conclude that external glycopeptide binding may reflect important functional properties in the MHC class-I system and that pH in different processing compartments might influence the expressed peptide repertoire.

Induction of cytotoxic T-cell response by optimal-length peptides does not require CD4+ T-cell help.

In several experimental models, synthetic peptides were shown to activate efficiently cytotoxic T-lymphocyte (CTL) responses and therefore represent an attractive strategy to develop new vaccines. However, the mechanisms by which they induce CTL responses are not yet fully understood. Several studies using 15 16-mer peptides previously demonstrated that CD4 helper T cells are required to induce optimal CTL responses with synthetic peptides. However, recently it was suggested that shorter 8 12-mer peptides could have an increased in vivo immunogenicity. In the present study, we therefore investigated if such optimal-length peptides still require CD4+ T-cell help to activate CTL responses. To address this question three synthetic peptides containing different viral CTL epitopes were injected into mice depleted of CD4+ or CD8+ T cells using specific monoclonal antibodies or into mice genetically deficient in those T-cell populations. Our results clearly established that activation of CTL responses by those short optimal peptides does not require CD4+ T-cell help and therefore suggested that high-density binding of peptides to major histocompatibility complex class I molecules on the surface of antigen-presenting cells is required for direct activation of CD8+ T cells, independently of CD4+ T-cell help.

Immunization with glycosylated Kb-binding peptides generates carbohydrate-specific, unrestricted cytotoxic T cells.

Cytotoxic T cells (CTL) recognize target proteins as short peptides presented by major histocompatibility complex (MHC) class I restriction elements. However, there is also evidence for peptide-independent T cell receptor (TCR) recognition of target proteins and non-protein structures. How such T cell responses are generated is presently unclear. We generated carbohydrate (CHO)-specific, MHC-unrestricted CTL responses by coupling di- and trisaccharides to Kb- or Db-binding peptides for direct immunization in mice. Four peptides and three CHO have been analyzed with the CHO either in terminal or central position on the carrier peptide. With two of these glycopeptides, with galabiose (Gal alpha 1-4Gal; Gal2) bound to a homocysteine (via an ethylene spacer arm) in position 4 or 6 in a vesicular stomatitis virus nucleoprotein-derived peptide (RGYVYQGL binding to Kb), CTL were generated which preferentially killed target cells treated with glycopeptide compared to those treated with the core peptide. Polyclonal CTL were also found to kill target cells expressing the same Gal2 epitope in a glycolipid. By fractionation of CTL, preliminary data indicate that glycopeptide-specific Kb-restricted CTL and unrestricted CHO-specific CTL belong to different T cell populations with regard to TCR expression. The results demonstrate that hapten-specific unrestricted CTL responses can be generated with MHC class I-binding carrier peptides. Different models that might explain the generation of such responses are discussed.

Major histocompatibility complex class I binding glycopeptides for the estimation of 'empty' class I molecules.

Different forms of major histocompatibility complex (MHC) class I heavy chains are known to be expressed on the cell surface, including molecules which are functionally 'empty'. Direct peptide binding to cells is obvious during sensitization of target cells in vitro for cytotoxic T lymphocyte killing and 'empty' MHC-I molecules are comparatively abundant on TAP-1/2 peptide transporter mutant cells. In the present work we have estimated the fraction of 'empty' MHC class I molecules using glycosylated peptides and cellular staining with carbohydrate specific monoclonal antibodies. Synthetic Db and Kb binding peptides were coupled at different positions with different di- or trisaccharides, using different spacing between the carbohydrate and the peptide backbone. Binding of sugar specific mAbs was compared in ELISA and cellular assays. An optimal Db binding glycopeptide was used for comparative staining with anti-Db and anti-carbohydrate monoclonal antibodies to estimate fractions of 'empty' molecules on different T lymphoid cells. On activated normal T cells, a large fraction of Db molecules were found to be 'empty'. The functional role of such 'empty' MHC class I molecules on T cells is presently unclear. However, on antigen presenting cells they might participate in the antigen presentation process.

Glycosylphosphatidylinositol-linked Db does not induce an influenza-specific cytotoxic T lymphocyte response or recycle membrane-bound peptides.

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I-bound peptides, are known to recycle between the cell surface and an undefined, endosomal-like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H-2Db molecule expressed in transgenic mice, either transmembranous (Db-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (Db-GPI). The recycling capacity of peptides bound to Db-tm and Db-GPI was investigated using glycosylated Db-binding glycopeptides, which were detected by flow cytometry. Only the tm form of Db was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366-374, ASNENMETM), only Db-tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I-bound peptides on antigen-presenting cells may be important for the generation of certain CTL responses.

Presentation of viral antigens restricted by H-2Kb, Db or Kd in proteasome subunit LMP2- and LMP7-deficient cells.

In the class II region of the major histocompatibility complex (MHC(, four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP1 and TAP2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP2 and LMP7) code for subunits of the proteasome. While TAP1 and TAP2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP1/2 and LMP2/7 genes, it was recently shown that expression of TAP1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP2/7, we transfected T2 cells with TAP1, TAP2 and either of the H-2Kb, Db or Kd genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP1/2 gene products, presented all tested viral antigens restricted through either the H-2Kb, Db or Kd class I molecules. We conclude that the proteasome subunits LMP2/7 as well as other gene products in the MHC class II region, except from TAP1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.

Major histocompatibility complex class I-binding peptides are recycled to the cell surface after internalization.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short, processed peptides bound to major histocompatibility complex class I (MHC-I) heavy and light chains (beta 2-microglobulin; beta 2-m). The heavy chain, which comprise the actual peptide binding alpha-1 and alpha-2 domains, can exist at the cell surface in different forms, either free, bound to beta 2-m or as a ternary complex with beta 2-m and peptides. MHC-I chains are also known to internalize, and recycle to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-I-bound peptides also can recycle is not known. We have investigated this by using both peptide transporter mutant RMA-S cells and EL4 cells loaded with Db-binding peptides, by two different approaches. First, peptides were covalently linked with galabiose (Gal alpha 4Gal) at a position which did not interfere with Db binding or immunogenicity, and peptide recycling tested with Gal2-specific monoclonal antibodies. By flow cytometry, a return of Gal2 epitopes to the cell surface was found, after cellular internalization and cell surface clearance by pronase treatment. This peptide recycling could be discriminated from free fluid-phase uptake and was inhibited by methylamine, chloroquine and low temperature (18 degrees C) but not by leupeptin. Second, specific CTL were reacted with peptide-loaded target cells after complete removal of surface Db molecules by pronase, and after different times of incubation at 37 degrees C to allow reexpression. By this procedure, reappearance of target cell susceptibility was confirmed. The results are in agreement with a model for optimizing peptide presentation by recycling through an intracellular compartment similar to early endosomes in certain antigen-presenting cells.

Regulation of MHC class I membrane expression by beta 2-microglobulin.

MHC-I binding peptides and beta 2 microglobulin (beta 2-m) can upregulate the MHC-I heavy chain expression on certain peptide transporter mutant cells. We have further studied this with normal cells and non-mutant cell lines. No MHC-I upregulation was seen with normal, resting or activated T cells. On mouse cell lines P815 and B16, both peptides and human beta 2-m gave an additive upregulation response. With the human small cell lung carcinoma H82, an optimal HLA.A2 binding peptide (GILGFVFTL) gave an upregulation response, whereas beta 2-m alone or in combination with this peptide had no effect. However, beta 2-m potentiated the response of H82 cells to a slightly longer peptide. Using mutant RMA-S cells, it was found that both Brefeldin A (BFA) and chloroquine, but not leupeptin, inhibited MHC-I upregulation response to both peptide and beta 2-m. In contrast to chloroquine, BFA also gave a reduction of background membrane MHC-I expression, presumably due to a block in Golgi transport. Human beta 2-m, which binds to RMA-S cells, and which is known to internalize into endosomes, did not reappear on the cell surface. When Db on RMA-S cells was upregulated by human beta 2-m, the sensitivity of these cells to Db restricted CTL cells increased. Even if beta 2-m did not upregulate the overall MHC-I expression on normal cells, it may still quantitatively increase the expression of optimally presented peptides and endosomal recycling many be important in this process.

In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.