Extensive diversity in the recognition of influenza virus hemagglutinin by murine T helper clones

A panel of H-2k class II-restricted Th clones were established from individual CBA mice primed by infection with X31 influenza virus. 27 clones, which showed specific recognition of the HA surface glycoprotein, were all H3N2 subtype specific, in contrast to a T cell line which was crossreactive and which may have other specificities. 20 distinct HA-specific clones recognized a tryptic cleavage fragment of X31 consisting of residue 28-328 of HA1 (tops) which includes all the Ab-combining regions of the HA molecule. Seven other HA-specific clones failed to respond to either tops or to aggregate (the remainder of the virus after tryptic cleavage of tops). The specificity of these clones has been mapped, tentatively, to a conformational determinant in the interface antibody-binding region of the HA trimer. Analysis of the fine specificity of the HA-specific clones against a panel of H3N2 natural variant viruses isolated from major virus epidemics from 1968 to 1984 revealed a hitherto unrecognized diversity in T cell recognition of a HA. A total of 12 specificity groupings were evident, and varied from groups of clones that recognized all natural variants to one clone that responded only to isolates from 1968 to 1972. Six out of eight clones from a major specificity group, which failed to recognize variants TX/77, BK/79, or CN/84, responded to two overlapping peptides (48-68 and 53-87), corresponding to a region of HA1 that includes part of two antibody combining sites. An examination of the amino acid sequences of natural variant viruses from this region of HA revealed that residues Asn53 and Asn54 and/or Ile62 were critical for recognition by these clones. We conclude that recognition of HA by Th cells is not restricted to a limited number of epitopes in the conserved regions of the molecule, but is extremely diverse and includes specificities that map to variable antibody-combining regions of the molecule. In addition, the sensitivity of the T cell clones to the amino acid substitutions occurring in HA1 of natural variant viruses suggests that Th may play a role in the immune pressure for antigenic variation in the HA molecule.     

Functional analysis of influenza-specific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin

We compared the effects of adoptively transferred Th cell clones specific for the influenza hemagglutinin (HA), matrix (M), or nucleoprotein (NP) on the antibody response of nude mice infected with A/PR/8/34 influenza virus. We show that the production of antibodies to the HA absolutely requires the presence of virus-specific Th cells. Further, transfer of a Th clone specific for the internal proteins, M or NP, was as effective as was transfer of an HA-specific clone in supporting an antibody response to the HA. With each of the clones, the kinetics of the response were accelerated by approximately 3 d compared with the antibody response of normal BALB/c mice. The HA- and M-specific clones supported an isotype switch from IgM to IgG and IgA similar to that which occurs during a normal antibody response. Finally, as shown by coinfection experiments, the response required a cognate T-B interaction whether the determinants recognized by the Th and B cell are located on the same viral protein or on different viral proteins within the same virus particle. The implications of these findings for understanding the T-B interactions that occur during an effective antiviral antibody response are discussed.     

Phosphatidyl choline is recognized by a series of Ly-1+ murine B cell lymphomas specific for erythrocyte membranes

Cells from 6 of 14 different Ly-1+ murine B cell lymphomas bound to synthetic liposomes encapsulating fluorescein. The liposomes were made from distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG), and cholesterol. In all cases, liposome binding was due to recognition of phosphatidyl choline by the surface IgM on the tumor cells. Liposome binding could be inhibited by DSPC but not by DSPG, and the number of liposomes bound per cell was directly related to the cell surface concentration of IgM. The IgM secreted by a hybridoma derived from one of the lymphomas, CH12, was shown to agglutinate liposomes, and was used in a solid-phase immunoassay to study inhibition of liposome binding by pure phospholipids; DSPC and sphingomyelin both inhibited, whereas DSPG did not. The Ig borne by the six lymphomas that bind phosphatidylcholine also bind to both SRBC and bromelain-treated mouse erythrocytes. The idiotypic of CH12 IgM is similar to that expressed by Ly-1+ normal splenic B cells of the same specificity. The significance of these data in relation to other commonly studied autoantigens, and to the restricted specificity of normal Ly-1+ B cells is discussed.     

Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.     

The cell surface molecule recognized by the erythrocyte receptor of T lymphocytes. Identification and partial characterization using a monoclonal antibody

A monoclonal antibody (mAb) to sheep red blood cells (SRBC), termed L180/1, is described that completely blocks rosette formation between SRBC and human or sheep T lymphocytes. L180/1 precipitated a minor glycoprotein of about approximately 42,000 mol wt from surface-labeled SRBC. This glycoprotein was partially affinity purified and found to block E rosette formation and to compete with anti-T11 mAb for the E receptor. The molecule detected by mAb L180/1 thus appears to be recognized by the E receptor and was given the preliminary name, T11 target structure (T11TS). Since the mAb to sheep T11TS blocks the binding of SRBC to both human and sheep T cells, and mAb to T11 blocks the binding of red cells from human and sheep to the human E receptor, we concluded that analogous receptor-ligand (T11-T11TS) systems exist in man and sheep that are crossreactive over the species barrier. The possibility is discussed that the E receptor, which is known to be involved in T cell activation, and T11TS function as complementary cell interaction molecules in T cell responses.     

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.     

Delayed-type hypersensitivity to influenza virus. Induction of antigen- specific suppressor T cells for delayed-type hypersensitivity to hemagglutinin during influenza virus infection in mice

Mice infected with A/England/939/69 X A/Puerto Rico/8/34 (Rec 31) influenza virus by aerosol develop significantly lower levels of delayed-type hypersensitivity (DTH) to A/Hong Kong/1/68 X A/Puerto Rico/8/34/ (X31) virus compared to uninfected mice. The suppression of DTH to the hemagglutinin appears to be mediated by suppressor T cells which carry Lyt-1 membrane antigen marker, and not by sy serum antibody. The suppressor T cells for DTH induced by Rec 31 virus (H3N1) infection suppress the DTH response to the variants of the H3 subtype of influenza viruses, but have no effect on the DTH responses to A/Puerto Rico/8/34 virus (H0N1), a B influenza virus or the matrix protein of type A influenza virus. Suppressor T cells for DTH appear 2 wk after infection and are detectable in the spleen for at least 40 d thereafter. T-helper cells for antibody response to hemagglutinin are induced concomitantly with the T-suppressor cells for DTH. Possible implications of the present findings on the regulation of the immune response to viral infection are discussed.     

Amino acid substitutions in the melanoma antigen recognized by T cell 1 peptide modulate cytokine responses in melanoma-specific T cells

Single amino acid substitutions in melanoma-associated peptides dramatically enhance T-cell cytotoxicity against target cells presenting the modified peptides (often referred to as heteroclitic peptides). The authors tried to determine whether peptide modifications influence other aspects of T-cell immunity toward malignant melanoma. A heteroclitic peptide, E26F, with an E to F substitution in melanoma antigen recognized by T cell 1 (MART-1)26-35, triggers an enhanced tyrosine phosphorylation response when compared with the native- and other-modified MART-1 peptides. Similarly, the E26F peptide enhances the production of mRNA for interleukin (IL)-5, IL-10, IL-13, IL-15, and interferon-gamma and significantly enhances release of IL-13 and IL-10 from anti-MART-1 cytotoxic T cells. Another heteroclitic peptide, 1L, with an A to L substitution in MART-1(27-35), also enhances the tyrosine phosphorylation response in anti-MART-1 cytotoxic CD8+ T cells. Yet, 1L does not enhance the production of T helper cell type 2-like cytokines (IL-10 and IL-13). Together these data show that minor amino acid modifications of immunodominant melanoma peptides profoundly influence the cytokine response in melanoma-specific T cells.     

The HLA-DRB1 locus as a genetic component in giant cell arteritis. Mapping of a disease-linked sequence motif to the antigen binding site of the HLA-DR molecule.

Giant cell arteritis (GCA) is a granulomatous vasculitis affecting persons over 50 years of age. The inflammatory infiltrate, which is targeted at the aorta and its proximal branches, includes activated CD4+ helper T cells, histiocytes, and giant cells. To investigate whether the genetic polymorphism of the HLA-DRB1 genes contributes to the local accumulation of activated T cells, we have analyzed both HLA-DRB1 alleles in a cohort of 42 patients with biopsy-proven GCA. The majority of patients (60%) expressed the B1*0401 or B1*0404/8 variant of the HLA-DR4 haplotype, both of which also represent the major genetic factors underlying the disease association in RA. GCA patients negative for the disease-linked HLA-DR4 alleles were characterized by a nonrandom distribution of HLA-DRB1 alleles. Sequence comparison among the allelic products identified in the GCA cohort demonstrated heterogeneity for the sequence polymorphism of the third hypervariable region (HVR), but homology for the polymorphic residues within the HVR2 of the HLA-DRB1 gene. The GCA patients shared a sequence motif spanning amino acid positions 28-31 of the HLA-DR beta 1 chain. In the structural model for HLA-DR molecules, this sequence motif can be mapped to the antigen-binding site of the HLA complex, suggesting a crucial role of antigen selection and presentation in GCA. In contrast, the sequence polymorphism linked to RA has been mapped to the HVR3 of the HLA-DRB1 gene and translates into a distinct domain of the HLA-DR molecule, the alpha-helical loop surrounding the antigen-binding groove. A consecutive case series study demonstrated that GCA and RA rarely co-occurred, supporting the interpretation that distinct functional domains of the HLA-DR molecule are implicated in the pathomechanisms of these two autoimmune diseases.     

Alteration at a single amino acid residue in the T cell receptor alpha chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper cell type 1 (Th1) to Th2

To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.     

Cure of progressive murine leishmaniasis: interleukin 4 dominance is abolished by transient CD4(+) T cell depletion and T helper cell type 1-selective cytokine therapy.

Progressive infection with Leishmania major in susceptible BALB/c mice is mediated by interleukin (IL)-4-producing T helper cell type 2 (Th2) CD4(+) T cells that, once established, become resistant to Th1-deviating therapies with recombinant (r)IL-12 and/or neutralizing anti-IL-4 antibodies. We sought to restore protective immunity in advanced leishmaniasis by depletion of Th2-biased CD4(+) populations and by cytokine-directed reconstitution of Th1 cellular responses during lymphocyte recovery. Treatment with cytolytic GK1.5 anti-CD4 mAb alone did not reverse disease in 3 wk-infected BALB/c mice, but GK1.5 combined with anti-IL-4 antibody and intralesional rIL-12 cured cutaneous lesions in 80% of mice and established a Th1-polarized cytokine response to L. major antigen protective against reinfection. The curative effects of GK1.5 were not replaced by cytotoxic anti-CD8 monoclonal antibody 2.43 or nondepleting anti-CD4 mAb YTS177, confirming that depletion of CD4(+) cells was specific and essential for therapeutic effect. Finally, combined CD4(+) depletion and IL-4 neutralization were curative, indicating that neither increased parasite burden nor altered accessory cell function independently biased towards Th2 reconstitution in advanced leishmaniasis. Advanced leishmaniasis can be cured by T cell depletion and cytokine-directed recovery of Th1 cellular responses, suggesting novel interventions for other immune-mediated diseases and identifying distinct roles for CD4(+) T cell and non-T cell in the maintenance of Th2 and Th1 phenotypes.     

T cell target 1 (TCT.1): a novel target molecule for human non-major histocompatibility complex-restricted T lymphocytes

We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.     

Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis.

Antigen-specific T helper type 1 (Th1) cells mediate protective immunity against a range of infectious diseases, including that caused by Bordetella pertussis. Distinct T cell subtypes that secrete interleukin (IL)-10 or tumor growth factor (TGF)-beta are considered to play a role in the maintenance of self-tolerance. However, the antigens recognized by these regulatory T cells in vivo have not been defined. Here we provide the first demonstration of pathogen-specific T regulatory type 1 (Tr1) cells at the clonal level and demonstrate that these cells are induced at a mucosal surface during an infection where local Th1 responses are suppressed. Tr1 clones specific for filamentous hemagglutinin (FHA) and pertactin were generated from the lungs of mice during acute infection with B. pertussis. The Tr1 clones expressed T1/ST2 and CC chemokine receptor 5, secreted high levels of IL-10, but not IL-4 or interferon (IFN)-gamma, and suppressed Th1 responses against B. pertussis or an unrelated pathogen. Furthermore, FHA inhibited IL-12 and stimulated IL-10 production by dendritic cells (DCs), and these DCs directed naive T cells into the regulatory subtype. The induction of Tr1 cells after interaction of a pathogen-derived molecule with cells of the innate immune system represents a novel strategy exploited by an infectious pathogen to subvert protective immune responses in vivo.     

Presentation of viral antigen to class I major histocompatibility complex-restricted cytotoxic T lymphocyte. Recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product

Class I major histocompatibility complex (MHC) restricted T lymphocytes preferentially recognize fragments of polypeptides processed through a nonendosomal presentation pathway. At present the intracellular compartment(s) in which polypeptide fragmentation occurs and factors which influence the formation of an antigenic epitope are not well understood. To assess the role of residues flanking an antigenic site in the generation of the antigenic moiety recognized by class I MHC restricted T lymphocytes we have moved the coding sequence for an immunodominant H-2Kd restricted site on the influenza A/JAPAN/57 hemagglutinin (residues 202-221) by site-directed mutagenesis to six different positions along the coding sequence of the hemagglutinin gene. We have found that all six classes of mutants are recognized by MHC class I restricted T cells as efficiently as the wild type hemagglutinin gene product. Thus neither N-terminal to C-terminal position within the translation product nor sequences flanking the antigenic site influence processing.     

Ethnic differences in the lymphocyte proliferative response induced by a murine IgG1 antibody, Leu-4, to the T3 molecule

The mitogenic effects of isotypically diverse antibodies to the T3 molecule were examined in genetically diverse population groups. Whereas the OKT3 antibody (IgG2a) was mitogenic for blood mononuclear cells from all individuals tested, the 38.1 antibody (IgM) was consistently nonmitogenic. In contrast, studies of the mitogenic effects of the Leu-4 antibody (IgG1) revealed striking ethnic differences. More than 80% of Caucasians and Negroes were good Leu-4 responders, whereas most individuals of Asian origin, including Indian, Japanese, and Chinese, were either Leu-4 nonresponders or Leu-4 low responders. However, the majority of American Indians, as well as a significant minority of Chinese, were good responders. Cell separation studies confirmed that monocytes govern the different mitogenic effects of the anti-T3 antibodies. The results reveal interesting ethnic differences in monocyte accessory function probably mediated via the Fc-gamma receptor, in the stimulation of T lymphocytes by an IgG1 antibody against the T3 molecule.     

Evidence for the failure of IgA specific T helper activity in a patient with immunodeficiency with hyper IgM.

A 28-year-old man with immunodeficiency with hyper IgM was studied. His serum immunoglobulins were characterized by the absence of IgA and low level of IgG associated with high level of IgM. The in vitro pokeweed mitogen (PWM)-induced immunoglobulin synthesis by his peripheral blood lymphocytes was depressed completely for IgA and moderately for IgG although normal numbers and proportions of IgA- and IgG-bearing lymphocytes (on the surface) were demonstrated in the peripheral blood. His T cells could not help IgA production by either normal or his own B cells, whereas they were more efficient helpers for IgM production by his own B cells than were normal T cells. In addition, his B cells produced no IgA and less IgG than normal B cells when co-cultured with normal T cells. This suggests that the failure of IgA specific T helper activity and the maturation arrest of B cells at the stage of the switchover from IgM- to IgG- and/or IgA-producing cells may be the major cause for the hypogammaglobulinaemia in this patient. It is uncertain whether the maturation arrest of B cells is secondary to the T cell defect in helper function for IgA production.     

Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells

Two antisera and a monoclonal antibody raised in BALB.K mice against cloned, major histocompatibility complex (MHC)-restricted, antigen-specific helper T cell lines are described. These antibodies are specific for individual cloned T cell lines and are potent inducers of T cell proliferation. The induction of T cell proliferation by these antibodies requires the presence of an adherent accessory cell. There is no H-2 restriction between this accessory cell and the cloned T cell, nor is this antibody-induced proliferation blocked by a monoclonal anti-Fc receptor antibody. The requirement for an accessory cell, however, is eliminated in the presence of an IL-1- or IL-2-rich supernatant. Thus this system allows the analysis of helper T cell activation with only a single cell type present. Anti-T cell sera also induce T cell-dependent B cell proliferation and immunoglobulin secretion. The induction of T cell-dependent B cell activation by these sera does not require H-2-matched T cells and B cells. The specificity of these antibodies and their ability to stimulate cloned helper T cells in the absence of antigen and antigen-presenting cells strongly suggest that these antibodies are directed against antigen and/or Ia recognition sites on the T cell.     

Influenza A (H1N1) virus resistance to cyanovirin-N arises naturally during adaptation to mice and by passage in cell culture in the presence of the inhibitor

Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR virus), as did virus passaged in cell culture in the presence of CVN (CVN-R virus). The CVN-R virus possessed a single amino acid change at position 94a (Asn94aAsp) of HA1 that eliminated this glycosylation site. The CVN-MR virus at mouse passage 7 was a mixture of clones, consisting of a single mutation (Asp225Gly) and double mutations (Asn63Ser+Asp225Gly or Asn94a+Asp225Gly), eliminating glycosylation sites. CVN did not bind well to the CVN-R and CVN-MR viruses. Propagating these viruses in cells treated with 1 mM deoxymannojirimycin (dMJ, mannosidase inhibitor) increased sensitivity to CVN, suggesting that glycans attached at other sites on HA1 that typically are not high-mannosidic became so due to dMJ treatment. Further evaluation showed that the Asp225Gly mutant virus was sensitive to the inhibitor and did not kill mice or induce weight loss. The CVN-R virus was also avirulent to mice. The double-mutant CVN-MR viruses were resistant to CVN and caused deaths and severe weight loss in mice. CVN-R virus subjected to mouse adaptation acquired the 225 mutation and a lethal phenotype. Thus, the 225 mutation in the HA receptor-binding site in combination with a loss of glycan at Asn (63 or 94a) are important for mouse adaptation in this virus. The mutations reported here causing resistance to CVN are consistent with its known mode of action.     

Correction of a rat model of Parkinson's disease by coexpression of tyrosine hydroxylase and aromatic amino acid decarboxylase from a helper virus-free herpes simplex virus type 1 vector

We previously reported long-term biochemical and behavioral correction of the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease (PD) by expression of tyrosine hydroxylase (TH) in the partially denervated striatum, using a herpes simplex virus type 1 (HSV-1) vector. This study had a number of limitations, including the use of a helper virus packaging system, limited long-term expression, and expression of only TH. To address these issues, we developed a helper virus-free packaging system, a modified neurofilament gene promoter that supports long-term expression in forebrain neurons, and a vector that coexpresses TH and aromatic amino acid decarboxylase (AADC). Coexpression of TH and AADC supported high-level (80%), behavioral correction of the 6-OHDA rat model of PD for 5 weeks. Biochemical correction included increases in extracellular dopamine and DOPAC concentrations between 2 and 4 months after gene transfer. Histologic analyses demonstrated neuronal-specific coexpression of TH and AADC at 4 days to 7 months after gene transfer, and cell counts revealed 1000 to 10,000 TH positive cells per rat at 2 months after gene transfer. This improved system efficiently corrects the rat model of PD.