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Background: Human immunodeficiency virus (HIV) is readily detectable in the lungs of infected subjects and leads to an accumulation of CD8(+) lymphocytes in the alveolar space. Although highly active antiretroviral therapy (HAART) is effective in reducing viremia, less is known about its effect on tissue compartments. The AIDS Clinical Trials Group Protocol 723 Team evaluated the effect of HAART on lung viral load and cellular constituents.Methods: Bronchoalveolar lavage (BAL) fluid and blood were collected before initiation of HAART and again at 4 and 24 weeks after initiation of therapy. The BAL cell differential was determined, lymphocyte phenotyping was performed, and acellular BAL fluid, plasma HIV RNA load, and BAL cell and peripheral blood mononuclear cell HIV RNA and DNA loads were measured.Results: HAART induced a rapid decrease in HIV that was detectable in acellular BAL fluid and a slower decrease in the HIV RNA and DNA loads in BAL cells. HAART was associated with a significant decrease in the absolute number and percentage of CD8(+) alveolar lymphocytes. There was a significant correlation between residual BAL cell DNA at 24 weeks and the absolute number of CD4(+) lymphocytes in the alveolar space.Conclusion: HAART is associated with a significant decrease in the pulmonary HIV burden and a return of alveolar cellular constituents to normal. [ABSTRACT FROM AUTHOR]

Strategies to limit life-long dependence on antiretroviral therapy (ART) are needed. We randomized 81 human immunodeficiency virus (HIV)-infected subjects to 4 interventional arms involving continued ART plus ALVAC vCP1452 (or placebo) with or without interleukin (IL)-2 infusions. Viral load rebound 12 weeks after ART interruption was then analyzed to assess immune control. Fifty-two subjects reached the study end point. ALVAC recipients had 0.5 log(10) lower virologic rebounds (P=.033). IL-2 plus vaccine boosted CD4(+) T cell counts (P<.001) but did not diminish viral rebound. Significant changes were not detected for HIV-specific lymphoproliferative responses in any arm. This exploratory protocol provides useful clinical data for future therapeutic immunization trial design. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Background. The ability to control human immunodeficiency virus (HIV) replication in vivo in the absence of antiretroviral therapy (ART) is a measure of the efficiency of antiviral immunity. In a study of patients with chronic, ART-suppressed HIV infection, AIDS Clinical Trials Group 5068 investigated the effects of immunization with an exogenous HIV vaccine and pulse exposure to the subject's unique viral epitopes, by means of structured treatment interruptions (STIs), on the dynamics of viral rebound during a subsequent analytical treatment interruption (ATI). Methods. Ninety-seven subjects receiving stable ART with an HIV-1 RNA load <50 copies/mL and CD4(+) T lymphocyte count >400 cells/mm(3) were randomized to undergo continued ART, STIs, ALVAC-HIV vCP1452 immunization, or STIs and ALVAC-HIV vCP1452 immunization. Results. Subjects in the 2 STI arms had a significantly longer median doubling time in the period of the initial rise of viral load, a significantly lower median peak viral load, a significantly lower median end-of-ATI viral load set point, and a greater proportion of subjects with an end-of-ATI viral load set point <1000 copies/mL, compared with the subjects in the 2 arms without STIs. With an immunization schedule of 3 sets of 3 weekly injections, ALVAC-HIV vCP1452 did not affect viral load measures. Conclusions. In this randomized, controlled study of intermittent STI as a therapeutic autoimmunization strategy, evidence of enhanced immunologic control of HIV replication was demonstrated. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4), a negative regulator of T-cell function, is increased in chronic HIV-1 infection. It was hypothesized that CTLA-4 blockade may enhance immune response to HIV-1 and result in better control of viremia. This open-label, multiple ascending dose study (NCT03407105)-the first to examine ipilimumab in participants with HIV-1 infection-assessed the safety, tolerability, and pharmacokinetics of ipilimumab, as well as whether ipilimumab enhanced immune response to HIV-1 and improved control of viremia. Twenty-four participants received 2 or 4 doses of ipilimumab (0.1, 1, 3, or 5 mg/kg) every 28 days. No serious adverse events (AEs) or dose-limiting toxicities were reported; one participant discontinued ipilimumab for an AE of grade 2 facial palsy. Twenty participants (83.3%) had ≥1 AE; all but 1 were grade 1 or 2. Eight participants (33.3%) had potentially immune-related AEs (7 had grade 1 diarrhea not requiring corticosteroids; 1 who had diarrhea also had transient antinuclear antibody positivity; 1 had grade 2 facial palsy requiring corticosteroids). Two participants (8.3%), one each in the 0.1- and 1-mg/kg dose groups, had a decrease from baseline HIV-1 RNA of 0.85 and 1.36 log10 copies/mL. Fourteen participants (58.3%) had an increase from baseline HIV-1 RNA (mean, 0.87 log10 copies/mL; range, 0.59-1.29). Of these 14 participants, all but 1 were in the higher ipilimumab dose groups (3 or 5 mg/kg). No pattern was noted regarding change from baseline in CD4 or CD8 T cells; ex vivo assessments of immune response were precluded because of inadequate cell viability. Serum concentration data for ipilimumab showed biphasic disposition, with steady state reached by dose 3. Ipilimumab treatment was well tolerated and was associated with variations in HIV-1 RNA in excess of expected repeat measures in most participants, but these were not related to combination antiretroviral therapy status or CD4 counts. The mechanism(s) underlying the increased variation in HIV-1 RNA is unclear and needs further study., Competing Interests: E.C. is a full-time employee and shareholder of Bristol-Myers Squibb. D. Grasela is a full-time employee and shareholder of Bristol-Myers Squibb. D. Gardiner is a full-time employee and shareholder of GlaxoSmithKline. R.P.B has no competing interests. B.V. is a full-time employee of Bristol-Myers Squibb. A.J.K. is a full-time employee of Bristol-Myers Squibb. I.L. is a stockholder of Bristol-Myers Squibb, which markets ipilimumab for cancer indications The authors confirm that the above disclosures do not alter our adherence to all PLOS ONE policies on sharing data and materials.

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigen-specific, TCR-transgenic for H2-A(b) /H2-K(d) 54-68 epitope (TCR75) CD4(+) T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4(+) T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4(+) T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4(+) T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4(+) T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4(+) T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4(+) T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4(+) T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(-) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(-) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcμR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcμR(-) mice compared with FcμR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcμR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcμR(-) and FcμR(+) B6/lpr mice. Collectively, these findings suggest that FcμR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background and Purpose: Improvement in axonal regeneration may lead to the development of longer nerve grafts and improved outcomes for patients with peripheral nerve injury. Although the use of acellular nerve grafts has been well documented (Groves et al, Exp Neurol. 2005;195:278-292; Krekoski et al, J Neurosci. 2001;21:6206-6213; Massey et al, Exp Neurol. 2008;209:426-445; Neubauer et al, Exp Neurol. 2007;207:163-170; Zuo et al, Exp Neurol. 2002;176:221-228), less is known about the ability of neurotrophic factors to enhance axonal regeneration. This study evaluates axonal ingrowth augmentation using acellular, chondroitinase-treated nerve grafts doped with nerve growth factor (NGF)., Methods: Acellular chondroitinase-treated murine nerve grafts were placed in experimental (NGF-treated grafts) and control (carrier-only grafts) rats. Five days after implantation, axonal regeneration was assessed by immunocytochemistry along with digital image analysis., Results: Higher axon count was observed throughout the length of the nerve in the NGF group (P < 0.0001), peaking at 3 mm from proximal repair (P = 0.02). Although the NGF group displayed a higher axon count per slice, the mean diameter of individual NGF axons was smaller (P < 0.0001), potentially consistent with induction of sensory axons (Rich et al, J Neurocytol. 1987;16:261-268; Sofroniew et al, Annu Rev Neurosci. 2001;24:1217-1128; Yip et al, J Neurosci. 1984;4:2986-2992)., Conclusion: The simple technique of doping acellular, chondroitinase-treated nerve grafts with NGF can augment axonal ingrowth and possibly preferentially induce sensory axons.

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., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-gamma) cytokine production (CP), single-cell IFN-gamma production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-gamma and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of <3,000 copies/ml (group B). The lymphocyte proliferation assay (LPA) did not significantly differentiate between the two groups. Using fresh peripheral blood mononuclear cells (PBMCs), the CP and ELISPOT assays for IFN-gamma detected the greatest differences between the two groups, specific for three of the five HIV-1 antigens, whereas significant differences were seen only in response to one antigen when cryopreserved cells were used. The strongest correlations were seen between the CP and ELISPOT assays. The ELISPOT B-LCL assay showed a cell concentration-dependent increase in IFN-gamma production compared to that shown by the standard ELISPOT assay but did not differentiate between the groups. In the ICC assay, greater numbers of IFN-gamma-producing T cells were seen in group B, and little or no detectable IL-2 production was seen in both groups. These studies highlight complexities of immunologic monitoring of T-cell responses in multisite clinical trials in HIV infection and outline considerations for optimizing these efforts.

AIMS: Prior studies have shown that HAART is associated with decreased HIV viral load in the lungs. The correlation between antiretroviral exposure in bronchoalveolar lavage (BAL) fluid and virologic response was evaluated in patients starting HAART and enrolled in The AIDS Clinical Trial Group Protocol 723. MATERIALS #ENTITYSTARTX00026; METHODS: A total of 24 subjects underwent blood and BAL sampling prior to starting HAART, and after 4 and 24 weeks of HAART. Drug concentrations and HIV RNA were measured in paired plasma and BAL samples. RESULTS: Antiretroviral drugs, including efavirenz, were detectable in BAL fluid of HIV-infected subjects beginning HAART. Efavirenz was also associated with a higher likelihood of clearing HIV RNA from the lungs. CONCLUSION: These results suggest the excellent pulmonary virologic response to antiretroviral therapy may, in part, be due to penetration of antiretroviral drugs into the alveolar compartment.

T cell depletion strategies are an efficient therapy for the treatment of acute rejections and are an essential part of tolerance induction protocols in various animal models; however, they are usually nonselective and cause wholesale T cell depletion leaving the individual in a severely immunocompromised state. So far it has been difficult to selectively delete alloreactive T cells because the majority of protocols either delete all T cells, subsets of T cells, or subpopulations of T cells expressing certain activation markers, ignoring the Ag specificity of the TCR. We have developed a model in which we were able to selectively deplete alloreactive T cells with an indirect specificity by targeting intact MHC molecules to quiescent dendritic cells using 33D1 as the targeting Ab. This strategy enabled us to inhibit the indirect alloresponse against MHC-mismatched skin grafts and hence the generation of IgG alloantibodies, which depends on indirectly activated T cells. In combination with the temporary abrogation of the direct alloresponse, we were able to induce indefinite skin graft survival. Importantly, the targeting strategy had no detrimental effect on CD4(+)CD25(+)FoxP3(+) T cells, which could potentially be used as an adjunctive cellular therapy. Transplantation tolerance depends on the right balance between depletion and regulation. For the former this approach may be a useful tool in the development of future tolerance induction protocols in non-sensitized patients.

Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV+keyhole limpet hemocyanin (KLH) (arm A, n=14) or CP-HIV+KLH alone (arm B, n=15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint < 5,000 c/mL during ATI (4/13 or 31% in arm A compared with 0/13 in arm B, p=0.096), but virologic control was transient. Subjects in arm A had a greater increase in KLH lymphoproliferative response than subjects in arm B; however, summed ELISPOT responses to HIV-1 antigens did not differ by treatment arm. We conclude that a DC-CP-HIV vaccine is well-tolerated in HIV-1-infected patients, but does not lower VL setpoint during ATI compared with CP-HIV alone. New methods to enhance the immunogenicity and antiviral efficacy of DC-based vaccines for HIV-1 infection are needed.

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.

This review focuses on the role of antigen-specific T cells that mediate active inhibition of immune responses over the past 35 years since their initial description. The field has experienced several changes in the accepted paradigm of such suppressor/regulatory T cells, from initial indications that such cells were CD8(+), to the view that such cells did not exist, to the identification of the transcription factor Foxp3 as a key orchestrator of inhibitory function. Although most Foxp3(+) cells in a resting animal are CD4(+)CD25(+) cells, Foxp3 expression and inhibitory function can be induced by antigens in the periphery by selective cytokine conditions, particularly TGF-beta. Such induced T cells occur within both the CD4 and the CD8 T-cell lineages and appear to mediate suppression by inhibiting the costimulatory activity of antigen-presenting cells and the production of inhibitory cytokines. Recent data generated by analysis of TCR Tg T cells that do not select many Foxp3-positive cells during thymic development are reviewed, emphasizing the pattern of "linked suppression" and focus of the relative potency of different mechanisms of suppression.

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Objective: Compare the initial phases of virologic decay when acute/early and advanced HIV-infected adults are administered the same treatment regimen., Design: Mathematical modeling of a previously completed prospective treatment pilot study involving treatment-naive patients with early and advanced immunosuppression., Methods: We analyzed data from a treatment protocol in which 18 individuals with acute or recent HIV-1 seroconversion and six patients with advanced AIDS were administered the same four-drug antiretroviral regimen. Initial treatment responses were compared by fitting a mathematical model to frequent viral load measurements in order to calculate the first and second phase kinetics of viral clearance, and also by comparing viral load suppression over 24 weeks. Patients were also comprehensively compared in terms of protease inhibitor drug levels, HIV-specific immune responses at baseline, and the presence of drug resistance-conferring mutations., Results: There was no statistically meaningful difference in first phase clearance of comparable high-level viremia in the two groups, whether protease inhibitor levels were inserted into the model or 100% antiviral drug effectiveness was assumed. In contrast, acute/early patients had inferior sustained responses than advanced patients, reflecting erratic adherence., Conclusions: Despite many years of intervening immune destruction, the initial virologic decay on therapy appears to be the same at the extremes of the HIV disease spectrum.

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.

Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor beta (TGFbeta). TGFbeta inhibited the expression of IFN-gamma, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5-15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFbeta. TGFbeta-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFbeta. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFbeta-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFbeta-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.

Although immunological memory is characterized by both an increase in the frequency of antigen-specific T cells and a qualitative change in the pattern of their subsequent response, it is not clear which of these components is more significant in the overall enhanced response to secondary stimulation. To address this question for the CD4+ T-cell response, T-cell receptor (TCR) Tg T cells were adoptively transferred to normal syngeneic mice that were immunized with the relevant peptide. After the initial expansion of TCR Tg T cells, the size of the subsequent memory population of T cells was approximately the same as the size of the starting population, independent of the number of TCR Tg cells initially transferred. This result was not caused by redistribution of memory cells into non-lymphoid tissues, although the relative frequency of antigen-specific T cells in these sites was increased after immunization. The fraction of the antigen specific TCR Tg cells that responded by production of either interleukin-2 or interferon-gammain vitro was substantially higher after immunization. Thus, the increased frequency of functionally responsive T cells was primarily caused by a higher fraction of responding T cells, rather than a substantial increase in the absolute number of antigen specific CD4+ TCR Tg T cells.

The aim of this study was to evaluate the tolerability and activity of intratumoral administered human interleukin 12 encoded by a vector derived from the canarypox virus (ALVAC-IL-12). Nine patients with surgically incurable metastatic melanoma who had subcutaneous nodules available for injection were enrolled. ALVAC-IL-12 was administered by intratumoral injection on days 1, 4, 8, and 11. Tumor nodules greater than 2 cm in diameter were injected with 2 x 10(6) median tissue culture infectious doses (TCID(50)), and smaller tumors were injected with 1 x 10(6) TCID(50). The total dose per patient per time point ranged from 1 x 10(6) to 4 x 10(6) TCID(50). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever associated with chills, myalgia, and fatigue. No dose-limiting toxicities occurred. Increases in IL-12 mRNA, and also increases in interferon gamma mRNA, were observed in ALVAC-IL-12-injected tumors compared with saline-injected control tumors in four of the nine patients. ALVAC-IL-12-injected tumors were also characterized by T cell infiltration. Three patients demonstrated increases in serum IL-12 and in interferon gamma levels. All patients developed neutralizing IgG antibody to the canarypox vector. One patient manifested a complete response of injected subcutaneous metastases and uninjected in-transit metastases. The intratumoral injection of ALVAC-IL-12 at these dose levels and according to this schedule was well tolerated and resulted in measurable biologic response in patients with metastatic melanoma.

Understanding the mechanisms of rejection of organs transplanted between unrelated individuals is confounded by the complexity of the alloantigens and the diversity of T cells responding to these alloantigens. To circumvent these problems, we developed a transgenic (Tg) C57BL/6 model system in which the T-cell receptor (TCR) expressed by CD4 T cells is specific for a defined allogeneic H-2Kd peptide and the cardiac donor expressed H-2Kd as a transgene on the C57BL/6 background (B6.Kd). These TCR Tg T cells were previously shown to mediate rapid rejection of a B10.D2 cardiac allograft when transferred to Rag1 recipients, demonstrating that the "indirect" pathway of allorecognition is sufficient for complete rejection in the absence of other T cells or antibody. Here, we report that B6.Kd hearts were rejected in an accelerated fashion by Rag1(-/-) TCR Tg T cells adoptively transferred to normal B6 recipients. Rejection in this model was associated with large myocardial infarcts and significant coronary artery inflammation. Moreover, transferred TCR Tg CD4+ cells mediated allograft injury without the requirement for cytotoxic function from recipient-derived CD8 T cells. A non-linear relationship was observed between the initial precursor frequency of the antigen-specific TCR Tg cells and the ultimate tempo of acute rejection, which is taken as evidence for cooperativity between components of the system.

T cells from TCR transgenic mice, expressing receptors specific for an allogeneic MHC class I peptide, were used to track T cell activation and migration in normal adoptive recipients that were subsequently transplanted with heterotopic hearts that were syngeneic except for a transgenic MHC class I antigen. T cells rapidly disappeared from the blood into the lymphoid tissues where they were activated within one day after transplantation. T cells initially formed discrete clusters in the spleen and lymph nodes. After proliferating for 2-4 days in lymphoid tissues, T cells reappeared in the blood and migrated to the heart and the intestines. The T cells underwent another round of proliferation in the heart, but not the intestines, and induced cardiac rejection uniformly on 6 day.

The control mechanisms that maintain a steady state viral load during chronic HIV-1 infection are critical to understanding the pathophysiology of HIV disease. The conceptual features of the two alternative models of viral control, referred to in this article as target cell limitation and immune control, are compared to the data regarding the viral and cellular dynamics of HIV-1 infection and the pattern of changes induced by effective antiretroviral drug therapy. The available data support the model that an antigen-driven immune response is the primary mechanism that limits viral growth in vivo.

The vast array of epitopes presented by allografts and the diversity of T cells responding to them complicates mechanistic studies of rejection. To minimize these problems, we developed a transgenic (Tg) model system limited to a single T-cell receptor (TCR)/peptide/major histocompatibility complex molecule. Two alloantigen-specific CD4 T-cell clones were used to isolate cDNA encoding the TCRalpha and TCRbeta chains that recognize the Kd54-68/I-Ab epitope. Two different TCR Tg lines were produced in C57BL/6 (B6) mice and crossed onto the B6.Rag1-/- background. B6.Rag1-/- recipients of T cells from TCR Tg Rag1-/-mice promptly rejected B10.D2, but not irrelevant B10.BR, cardiac grafts. Thus, a single allogeneic epitope presented by self-major histocompatibility complex class II is sufficient to activate TCR Tg T cells and serve as a target for rejection.

The chronically HIV-infected cellular reservoir in lymphoid tissue (LT) represents a formidable obstacle to the long-term success of antiretroviral therapy. Cytoreductive chemotherapy with cyclophosphamide (CTX) reduces cells in LT, and we hypothesized that coadministration of antiretroviral therapy with CTX may diminish the cellular reservoir over time. Ten antiretroviral treatment-naive subjects were recruited, and they received stavudine, lamivudine and nelfinavir (antiretroviral therapy, ART) until 2 consecutive plasma HIV RNA levels measured < 50 copies/ml (step 1). Five subjects then received ART alone, whereas five subjects received ART plus three escalating doses of CTX (step 2). Viral DNA was measured in LT obtained by excisional lymph node biopsy and peripheral blood mononuclear cells (PBMCs), using quantitative polymerase chain reaction at three time points in both groups (before steps 1 and 2, and after CTX). Viral DNA declined in both groups after the initiation of ART alone in step 1. During step 2 both groups experienced a modest decline compared with step 1. However, no significant differences were observed in viral DNA in LT or PBMCs between the ART alone and the ART plus CTX groups. Suppression of plasma HIV RNA levels < 50 copies/ml was not maintained in the ART plus CTX group, perhaps because of inadequate medication adherence. The group receiving ART plus CTX had lower CD4(+) lymphocyte counts and absolute total lymphocytes compared with the ART alone group. We conclude that the addition of CTX to ART did not diminish the cellular reservoir in HIV-infected persons.

Vitamin A deficiency diminishes Th2-mediated Ab responses, and high-level dietary vitamin A or treatment with the vitamin A metabolite retinoic acid (RA) enhances such responses. To identify a potential mechanism(s) underlying this in vivo activity of vitamin A, we examined the effects of all-trans and 9-cis RA on development of Th1 and Th2 cell populations using in vitro stimulation of Ag-naive Th0 cells from the DO11.10 TCR-transgenic mouse. Treatment with 9-cis, but not with all-trans RA, at primary stimulation strongly enhanced Th2 development. IL-4-neutralizing Ab blocked this activity, but IL-12- and IFN-gamma-neutralizing Ab did not. Because 9-cis RA regulates gene transcription via either RA receptors or retinoid X receptors (RXRs), we tested the Th2-enhancing activities of the RXR- and RA receptor-selective agonists AGN194204 and 4-((E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB). AGN194204 strongly enhanced Th2 development, whereas TTNPB did not. This RXR agonist also enhanced Th2 development when purified, naive Th0 cells (L-selectin(high)/CD4(+)) were stimulated with CD3 and CD28 Abs in the absence of APCs. During primary antigenic stimulation of naive Th0 cells from DO11.10 mice, AGN194204 increased IL-4 and IL-5 production, decreased IFN-gamma production, increased mRNA in responding T cells for genes involved in Th2 development (IL-4, GATA-3, and c-maf), and decreased mRNA for genes involved in Th1 development (IFN-gamma, T-bet, and IL-12R). These data show that stimulation of the RXR pathway enhances Th2 development, perhaps by affecting the relative expression of pertinent transcription factors, cytokines, and cytokine receptors.

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.

Highly active antiretroviral therapy (HAART) of HIV disease is associated with effective virologic control, immune reconstitution, and clinical improvements. This study addresses the potential for improvements in lymphocyte phenotype and virologic responses of HIV-infected persons with extensive experience with dual nucleoside reverse transcriptase (NRTI) treatment and advanced HIV disease after a change to a potent antiretroviral therapy (NRTI + protease inhibitor). The majority of participants achieved virologic success. There was a median rise in CD4+ lymphocytes of 99 cells/mm(3) by 48 weeks, because of an increase in memory CD4+ cells at 4 and 16 weeks, followed by a later increase in naive CD4+ cells between weeks 16 and 48. The proportion of activated, DR+ CD38+ CD8+ lymphocytes decreased during the 48 weeks of follow-up. The immunologic findings (increased memory and naive T cells and reduced activation levels) were significantly improved in participants with persistent suppression of viral replication over the 48 weeks of the study. Baseline HIV RNA copy number was lower (median, 14,784 copies/ml) in persons who responded virologically than in those not suppressing viral replication (median, 49,454 copies/ml). CD4+ cell counts above the median (125/mm(3)) at time 0 for the participants, was the only baseline immunologic marker significantly associated with viral suppression at week 48. Participants older than 40 years of age demonstrated less immunologic recovery. The results of the study show that patients with extensive experience with NRTIs respond both virologically and immunologically during the first 48 weeks of therapy with a potent antiretroviral regimen.

The control mechanisms that maintain a steady-state viral load during chronic HIV-1 infection are critical to understanding the pathophysiology of HIV disease. This paper compares the conceptual features of the two alternative models of viral control, target cell limitation and immune control, with available data on the viral and cellular dynamics of HIV-1 infection and the pattern of changes induced by effective antiretroviral drug therapy. These data suggest that an antigen-driven immune response is the primary control mechanism for in vivo viral growth.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Objective: To investigate the safety, tolerability, pharmacokinetics, and immunologic activity of single intravenous infusions (0.2-10 mg/kg) of Orthoclone OKTcdr4a, a nondepleting humanized anti-CD4 monoclonal antibody (Mab) in patients with rheumatoid arthritis., Methods: Eighteen patients were treated with a single intravenous dose of Mab. Three patients each received 0.2, 0.5, 1.0, 2.0, 5.0, or 10.0 mg/kg of OKTcdr4a., Results: No patient had a significant change in CD4+ T cells in peripheral blood after treatment. No human antimurine antibodies were detected. At > or = 1.0 mg/kg dose level, CD4 receptor saturation was > or = 95% 24 h after infusion. At 5.0 mg/kg, CD4 receptor occupancy was a mean of 88% at 6 days after infusion. At 10 mg/kg, CD4 receptor occupancy was still a mean of 79% 2 weeks after infusion. No significant infusion related adverse events occurred. Two subjects had headaches at the time of drug administration. Two subjects were hospitalized for infections (pneumonia, Day 45; cellulitis, Day 14), which resolved with antibiotic therapy., Conclusion: OKTcdr4a was well tolerated at the doses used and saturation of CD4 receptors in peripheral blood could be routinely obtained for over one week with a single infusion of Mab.