Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice.

One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1-day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus-grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4-8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single-positive cells and the maintenance of thymocyte viability during the double-positive stage of intrathymic differentiation.

Correlates of latent and productive HIV type-1 infection in tonsillar CD4+ T cells

Correlates of virus load and characteristics of virus-producing cells in tonsillar tissue were investigated. Our results suggest that when less than 1:100 tonsillar CD4+ T cells from individuals infected with HIV type-1 (HIV-1) contain replication competent provirus, the level of CD4+ T cells in tonsils is comparable to that observed in uninfected individuals. Virus load at or above this level was associated with low CD4 cell numbers in tonsillar tissue. Only a few percent of all infected T cells in tonsillar tissue were active virus producers, with minor differences observed between individuals. Plasma viremia was found to correlate with infectious virus load in tonsillar tissue. With less than 1:1,000 of CD4 cells in lymphoid tissues being involved in active virus production, direct cytopathic effect by HIV-1 on infected CD4 cells is unlikely to fully explain the immunodeficiency seen in AIDS.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.

Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+ cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+ cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+ cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+ cell ratio of 0.1. The CD8+ cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+ cell ratio of 0.25; the subject’s peripheral blood CD8+ cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+ cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+ cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+ cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIVDH12-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIVDH12 infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4+ T lymphocytes. Although the CD4+ T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIVDH12R, induced marked and rapid CD4+ cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIVDH12R indicated: (i) the input cloned SHIV remained the predominant virus during the first 5–7 months of infection; (ii) variants bearing only a few of the SHIVDH12R consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIVDH12R ever developed.

Chronic elevation of plasma thioredoxin: Inhibition of chemotaxis and curtailment of life expectancy in AIDS

Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like and chemokine-like activities. We show here that, although plasma Trx levels are unrelated to survival of HIV-infected individuals with CD4 cell counts above 200/μl blood, survival is significantly impaired (P = 0.003) when plasma Trx is chronically elevated in HIV-infected subjects with CD4 T cell counts below this level (i.e., with Centers for Disease Control (CDC)-defined AIDS). Relevant to the mechanism potentially underlying this finding, we also present data from experimental studies in mice showing that elevated plasma Trx efficiently blocks lipopolysaccharide (LPS)-induced chemotaxis, an innate immune mechanism that is particularly crucial when adaptive immunity is compromised. Thus, we propose that elevated plasma Trx in HIV-infected individuals with low CD4 T cell counts directly impairs survival by blocking pathogen-induced chemotaxis, effectively eliminating the last (innate) barrier against establishment of opportunistic and other infections in these immunodeficient individuals.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor alpha, and human immunodeficiency virus infection.

We tested the hypothesis that increases in tumor necrosis factor alpha (TNF-alpha) induced by human immunodeficiency virus (HIV) are associated with the increases in slow-wave sleep seen in early HIV infection and the decrease with sleep fragmentation seen in advanced HIV infection. Nocturnal sleep disturbances and associated fatigue contribute to the disability of HIV infection. TNF-alpha causes fatigue in clinical use and promotes slow-wave sleep in animal models. With slow progress toward a vaccine and weak effects from current therapies, efforts are directed toward extending productive life of HIV-infected individuals and shortening the duration of disability in terminal illness. We describe previously unrecognized nocturnal cyclic variations in plasma levels of TNF-alpha in all subjects. In 6 of 10 subjects (1 control subject, 3 HIV-seropositive patients with CD4+ cell number > 400 cells per microliters, and 2 HIV-positive patients with CD4+ cell number < 400 cells per microliters), these fluctuations in TNF-alpha were coupled to the known rhythm of electroencephalogram delta amplitude (square root of power) during sleep. This coupling was not present in 3 HIV-positive subjects with CD4+ cell number < 400 cells per microliters and 1 control subject. In 5 HIV subjects with abnormally low CD4+ cell counts ( < 400 cells per microliters), the number of days since seroconversion correlated significantly with low correlation between TNF-alpha and delta amplitude. We conclude that a previously unrecognized normal, physiological coupling exists between TNF-alpha and delta amplitude during sleep and that the lessened likelihood of this coupling in progressive HIV infection may be important in understanding fatigue-related symptoms and disabilities.

Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells

AIDS is characterized by a progressive decrease of CD4+ helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56lck and Giα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4+ but not in CD8+ T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.

Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline

Evolution of HIV-1 env sequences was studied in 15 seroconverting injection drug users selected for differences in the extent of CD4 T cell decline. The rates of increase of either sequence diversity at a given visit or divergence from the first seropositive visit were both higher in progressors than in nonprogressors. Viral evolution in individuals with rapid or moderate disease progression showed selection favoring nonsynonymous mutations, while nonprogressors with low viral loads selected against the nonsynonymous mutations that might have resulted in viruses with higher levels of replication. For 10 of the 15 subjects no single variant predominated over time. Evolution away from a dominant variant was followed frequently at a later time point by return to dominance of strains closely related to that variant. The observed evolutionary pattern is consistent with either selection against only the predominant virus or independent evolution occurring in different environments within the host. Differences in the level to which CD4 T cells fall in a given time period reflect not only quantitative differences in accumulation of mutations, but differences in the types of mutations that provide the best adaptation to the host environment.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4+, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Rα−/− mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Rα−/−, β2-microglobulin−/− double homozygous mice produced less IL-4 than did either IL-4Rα−/− or wild-type mice. These results indicate that an IL-4-independent, β2-microglobulin-dependent pathway exists through which the CD62L-low CD4+ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.