Antibody-mediated rejection of single class I MHC-disparate cardiac allografts.

Murine CCR5(-/-) recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2(b)) and B6.CCR5(-/-) recipients. Donor-specific antibody titers in CCR5(-/-) recipients were 30-fold higher than in wild-type recipients. B6.K(d) allografts survived longer than 60 days in wild-type recipients whereas CCR5(-/-) recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K(d) allografts were rejected by CD8(-/-)/CCR5(-/-), but not µMT(-/-)/CCR5(-/-), recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5(-/-) and CD8(-/-)/CCR5(-/-) recipients and from RAG-1(-/-) allograft recipients injected with anti-K(d) antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity.

The potency of allospecific Tregs cells appears to correlate with T cell receptor functional avidity.

CD4(+) CD25(+) regulatory T cells (T(reg) cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T-cell receptor (TcR) activation is critical for T(reg) function, suggesting that the TcR avidity of T(reg) cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T(reg) lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T(reg) lines generated from CD4(+)CD25(+) T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T(reg) lines was confirmed in vitro. T(reg) lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T(reg) lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T(reg) cells, together with anti-CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T(reg) lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T(reg) function. It highlights the fact that strategies to select T(reg) with higher functional avidity might be beneficial for immunotherapy in transplantation.

Preferential priming of alloreactive T cells with indirect reactivity.

The relative contributions of the direct and indirect pathways in alloimmune responses have not been fully elucidated. We report a novel murine TCR transgenic system that can simultaneously track the CD4-direct (CD4-d), CD4-indirect (CD4-i) and CD8-direct (CD8-d) pathways after transplantation. Using this system, we have observed a profoundly greater proliferation of CD4-i T cells relative to CD4-d and CD8-d T cells after transplantation. Furthermore, a much larger proportion of CD4-i T cells attain an effector phenotype. We also analyzed endogenous, wild-type T cells using enzyme-linked immunospot analysis. In naïve mice, T cells with indirect reactivity were undetectable, but T cells with direct reactivity were abundant. However, 10 days after skin or heterotopic heart transplantation, CD4-i T cells comprised approximately 10% of the CD4+ response. Consistent with increased priming of the CD4-i pathway, we observed that the CD4-i T cells were further enriched in the effector cells migrating to the allograft and in memory-like T cells persisting after rejection. Thus, priming of the CD4-i pathway is favored after transplantation, allowing a rare population to rapidly become a major component of the CD4+ T-cell response in acute allograft rejection. The generalizability of this observation to other models remains to be determined.

Immune function in patients with rheumatoid arthritis treated with etanercept.

Etanercept, a recombinant human tumor necrosis factor (TNF) inhibitor that binds both soluble and cell-bound TNF, has been shown to reduce disease activity and inhibit joint destruction when administered to patients with rheumatoid arthritis (RA). Because TNF receptors are found on many types of cells that modulate the immune response, we evaluated the general immune function of a subset of RA patients in a blinded clinical study. No significant differences were seen between patients treated with etanercept or placebo in the surface antigen phenotypes of peripheral blood leukocytes, T cell proliferative responses, neutrophil function, delayed-type hypersensitivity (DTH) reactions, serum immunoglobulin levels, or incidence of infections. Although this observational study was relatively small and could detect only major changes in immunological status, the stability of immune function over time in patients receiving etanercept corroborates the findings in clinical studies, which suggest that etanercept does not alter overall global immune function.

Perspectives on inducing efficient immune control of HIV-1 replication--a new goal for HIV therapeutics?

OBJECTIVES: A goal for long-term therapy of HIV infection is immune control of virus replication rather than the somewhat unrealistic aim of complete viral elimination. This paper will review the evidence that the control of viral infection can be achieved by an active CD8+ T-cell-mediated response. DESIGN: This review will draw on both experimental and clinical sources to discuss the potential mechanisms of the immune control. RESULTS: Data indicate that HIV infection can be effectively controlled by HIV-specific CD8+ T-cell-mediated responses. In infected individuals, the development of active cytotoxic T lymphocytes (CTLs, as measured by lytic activity) is associated with the control of viral replication. Within the simian immunodeficiency virus infection model in rhesus macaques, strong CTL responses are similarly associated with effective viral control. In addition, depletion by antibodies of CD8+ T cells within infected macaques results in rapid increases in viral load. However, in most HIV-infected individuals, the CD8+ T-cells response is inefficient at low antigen dose, probably due to the lack of an effective H V-specific CD4+ T-cell response. If this CD4+ T-cell response is lost due to viral induced anergy, rather than clonal deletion, such responses may be generated by interruptions in antiretroviral treatment, and/or therapeutic immunization in chronically infected patients. A strong immune response stimulated at low-antigen dose early during viral rebound may be critical in preventing accumulation of toxic viral products that might inhibit effective CD4+ T-cell responses. CONCLUSION: Immune control of HIV infection is a realistic goal. Understanding both the basic immune mechanisms of in vivo viral replication and identifying practical therapeutic regimens to activate HIV CD4+ and CD8+ T-cell responses may allow the development of efficient immune control of HIV replication in chronically infected patients.

Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe.

Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC--PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays. As few as ten copies of vDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR.

Heterogeneity of T cell clones specific for a single indirect alloantigenic epitope (I-Ab/H-2Kd54-68) that mediate transplant rejection.

BACKGROUND: One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells. To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2Kd molecule, recognized by H-2b CD4 T cells in the context of syngeneic I-Ab. METHODS: To identify a stimulatory peptide derived from the H-2Kd molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR Vbeta usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. RESULTS: The H-2Kd54-68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2b) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-gamma production by peptide stimulation. Furthermore, direct immunization with synthetic H-2Kd54-68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2d) mice, but not 3rd party B10.BR (H-2k) grafts. A panel of 15 distinct CD4+ clones specific for H-2Kd54-68 peptide were established and shown to utilize a variety of TCR Vbeta and different apparent TCR avidities to H-2Kd54-68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2Kd54-68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H.2b and I-Abeta chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. CONCLUSIONS: These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.

Recurrence of the acute HIV syndrome after interruption of antiretroviral therapy in a patient with chronic HIV infection: A case report.

BACKGROUND: Clinical and virologic consequences of temporary interruption of HIV therapy are incompletely understood. OBJECTIVE: To describe a febrile illness that was consistent with the acute HIV syndrome and occurred after interruption of antiretroviral therapy. DESIGN: Case report. SETTING: University clinic. PATIENT: HIV-infected man. MEASUREMENTS: Plasma viral load, lymphocyte subsets, diagnostic evaluation (including cultures and serologic tests), and analysis of lymph node tissue. RESULTS: The patient began antiretroviral therapy 3 months after initial HIV exposure and had sustained viral suppression, except during a brief scheduled treatment interruption. One hundred sixty-nine days after resuming therapy, the patient discontinued it again immediately following an influenza vaccination. Eleven days later, he presented with a febrile mononucleosis-like syndrome associated with dramatic shifts in plasma HIV RNA level (<50 to >1 000 000 copies/mL) and CD4 cell count (0.743 x 10(9) cells/L to 0.086 x 10(9) cells/L). Evaluation for alternative causes of fever was unrevealing. Symptoms resolved rapidly with resumption of HIV therapy. CONCLUSION: Therapeutic interruption may be associated with profound viral rebound and recurrence of the acute HIV syndrome.

Stability in the HIV vDNA pool in peripheral CD4+ T cells of untreated patients by single tube quantitative PCR.

HIV infection leads to loss of CD4 T cells and development of AIDS in most individuals without treatment. While disease progression during HIV infection correlates with the plasma viral load, much less is known about the levels of HIV vDNA. This paper describes the development and validation of a sensitive, quantitative PCR assay for the assessment of HIV vDNA. The system uses novel single tube, multiply competitive PCR technology, which allows five-point competitor competition in a single PCR reaction. The reproducibility and performance characteristics of the assay are extensively studied, which indicate that the system performs well in high DNA backgrounds. Using this assay system on a cohort of protease naïve patients, HIV vDNA was assessed from PBMCs over an average follow-up period of 5 years. The data indicate that the HIV vDNA pool does not appreciably accumulate over the follow-up period, with many of the patients followed for up to 8 years. A reliable, quantitative assessment of vDNA pools will allow a better understanding of the dynamics of HIV pathogenesis.

Effect of highly active antiretroviral therapy and thymic transplantation on immunoreconstitution in HIV infection.

The purpose of this study was to determine whether thymic transplantation in addition to highly active antiretroviral therapy (HAART) will restore T cell function in HIV infection. Eight treatment-naive HIV-infected patients with CD4+ T cell counts of 200-500/mm3 were randomized into thymic transplantation and control arms. All patients received HAART (zidovudine, lamivudine, and ritonavir) for 6 weeks prior to transplantation. Thymic transplantation was done without immunosuppression, using postnatal HLA-unmatched cultured allogeneic thymus tissue. Patients were immunized every 6 months with the neoantigen keyhole limpet hemocyanin (KLH) and the recall antigen tetanus toxoid (TT). T cell phenotype and function and T cell receptor rearrangement excision circles (TRECs) were assessed. Thymic allografts were biopsied at 2 months. Six HIV-infected patients completed the study. Four patients received cultured allogeneic postnatal thymic grafts, two others were controls. CD4+ T cell counts increased and T cell-proliferative responses to Candida antigen and TT normalized in all patients. Proliferative responses to KLH developed in three of four transplant recipients and one of two controls. Patients responding to KLH after secondary immunization had greater TREC increases compared with the patients who did not respond. All thymic allografts were rejected within 2 months. In summary, four of six patients developed T cell-proliferative responses to the neoantigen KLH over the first 2 years of HAART. The transplanted thymus tissue, however, was rejected. There was no clear difference in restoration of T cell function in the transplant recipients compared with the controls. Increases in TRECs after initiation of HAART may correlate with improved immune function.

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

The Arthus reaction in rodents: species-specific requirement of complement.

We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.

Evaluation of distinct blood lymphocyte populations in human immunodeficiency virus type 1-infected subjects in the absence or presence of effective therapy.

Virus reservoirs can persist in human immunodeficiency virus type 1 (HIV-1)-infected subjects despite effective plasma virus suppression. To compare viral dynamics in the absence and presence of antiretroviral therapy, blood mononuclear cells from 19 subjects with high plasma RNA levels and 18 subjects following prolonged virus suppression were examined, by use of in situ hybridization, to detect virus RNA expression before and after in vitro T cell activation. This approach reveals circulating lymphocytes expressing HIV-1 RNA before activation and an increase in cells with detectable HIV-1 RNA transcription after in vitro activation. The frequencies of these 2 cell populations are strongly correlated with plasma virus load and appear to be stable once a new steady state is established during therapy. The frequency of viral RNA-positive cells is equivalent to the frequency of cells that produce infectious virus. Thus, in HIV-1-infected subjects there are distinct virus reservoirs comprising both latent and replication-active cells.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Correlation between circulating stromal cell-derived factor 1 levels and CD4+ cell count in human immunodeficiency virus type 1-infected individuals.

Stromal cell-derived factor 1 (SDF-1) is the natural ligand that recognizes CXCR4, which also serves as a coreceptor for some strains of HIV-1. In this study, we explored SDF-1 blood levels among HIV-1-infected individuals exhibiting a wide range of CD4+ cell counts. Plasma or serum concentrations of SDF-1 protein were measured by ELISA in samples from 31 HIV-1-seronegative individuals and 79 HIV-1-infected subjects. Although SDF-1 protein levels were stable for months among seronegative individuals (mean intrasubject variation, 17%), the absolute values varied widely (0.28 to 106.5 ng/ml; mean, 25.6 ng/ml). In HIV-1-infected subjects, there was a direct correlation between SDF-1 level and CD4+ cell count. Subjects with fewer than 50 CD4+ cells per cubic microliter of blood had significantly lower mean SDF-1 levels (+/-SD) than did either HIV-1-infected subjects with higher CD4+ cell counts or uninfected controls: CD4+ cell count <50, mean SDF-1 level of 10.7+/-33.7, 50 < CD4+ cell count <200, mean SDF-1 level of 12.9+/-19.0, 200 < CD4+ cell count <500, mean SDF-1 level of 19.3+/-36.8; CD4+ cell count >500, mean SDF-1 level of 18.5+/-25.2; uninfected control mean SDF-1 level, 25.6+/-34.7. No significant change in SDF-1 level was detected after administration of antiretroviral therapy in nine subjects with advanced disease (mean intrasubject variation, 43%). Analysis of SDF-1 mRNA expression in lymph nodes from HIV-1-infected subjects at different disease stages revealed that the medullary cords contained stromal cells that express SDF-1 mRNA. This preliminary analysis suggests a possible link between lower SDF-1 levels and disease progression.

The zinc finger protein GLI induces cellular sensitivity to the mTOR inhibitor rapamycin.

The protein synthetic machinery is activated by diverse genetic alterations during tumor progression in vivo and represents an attractive target for cancer therapy. We show that rapamycin inhibits the induction of transformed foci in vitro by GLI, a transcription factor that functions in the sonic hedgehog-patched pathway in tumors. In control cells, which were nontransformed epithelioid RK3E cells and derivative c-MYC- or RAS-transformed sister cell lines, rapamycin inhibits mTOR and mTOR-dependent activities but increases global protein synthesis, perhaps by activating a feedback mechanism. In GLI-transformed cells, rapamycin inhibits global protein synthesis and turnover and prevents cellular proliferation. In contrast to its effects on protein synthesis, rapamycin affects bromodeoxyuridine incorporation and cell cycle occupancy of GLI cells and control cells to a similar extent. Rare, variant GLI cells isolated by selection in rapamycin are also drug-resistant for protein metabolism and for cell cycle progression through G1. Our results indicate that sensitivity to rapamycin can be induced by a specific oncogene and that inhibition of global protein metabolism is linked to the rapamycin-sensitive phenotype.

Initial increase in blood CD4(+) lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues.

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA.

Quantitative analysis of the relationship between virus expression and disease outcome has been critical for understanding HIV-1 pathogenesis. Yet the amount of viral RNA contained within an HIV-expressing cell and the relationship between the number of virus-producing cells and plasma virus load has not been established or reflected in models of viral dynamics. We report here a novel strategy for the coordinated analysis of virus expression in lymph node specimens. The results obtained for patients with a broad range of plasma viral loads before and after antiretroviral therapy reveal a constant mean viral (v)RNA copy number (3.6 log10 copies) per infected cell, regardless of plasma virus load or treatment status. In addition, there was a significant but nonlinear direct correlation between the frequency of vRNA+ lymph node cells and plasma vRNA. As predicted from this relationship, residual cells expressing this same mean copy number are detectable (frequency <2/10(6) cells) in tissues of treated patients who have plasma vRNA levels below the current detectable threshold (<50 copies/ml). These data suggest that fully replication-active cells are responsible for sustaining viremia after initiation of potent antiretroviral therapy and that plasma virus titers correlate, albeit in a nonlinear fashion, with the number of virus-expressing cells in lymphoid tissue.

Heterogeneity in the clonal T cell response. Implications for models of T cell activation and cytokine phenotype development.

The T cell can be defined in the context of two properties--the recognition specificity of the T cell receptor (TCR) heterodimer and the functional response of the T cell after TCR stimulation. Once a particular TCR heterodimer is expressed and successfully selected during thymic development, the antigen specificity is fixed for all the clonal progeny of that cell. In contrast, the potential functional responses that may be generated in response to specific antigen in the postthymic environment are quite extensive. These range from programmed cell death to initiation of alternate programs of phenotype development that generate effector populations with distinct cytokine expression patterns and regulatory properties. Recent advances in analytical methods that have permitted multiparametric characterizations of the T cell response at the single cell, rather than population level, have necessitated a modified view of T cell activation and the clonal T cell response, and have generated new insights into the regulation of immunity. In this brief review, we highlight studies that have characterized heterogeneity of the CD4+ T cell clonal response based on single-cell analyses, and discuss implications for models of T cell activation and cytokine phenotype development.