peptide-MHC Class Research Articles

Suppression of ongoing T cell-mediated autoimmunity by peptide-MHC class II dimer vaccination.

Tissue-specific autoimmune diseases such as type 1 diabetes (T1D) are characterized by T cell-driven pathology. Administration of autoantigenic peptides provides a strategy to selectively target the pathogenic T cell response. Indeed, treatment with beta cell peptides effectively prevents T1D in NOD mice. However, the efficacy of peptide immunotherapy generally wanes as beta cell autoimmunity progresses and islet inflammation increases. With the goal of enhancing the efficacy of peptide immunotherapy, soluble (s)IA(g7)-Ig dimers covalently linked to beta cell autoantigen-derived peptides were tested for the capacity to suppress late preclinical T1D. NOD female mice with established beta cell autoimmunity were vaccinated i.v. with a short course of sIA(g7)-Ig dimers tethered to peptides derived from glutamic acid decarboxylase (GAD)65 (sIA(g7)-pGAD65). Treatment with sIA(g7)-pGAD65 dimers and the equivalent of only approximately 7 microg of native peptide effectively blocked the progression of insulitis and the development of diabetes. Furthermore, suppression of T1D was dependent on beta cell-specific IL-10-secreting CD4+ T cells, although the frequency of GAD65-specific FoxP3-expressing CD4+ T cells was also increased in sIA(g7)-pGAD65 dimer vaccinated NOD mice. These results demonstrate that MHC class II-Ig dimer vaccination is a robust approach to suppress ongoing T cell-mediated autoimmunity, and may provide a superior strategy of adjuvant-free peptide-based immunotherapy to induce immunoregulatory T cells.

HLA-DM mediates peptide exchange by interacting transiently and repeatedly with HLA-DR1

The peptide editor HLA-DM (DM) catalyzes the exchange of peptides bound to MHC class II molecules within antigen presenting cells by generating a “peptide-receptive” MHC class II conformation (MHCreceptive) to which peptides readily bind and rapidly unbind. While recent work has uncovered the determinants of DM recognition and effector functions, the nature of MHCreceptive and its interaction with DM remains unclear. Here, we show that DM induces but does not stabilize MHCreceptive in the absence of peptides. We demonstrate that DM is out-competed by certain superantigens, and increasing solvent viscosity inhibits DM-induced peptide association. We suggest that DM mediates peptide exchange by interacting transiently and repeatedly with MHC class II molecules, continually generating MHCreceptive. The simultaneous presence of peptide and DM in the milieu is thus crucial for the efficient generation of specific peptide–MHC class II complexes over time.

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.

Basophils contribute to TH2-IgE responses in vivo via IL-4 production and presentation of peptide–MHC class II complexes to CD4+ T cells

Basophils express major histocompatibility complex class II, CD80 and CD86 and produce interleukin 4 (IL-4) in various conditions. Here we show that when incubated with IL-3 and antigen or complexes of antigen and immunoglobulin E (IgE), basophils internalized, processed and presented antigen as complexes of peptide and major histocompatibility complex class II and produced IL-4. Intravenous administration of ovalbumin-pulsed basophils into naive mice 'preferentially' induced the development of naive ovalbumin-specific CD4+ T cells into T helper type 2 (T(H)2) cells. Mice immunized in this way, when challenged by intravenous administration of ovalbumin, promptly produced ovalbumin-specific IgG1 and IgE. Finally, intravenous administration of IgE complexes rapidly induced T(H)2 cells only in the presence of endogenous basophils, which suggests that basophils are potent antigen-presenting cells that 'preferentially' augment T(H)2-IgE responses by capturing IgE complex.

The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation

Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance.

The absence of ER trimming alters peptides presented by non-classical MHC class I molecules (78.8)

Abstract The nature, function and pathways used to produce peptides for presentation by non-classical MHC class I molecules is poorly understood. Here we show that cells from mice lacking the ER aminopeptidase associated with antigen processing (ERAAP) present an immunologically distinct peptide-MHC class I repertoire. Wild-type mice immunized with ERAAP-deficient cells mount robust CD8 T cell responses specific for peptides presented by classical as well as non-classical MHC I molecules. These CD8 T cells produce pro-inflammatory cytokines and effectively kill ERAAP-deficient target cells in vitro as well as in vivo. Further, peptides presented by non-classical MHC class I molecules are expressed in a tissue-specific manner and elicit CD8 T cell responses both by direct and cross-priming. Thus, ERAAP trims the final peptides presented by classical as well as non-classical MHC class I molecules, and may be involved in trimming peptides for cross-presentation. This research was supported by grants from the NIH (AI060040) to N.S.; N.A.N. is supported by the Irvington Institute Fellowship Program of the Cancer Research Institute.

Associations of Erosive Arthritis with Anti-Cyclic Citrullinated Peptide Antibodies and MHC Class II Alleles in Systemic Lupus Erythematosus

Associations of Erosive Arthritis with Anti-Cyclic Citrullinated Peptide Antibodies and MHC Class II Alleles in Systemic Lupus Erythematosus

Sequence Dependent Efficiency of Cross-Presentation in MHC Class I Requires Rational Design of Long Synthetic Peptides for Vaccination or Ex Vivo Activation

For the induction or boosting of antigen-specific CD8+ T cell responses, long synthetic peptides have been used in vaccination studies. Superior in vivo CD8+ T cell responses have been reported following vaccination with long peptides compared with minimal peptides, which was attributed to selective uptake and cross-presentation by professional antigen-presenting cells. Furthermore, to generate antigen-specific T cell lines for adoptive immunotherapy or to measure antigen-specific T cell responses, protein-spanning pools of overlapping long synthetic peptides can be used to simultaneously activate CD8+ and CD4+ T cells in peripheral blood mononuclear cells (PBMC) ex vivo. Although exogenous antigen is predominantly presented in MHC class II, it has been suggested that cross-presentation of long peptides in MHC class I can occur. However, the mechanism of cross-presentation of exogenous long peptides in MHC class I is not clear. Various models for cross-presentation have been described following uptake of soluble antigen in endosomes, among whichantigen transport over the endosomal membrane followed by the classical proteasome- and TAP-dependent route, andentrance of MHC class I in the recycling endocytic MHC class II pathway where peptidase-trimmed exogenous antigens can exchange with peptides in the MHC class I molecules, resulting in TAP- and proteasome-independent cross-presentation.To improve the design of peptides for the in vivo or ex vivo activation of CD8+ T cells we investigated the mechanism and efficiency of cross-presentation of long peptides. We observed that antigen-presenting cells in peripheral blood, in particular monocytes, loaded with 15-mer peptides, 31-mer peptides or full length protein containing the NLV epitope were able to very efficiently induce IFNg production by cytomegalovirus (CMV) pp65 NLV-specific T cells. Specific T cells were most efficiently activated by N-terminally extended variants of the minimal epitope, while the use of C-terminally extended variants resulted in a 10-fold reduction of activation efficiency. Purification of these antigens by high performance liquid chromatography (HPLC) followed by mass spectrometry demonstrated that activation was not caused by contamination with the minimal epitope sequence. Also CD8+ T cells specific for other CMV and minor histocompatibility antigen (mHag) epitopes were activated by monocytes loaded with 15-mer or 20-mer peptides. Again N-terminally extended variants of minimal epitopes very efficiently induced activation, while the use of C-terminally variants or full length protein resulted in highly variable efficiency of activation, ranging from 10-fold reduction to complete absence of activation. Interestingly, TAP-deficient T2 cells loaded with CMV pp65 NLV antigens also efficiently activated NLV-specific T cells, indicating that the route of presentation was TAP-independent. Addition of lactacystin during loading of monocytes with CMV pp65 NLV 15-mer did not affect activation of specific T cells, suggesting that cross-presentation was proteasome-independent. Addition of primaquine reduced activation of specific T cells by the NLV 15-mer peptide, but not by the minimal NLV 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. To compare cross-presentation with presentation of endogenously synthesized antigen, TAP-competent T1 and TAP-deficient T2 cells were retrovirally transduced with the CMV pp65 gene. CMV pp65-specific T cells were activated by CMV pp65 transduced T1 but not T2 cells, indicating that endogenously synthesized CMV pp65 required processing and presentation by the classical proteasome- and TAP-dependent route. These data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling MHC class I molecules. Not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. As the efficiency of cross-presentation of long synthetic peptides may depend on the sequence of the C-terminal extension, a rational design of peptides is crucial for efficient activation of CD8+ T cells in approaches of vaccination, adoptive transfer and immune monitoring.

Cutting Edge: TLR Ligands Increase TCR Triggering by Slowing Peptide-MHC Class I Decay Rates

TLR ligands are among the key stimuli driving the optimal dendritic cell (DC) maturation critical for strong and efficacious T cell priming. In this study, we show that part of this effect occurs via increased TCR triggering. Pretreatment of DCs with TLR ligands resulted in the triggering of many more TCRs in responding CD8(+) T cells. Importantly, even when DCs expressed the same amount of cognate peptide-MHC (pMHC) molecules, TLR ligand treatment resulted in down-regulation of larger numbers of TCR molecules. This was independent of the up-regulation of costimulatory, adhesion or cytokine molecules or the amount of noncognate pMHCs. Rather, DCs pretreated with TLR ligands exhibited increased stability of cognate pMHCs, enabling extended TCR triggering. These findings are of potential importance to T cell vaccination.

Improving peptide-MHC class I binding prediction for unbalanced datasets

BackgroundEstablishment of peptide binding to Major Histocompatibility Complex class I (MHCI) is a crucial step in the development of subunit vaccines and prediction of such binding could greatly reduce costs and accelerate the experimental process of identifying immunogenic peptides. Many methods have been applied to the prediction of peptide-MHCI binding, with some achieving outstanding performance. Because of the experimental methods used to measure binding or affinity between peptides and MHCI molecules, however, available datasets are enriched for nonbinders, and thus highly unbalanced. Although there is no consensus on the ideal class distribution for training sets, extremely unbalanced datasets can be detrimental to the performance of prediction algorithms.ResultsWe have developed a decision-theoretic framework to construct cost-sensitive trees to predict peptide-MHCI binding and have used them to 1) Assess the impact of the training data's class distribution on classifier accuracy, and 2) Compare resampling and cost-sensitive methods as approaches to compensate for training data imbalance. Our results confirm that highly unbalanced training sets can reduce the accuracy of classifier predictions and show that, in the peptide-MHCI binding context, resampling methods do not improve the classifier performance. In contrast, cost-sensitive methods significantly improve accuracy of decision trees. Finally, we propose the use of a training scheme that, when the training set is enriched for nonbinders, consistently improves the overall classifier accuracy compared to cost-insensitive classifiers and, in particular, increases the sensitivity of the classifiers. This method minimizes the expected classification cost for large datasets.ConclusionOur method consistently improves the performance of decision trees in predicting peptide-MHC class I binding by using cost-balancing techniques to compensate for the imbalance in the training dataset.

Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses.

Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.

Antigen Specificity Acquisition of Adoptive CD4+ Regulatory T Cells via Acquired Peptide-MHC Class I Complexes

The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.

Prediction of MHC Binding Peptides and Epitopes from Groundnut Bud Necrosis Virus (GBNV)

A virus disease caused of crop losses to groundnut in Andhra Pradesh during kharif in the year 2000. It was presumed to be caused by Groundnut bud necrosis virus (GBNV), a virus known to be widely distributed in India. It is one among the important infestations affecting groundnut cultivation especially during summer. Nucleocapsid peptides of groundnut bud necrosis virus are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. Analysis shows MHC class II binding peptides of coat protein from SMV are important determinant for protection of many plants form viral infection. In this assay we predicted the binding affinity of SMV genome polyprotein having 276 amino acids, which shows 268 nonamers. In this analysis, we found the SVM based MHCII-IAb peptide regions, 171- PLAYYQNVK, 95- RTEAFIRTK, 90- DWTFKRTEA, 37- KAFYDANKT, (optimal score is 1.059); MHCII-IAd peptide regions, 159- LSSMTGLAP, 68- KSGKYVFCG, 11- KIKELLAGG, 49- TFTNCLNIL, (optimal score is 0.702); MHCIIIAg7 peptide regions, 128- PLVAAYGLN, 127- LPLVAAYGL, 18- GGSADVEIE, 39- FYDANKTLE, (optimal score is 1.551); and MHCII- RT1.B peptide regions, 49- TFTNCLNIL, 247- DKAFSASLS, 110- KSKNDAAKQ, 92- TFKRTEAFI , (optimal score is 1.092) which represented predicted binders from nucleocapsid protein. The method integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency of the nucleocapsid protein of GBNV. Thus a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines.

Vaccine Adjuvants Alter TCR-Based Selection Thresholds

How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.

A Role for CD8 in the Developmental Tuning of Antigen Recognition and CD3 Conformational Change

TCR engagement by peptide-MHC class I (pMHC) ligands induces a conformational change (Deltac) in CD3 (CD3Deltac) that contributes to T cell signaling. We found that when this interaction took place between primary T lineage cells and APCs, the CD8 coreceptor was required to generate CD3Deltac. Interestingly, neither enhancement of Ag binding strength nor Src kinase signaling explained this coreceptor activity. Furthermore, Ag-induced CD3Deltac was developmentally attenuated by the increase in sialylation that accompanies T cell maturation and limits CD8 activity. Thus, both weak and strong ligands induced CD3Deltac in preselection thymocytes, but only strong ligands were effective in mature T cells. We propose that CD8 participation in the TCR/pMHC interaction can physically regulate CD3Deltac induction by "translating" productive Ag encounter from the TCR to the CD3 complex. This suggests one mechanism by which the developmentally regulated variation in CD8 sialylation may contribute to the developmental tuning of T cell sensitivity.

Epitope-specific TCRβ repertoire diversity imparts no functional advantage on the CD8+T cell response to cognate viral peptides

TCR repertoire diversity has been convincingly shown to facilitate responsiveness of CD8+ T cell populations to mutant virus peptides, thereby safeguarding against viral escape. However, the impact of repertoire diversity on the functionality of the CD8+ T cell response to cognate peptide-MHC class I complex (pMHC) recognition remains unclear. Here, we have compared TCRbeta chain repertoires of three influenza A epitope-specific CD8+ T cell responses in C57BL/6 (B6) mice: D(b)NP(366-374), D(b)PA(224-233), and a recently described epitope derived from the +1 reading frame of the influenza viral polymerase B subunit (residues 62-70) (D(b)PB1-F2(62)). Corresponding to the relative antigenicity of the respective pMHCs, and irrespective of the location of prominent residues, the D(b)PA(224)- and D(b)PB1-F2(62)-specific repertoires were similarly diverse, whereas the D(b)NP(366) population was substantially narrower. Importantly, parallel analysis of response magnitude, cytotoxicity, TCR avidity, and cytokine production for the three epitope-specific responses revealed no obvious functional advantage conferred by increased T cell repertoire diversity. Thus, whereas a diverse repertoire may be important for recognition of epitope variants, its effect on the response to cognate pMHC recognition appears minimal.

Defining the directionality and quality of influenza virus–specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus

Cross-reactivity of murine and recently human CD8(+) T cells between different viral peptides, i.e., heterologous immunity, has been well characterized. However, the directionality and quality of these cross-reactions is critical in determining their biological importance. Herein we analyzed the response of human CD8(+) T cells that recognize both a hepatitis C virus peptide (HCV-NS3) and a peptide derived from the influenza neuraminidase protein (Flu-NA). To detect the cross-reactive CD8(+) T cells, we used peptide-MHC class I complexes (pMHCs) containing a new mutant form of MHC class I able to bind CD8 more strongly than normal MHC class I complexes. T cell responses against HCV-NS3 and Flu-NA peptide were undetectable in normal donors. In contrast, some responses against the Flu-NA peptide were identified in HCV(+) donors who showed strong HCV-NS3-specific reactivity. The Flu-NA peptide was a weak agonist for CD8(+) T cells in HCV(+) individuals on the basis of novel pMHCs and functional assays. These data support the idea of cross-reactivity between the 2 peptides, but indicate that reactivity toward the Flu-NA peptide is highly CD8-dependent and occurs predominantly after priming during HCV infection. Our findings indicate the utility of the novel pMHCs in dissecting cross-reactivity and suggest that cross-reactivity between HCV and influenza is relatively weak. Further studies are needed to relate affinity and functionality of cross-reactive T cells.

PRDM1/BLIMP-1 Modulates IFN-γ-Dependent Control of the MHC Class I Antigen-Processing and Peptide-Loading Pathway

A diverse spectrum of unique peptide-MHC class I complexes guides CD8 T cell responses toward viral or stress-induced Ags. Multiple components are required to process Ag and facilitate peptide loading in the endoplasmic reticulum. IFN-gamma, a potent proinflammatory cytokine, markedly up-regulates transcription of genes involved in MHC class I assembly. Physiological mechanisms which counteract this response are poorly defined. We demonstrate that promoters of functionally linked genes on this pathway contain conserved regulatory elements that allow antagonistic regulation by IFN-gamma and the transcription factor B lymphocyte-induced maturation protein-1 (also known as PR domain-containing 1, with ZNF domain (PRDM1)). Repression of ERAP1, TAPASIN, MECL1, and LMP7 by PRDM1 results in failure to up-regulate surface MHC class I in response to IFN-gamma in human cell lines. Using the sea urchin prdm1 ortholog, we demonstrate that the capacity of PRDM1 to repress the IFN response of such promoters is evolutionarily ancient and that dependence on the precise IFN regulatory factor element sequence is highly conserved. This indicates that the functional interaction between PRDM1 and IFN-regulated pathways antedates the evolution of the adaptive immune system and the MHC, and identifies a unique role for PRDM1 as a key regulator of Ag presentation by MHC class I.

The Absence of Vasoactive Intestinal Peptide Augments Allo-Reactivity and the Anti-Viral Response in Bone Marrow Transplantation.

Background: The severe morbidity and mortality associated with bone marrow transplantation (BMT) is caused by uninhibited immune responses to alloantigen and suppressed immune responses to pathogens. Vasoactive Intestinal Peptide (VIP) is an immunomodulatory neuropeptide produced by T-cells and nerve fibers in peripheral lymphoid organs that suppresses immune responses by induction of tolerogenic dendritic cells. In order to determine the immunoregulatory effects of VIP, we examined T-cell immune responses to allo- and viral-antigens in VIP knockout (KO) mice and mouse BMT recipients of hematopoietic cells from VIP KO donors.Methods: VIP KO mice and VIP WT littermates were infected with lethal or sub-lethal doses (5 × 104− 5 × 105 PFU) of murine cytomegalovirus (mCMV) and the T-cell response to viral antigen was measured by flow cytometry for mCMV peptide-MHC class 1-tetramer+ CD8+ T-cells. We transplanted 5 × 106 BM plus 1 × 106 splenocytes (SP) either from VIP KO or VIP WT donors in an C57BL/6 to F1(BL/6 × Balb/c) allo-BMT model and assessed survival, GvHD, donor T-cell expansion, chimerism, and response to mCMV vaccination and mCMV infection.Results: B-cell, αβ and γδ T-cell, CD8+ T-cell, CD11b+ myeloid cell, and dendritic cell numbers were equivalent between VIP KO and WT mice, while VIP KO mice had higher number of CD4+ and CD4+CD62L+CD25+ T-cells. Non-transplanted VIP KO mice survived mCMV infection better compared to VIP WT, with a brisker anti-viral T-cell response in the blood. In the allogeneic BMT setting, recipients of VIP KO BM plus VIP KO SP had more weight loss and lower (40%) 100 day post-transplant survival compared to the recipients of VIP KO BM plus WT SP (80% survival), recipients of WT BM plus KO SP (100% survival), and recipients of WT BM plus WT SP (80% survival). Recipients of VIP KO grafts had a significantly greater anti-mCMV response that peaked four days earlier than the tetramer response of mice transplanted with WT cells. This increased anti-viral response to vaccination correlated with a greater and more rapid T-cell response to secondary viral challenge.Conclusions: These experiments suggest that the absence of all VIP in the body, or the absence of VIP in a transplanted immune system, enhances anti-viral immunity and allo-immune responses. Modulation of the VIP pathway is a novel method to regulate post-transplant immunity. [Display omitted]

Trace Capsid DNA Impurities in AAV2 Vectors Are Not Transcribed and Are Unlikely To Contribute to Capsid Peptide-MHC Class I Complexes

Cytotoxic T cell responses directed against adeno-associated virus (AAV) capsid may limit long-term therapeutic gene expression in human subjects following AAV vector administration to liver (Manno et al, 2006, Nature Med). Possible mechanisms for presentation of AAV capsid-derived peptide epitopes on the surface of transduced hepatocytes include:1.degradation of the pre-formed capsid component of the vector and presentation of capsid-derived peptides on the surface of transduced cells; and2.transcription and translation of trace levels of AAV capsid DNA (an impurity), resulting in cell-surface presentation of de novo-synthesized capsid-derived peptides via the endogenous antigen presentation pathway.Determination of which mechanism occurs is critical to implement effective strategies to achieve long-term therapeutic gene expression. Herein we investigate the endogenous pathway mechanism involving transcription of trace impurity capsid DNA by evaluating capsid mRNA levels following transduction using highly sensitive real time quantitative reverse transcriptase PCR (Q-RTPCR). In in vitro studies, a human hepatocyte cell line (HHL5) was transduced at doses of 1k, 10k or 100k vg/cell with AAV2-hFIX (recombinant AAV2 expressing human coagulation factor IX). Following transduction, RNA was purified from harvested HHL5 cells, and levels of AAV2 capsid mRNA assessed by Q-RTPCR using primers / probes designed to detect four AAV2 capsid epitopes (TTSTRTWAL, VPQYGYLTL, SADNNNSEY, YHLNGRDSL) recognized by human CTL's (Mingozzi et al, 2007, Blood). Using a primer / probe corresponding to hFIX as a positive control, hFIX mRNA levels were positive and dose-dependent. However, AAV2 capsid mRNA levels using primer / probes corresponding to the four capsid epitopes were not detected at any vector dose. The limit of quantitation of the Q-RTPCR was 20 transcripts per 100ng RNA input (10,000 cells). In in vivo studies, C57Bl/6 mice were administered 6x1013 or 1.2x1014 vg/kg of AAV2-hFIX vector, and 3 animals euthanized at 2, 4 or 6 weeks post vector administration. Supraphysiological levels (20–50 μg/mL) of hFIX protein were measured in mouse blood samples taken at the time of euthanization. RNA extracted from livers from vector-treated mice demonstrated 80,000–400,000 fold increased hFIX mRNA over negative (PBS-treated) controls. However, all mice were negative for capsid mRNA measured by Q-RTPCR using each of the four capsid primer / probe sets at all time points. Combined, the results support that AAV2 capsid DNA present as a trace impurity in purified recombinant AAV2 vectors (Nony et al, 2003, J Virol), is not transcribed. Therefore, MHC Class I presentation of AAV2 capsid-derived peptides via the endogenous pathway is unlikely. Rather, processing and presentation of the pre-formed capsid component of the gene transfer vector, through less efficient alternative pathways, is the likely source of MHC Class I - associated AAV capsid peptides on the surface of transduced human hepatocytes. Therapeutic strategies that prevent CTL recognition of transduced cells following clinical gene therapy vector administration until clearance of pre-formed capsid protein occurs (e.g. through intracellular metabolic pathways) are predicted to increase the duration of therapeutic gene expression.