Using the emerging Collaborative Cross to probe the immune system.

The Collaborative Cross (CC) is an emerging panel of recombinant inbred (RI) mouse strains. Each strain is genetically distinct but all descended from the same eight inbred founders. In 66 strains from incipient lines of the CC (pre-CC), as well as the 8 CC founders and some of their F1 offspring, we examined subsets of lymphocytes and antigen-presenting cells. We found significant variation among the founders, with even greater diversity in the pre-CC. Genome-wide association using inferred haplotypes detected highly significant loci controlling B-to-T cell ratio, CD8 T-cell numbers, CD11c and CD23 expression. Comparison of overall strain effects in the CC founders with strain effects at QTL in the pre-CC revealed sharp contrasts in the genetic architecture of two traits with significant loci: variation in CD23 can be explained largely by additive genetics at one locus, whereas variation in B-to-T ratio has a more complex etiology. For CD23, we found a strong QTL whose confidence interval contained the CD23 structural gene Fcer2a. Our data on the pre-CC demonstrate the utility of the CC for studying immunophenotypes and the value of integrating founder, CC and F1 data. The extreme immunophenotypes observed could have pleiotropic effects in other CC experiments.

Allelic diversity at the DLA-88 locus in Golden Retriever and Boxer breeds is limited.

In the dog, previous analyses of major histocompatibility complex class I genes suggest a single polymorphic locus, dog leukocyte antigen (DLA)-88. While 51 alleles have been reported, estimates of prevalence have not been made. We hypothesized that, within a breed, DLA-88 diversity would be restricted, and one or more dominant alleles could be identified. Accordingly, we determined allele usage in 47 Golden Retrievers and 39 Boxers. In each population, 10 alleles were found; 4 were shared. Seven novel alleles were identified. DLA-88*05101 and *50801 predominated in Golden Retrievers, while most Boxers carried *03401. In these breeds, DLA-88 polymorphisms are limited and largely non-overlapping. The finding of highly prevalent alleles fulfills an important prerequisite for studying canine CD8+ T-cell responses.

Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice.

Immune responses leading to antibody-mediated elimination of the transgenic protein are a concern in gene replacement for congenital protein deficiencies, for which hemophilia is an important model. Although most hemophilia B patients have circulating non-functional but immunologically crossreactive factor IX (FIX) protein (CRM+ phenotype), inciting factors for FIX neutralizing antibody (inhibitor) development have been studied in crossreactive material-negative (CRM-) animal models. For this study, determinants of FIX inhibitor development were compared in hemophilia B mice, in which circulating FIX protein is absent (CRM- factor IX knockout (FIXKO) model) or present (CRM+ missense R333Q-hFIX model) modeling multiple potential therapies. The investigations compare for the first time different serotypes of adeno-associated virus (AAV) vectors (AAV2 and AAV1), each at multiple doses, in the setting of two different FIX mutations. The comparisons demonstrate in the FIXKO background (CRM- phenotype) that neither vector serotype nor vector particle number independently determine the inhibitor trigger, which is influenced primarily by the level and kinetics of transgene expression. In the CRM+ missense background, inhibitor development was never stimulated by AAV gene therapy or protein therapy, despite the persistence of lymphocytes capable of responding to FIX with non-inhibitory antibodies. This genotype/phenotype is strongly protective against antibody formation in response to FIX therapy.

Identification of T-cell epitopes in clotting factor IX and lack of tolerance in inbred mice.

Immune responses to the factor IX protein pose problems for hemophilia B patients who develop antibodies against factor IX and for potential future treatment with gene therapy. To better define the response to human factor IX, we analyzed T-cell responses to human factor IX in factor IX knockout mice on BALB/c and C57BL/6 (B6) backgrounds, both strains having CD4+ T cells that proliferate in response to human factor IX. Surprisingly, wild-type mice have similar factor IX-recognizing CD4+ T cells. We defined a dominant CD4+ epitope for each strain (CVETGVKITVVAGEH for BALB/c and LLELDEPLVLNSYVTPIC for B6) that was recognized by both factor IX knockout and wild-type mice. While human factor IX did not cross-react with the mouse homologs of these epitopes, immunization with peptides from murine factor IX stimulated proliferation in factor IX knockout mice and wild-type mice, demonstrating a failure to delete murine factor IX-specific T cells in normal mice.

Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts.

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP(+) vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-gamma). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.

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.

Intracellular cytokine staining for TGF-beta.

TGF-beta is a well-described immunoregulatory molecule that is produced by most cell types. Many studies have been aimed at investigating the role played by TGF-beta in different cell types and situations. Most methods of measuring TGF-beta have previously relied on enzyme-linked immunosorbent assay (ELISA) or assays of its anti-proliferative effects on various cell lines. Both assays, though useful, cannot be used to effectively identify the cells that are producing TGF-beta in a mixture of cells. It is especially important to know the source and dynamics of TGF-beta secretion in cell culture studies since most cell types are known to be capable of producing TGF-beta. We describe here a technique of qualitative and quantitative measurement of TGF-beta production using flow cytometry. Previous work by others has led to the production of polyclonal and monoclonal antibodies to human and other mammalian TGF-beta. We have developed an intracellular cytokine staining for human TGF-beta using a monoclonal antibody, TB21.

Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers.

Soluble MHC/peptide tetramers that can directly bind the TCR allow the direct visualization and quantitation of Ag-specific T cells in vitro and in vivo. We used HY-D(b) tetramers to assess the numbers of HY-reactive CD8+ T cells in HYTCR-transgenic mice and in naive, wild-type C57BL/6 (B6) mice. As expected, tetramer staining showed the majority of T cells were male-specific CD8+ T cells in female HY-TCR mice. Staining of B6 mice showed a small population of male-specific CD8+ T cells in female mice. The effect of administration of soluble MHC class I tetramers on CD8+ T cell activation in vivo was unknown. Injection of HY-D(b) tetramer in vivo effectively primed female mice for a more rapid proliferative response to both HY peptide and male splenocytes. Furthermore, wild-type B6 female mice injected with a single dose of HY-D(b) tetramer rejected B6 male skin grafts more rapidly than female littermates treated with irrelevant tetramer. In contrast, multiple doses of HY-D(b) tetramer did not further decrease graft survival. Rather, female B6 mice injected with multiple doses of HY-D(b) tetramer rejected male skin grafts more slowly than mice primed with a single injection of tetramer or irradiated male spleen cells, suggesting clonal exhaustion or anergy. Our data highlight the ability of soluble MHC tetramers to identify scarce alloreactive T cell populations and the use of such tetramers to directly modulate an Ag-specific T cell response in vivo.

CD8(+) T cells express a T-helper 1--like phenotype after burn injury.

BACKGROUND: Previous studies suggest that CD8(+) T cells are immunosuppressive after burn injury, but recent reports indicate that CD8(+) T cells have several functions similar to CD4(+) T cells, including the secretion of cytokines. This study uses HY male antigen in transgenic HY female mice to determine the antigen-specific response of activated CD8(+) T cells after burn injury. METHODS: HY TCR transgenic female mice underwent burn or sham injury. Seventy-two hours after the burn, splenocytes were stimulated with 20 micromol/L HY peptide for 16, 48, and 64 hours; cellular proliferation, intracellular interferon-gamma and interleukin-2, and apoptosis were measured. RESULTS: Burn injury significantly impaired proliferation to HY antigen (P < or =.05). Activated CD8(+) T cells from burned mice showed increased intracellular interferon-gamma and interleukin-2 16 hours after stimulation compared with sham (P < or =.05) and at no time was less than control mice. The percent of CD8(+) T cells decreased with the time of stimulation but was not due to apoptosis by Annexin V staining. CONCLUSIONS: Activated CD8(+) T cells express a T(h1)-like phenotype after burn injury. This provides evidence that CD8(+) T cells are not simply suppressive and that is consistent with data that CD4(+) T cells are primed for a T(h1) response after burn injury.

Dendritic cells transduced with HIV Nef express normal levels of HLA-A and HLA-B class I molecules.

HIV Nef protein is important for viral pathogenesis and disease progression. Nef downregulates CD4 and major histocompatibility antigens on the surface of HIV-infected T cells. HIV also infects dendritic cells. We wanted to determine if Nef had a similar function in professional antigen-presenting cells, where downregulation of Class I could have important effects on the initiation of HIV specific cytolytic T cell responses. We infected human dendritic cells with adenovirus expressing Nef. In contrast to T cells and Hela cells, HLA-A and HLA-B molecules are not downregulated nor are other class I molecules increased. We show that, in dendritic cells, HIV Nef has little effect on CD4 or Class I expression.

Multiple paths for activation of naive CD8+ T cells: CD4-independent help.

CD8(+) CTLs play a pivotal role in immune responses against many viruses and tumors. Two models have been proposed. The "three-cell" model focuses on the role of CD4(+) T cells, proposing that help is only provided to CTLs by CD4(+) T cells that recognize Ag on the same APC. The sequential "two-cell" model proposes that CD4(+) T cells can first interact with APCs, which in turn activate naive CTLs. Although these models provide a general framework for the role of CD4(+) T cells in mediating help for CTLs, a number of issues are unresolved. We have investigated the induction of CTL responses using dendritic cells (DCs) to immunize mice against defined peptide Ags. We find that help is required for activation of naive CTLs when DCs are used as APCs, regardless of the origin or MHC class I restriction of the peptides we studied in this system. However, CD8(+) T cells can provide self-help if they are present at a sufficiently high precursor frequency. The important variable is the total number of T cells responding, because class II-knockout DCs pulsed with two noncompeting peptides are effective in priming.

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

T cell activity after dendritic cell vaccination is dependent on both the type of antigen and the mode of delivery.

Previous work in both human and animal models has shown that CTL responses can be generated against proteins derived from tumors using either peptide-pulsed dendritic cells (DCs) or nucleic acids from the tumor transfected into autologous DCs. Despite the efficacy of this approach for vaccine therapy, many questions remain regarding whether the route of administration, the frequency of administration, or the type of Ag is critical to generating T cell responses to these Ags. We have investigated methods to enhance CTL responses to a peptide derived from the human proto-oncogene HER-2/neu using mice containing a chimeric HLA A2 and H2Kb allele. Changes in amino acids in the anchor positions of the peptide enhanced the binding of the peptide to HLA-A2 in vitro, but did not enhance the immunogenicity of the peptide in vivo. In contrast, when autologous DCs presented peptides, significant CTL activity was induced with the altered, but not the wild-type, peptide. We found that the route of administration affected the anatomic site and the time to onset of CTL activity, but did not impact on the magnitude of the response. To our surprise, we observed that weekly administration of peptide-pulsed DCs led to diminishing CTL activity after 6 wk of treatment. This was not found in animals injected with DCs every 3 wk for six treatments or in animals initially given DCs weekly and then injected weekly with peptide-pulsed C1R-A2 transfectants.

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells.

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.

Hemophilia factor VIII therapy. Immunological tolerance. A clinical perspective.

Human alloantibodies to factor VIII occur in 15-50% of patients with severe hemophilia A. The development of these inhibitory alloantibodies prevents treatment with replacement factor VIII and places the patient at higher risk for complications from bleeding. Immune tolerance to factor VIII can be achieved in patients with inhibitors through the regular administration of factor VIII. The mechanism by which immune tolerance is achieved is unclear but the regular administration of factor VIII is believed to interrupt the normal immune mechanisms, perhaps by altering antigen presentation and inducing T or B cell anergy. The development of immune tolerance in hemophilia can be seen as a potential model for the development of methods for inducing tolerance to other alloimmune and autoimmune disorders.

Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles.

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.

The structural basis for the increased immunogenicity of two HIV-reverse transcriptase peptide variant/class I major histocompatibility complexes.

Designing altered peptide ligands to generate specific immunological reactivity when bound to class I major histocompatibility complexes is important for both therapeutic and prophylactic reasons. We have previously shown that two altered peptides, derived from human immunodeficiency virus (HIV)-reverse transcriptase (RT) residues 309-317, are more immunogenic in vitro than the wild-type peptide. One peptide variant, I1Y, was able to stimulate RT-specific cytotoxic T cells from the blood of three HIV-infected individuals better than the wild-type RT peptide. Both I1Y and I1F peptide variants increase the cell surface half-life of the peptide-class I complex approximately 3-fold over that of the RT peptide but have different immunological activities. These peptides are candidates for the design of vaccines for HIV due to their increased immunogenicity. To understand the basis for the increased cell surface stability compared with wild-type peptide and to understand the differences in T cell recognition between I1Y and I1F, we determined the x-ray crystal structures of the two class I MHC-peptide complexes. These structures indicate that the increased cell surface half-life is due to pi-pi stacking interactions between Trp-167 of HLA-A2.1 and the aromatic P1 residues of I1F and I1Y. Comparison of the structures and modeling potential T cell receptor (TCR) interactions suggests that T cell interactions and immunogenicity are different between I1Y and I1F for two reasons. First, subtle changes in the steric and polar properties of the I1Y peptide affect TCR engagement. Second, water-mediated hydrogen bond interactions between the P1-Tyr and the P4-Glu peptide residues increase peptide side chain rigidity of residues critical for TCR engagement.

Analysis of the mutant HLA-A*0201 heavy chain H74L: impaired TAP-dependent peptide loading.

A mutation of the HLA-A*0201 heavy chain at position 74 from histidine to leucine (H74L) resulted in a molecule with an interesting phenotype. H74L-expressing targets were recognized by peptide-specific HLA-A*0201-restricted cytotoxic T lymphocytes at lower peptide concentrations than wild type HLA-A*0201. H74L's improved ability to sensitize cells for tysis was due to its enhanced capability to bind exogenous peptide. Furthermore, this phenotype of improved exogenous binding and functional recognition was not peptide-specific. In contrast, the H74L molecule failed to present the HIV- HLA-A2-restricted pol peptide when expressed and processed endogenously. The inability to bind endogenous pol could be rescued by preceding the pol peptide with a signal sequence. The defect affecting endogenous presentation, therefore, appeared to be limited to the TAP-dependent pathway. Surprisingly, the H74L heavy chain was able to enter the defined MHC class I pathway and associate with beta2M, calreticulin, tapasin, and TAP. Despite the presence of the H74L heavy chain at the TAP complex, H74L was functionally inefficient at loading TAP-dependent peptides. H74L may help elucidate further steps in the process of loading TAP-dependent peptides into the class I cleft.

Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1-infected long-term survivors.

HIV-1-specific cytotoxic T cell (CTL) activity has been suggested to correlate with protection from progression to AIDS. We have examined the relationship between HIV-specific CTL activity and maintenance of peripheral blood CD4+ T lymphocyte counts and control of viral load in 17 long-term survivors (LTSs) of HIV-1 infection. Longitudinal analysis indicated that the LTS cohort demonstrated a decreased rate of CD4+ T cell loss (18 cells/mm3/year) compared with typical normal progressors (approximately 60 cells/mm3/year). The majority of the LTSs had detectable, variable, and in some individuals, quite high (>10(4) RNA copies/ml) plasma viral load during the study period. In a cross-sectional analysis, HIV-specific CTL activity to HIV Gag, Pol, and Env proteins was detectable in all 17 LTSs. Simultaneous analysis of HIV-1 Gag-Pol, and Env-specific CTLs and virus load in protease inhibitor-naive individuals showed a significant inverse correlation between Pol-specific CTL activity and plasma HIV-1 RNA levels (p = 0.001). Furthermore, using a mixed linear effects model the combined effects of HIV-1 Pol- and Env-specific CTL activity on the viral load were significantly stronger than the effects of HIV-1 Pol-specific CTL activity alone on predicted virus load. These data suggest that the presence of HIV-1-specific CTL activity in HIV-1-infected long-term survivors is an important component in the effective control of HIV-1 replication.

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy.

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.