IL-15 is superior to IL-2 in the generation of long-lived antigen specific memory CD4 and CD8 T cells in rhesus macaques.

Using tetanus toxoid (TT) and influenza (Flu) immunization of rhesus macaques as a model, the effect of IL-2 and IL-15 on the generation and maintenance of antigen specific memory T cells was evaluated following primary and secondary immunization. Daily cytokine administration expanded primarily effector but not memory cells, while spacing cytokine administration to q3-7 days markedly enhanced TT and Flu specific memory responses. Following primary immunization, TT specific CD4 and influenza matrix protein (Flu-MP) specific CD8 effector responses were enhanced by IL-2 administration but CD8 specific memory responses were no different from cytokine non-treated monkeys. In contrast, expansion of Flu specific CD8 cells with IL-15 was only modest but resulted in significantly elevated levels of memory cells at 6 months. IL-15 also significantly enhanced early and late TT specific CD4 responses. The highest levels of primary effector and memory T cells were observed following alternate administration of both IL-2 and IL-15. Following booster immunization, however, only IL-15 appeared able to enhance CD8 T cell responses while IL-2 or IL2/IL-15 administration were less effective.

Administration of recombinant rhesus interleukin-12 during acute simian immunodeficiency virus (SIV) infection leads to decreased viral loads associated with prolonged survival in SIVmac251-infected rhesus macaques.

The ability of recombinant rhesus interleukin-12 (rMamu-IL-12) administration during acute simian immunodeficiency virus SIVmac251 infection to influence the quality of the antiviral immune responses was assessed in rhesus macaques. Group I (n = 4) was the virus-only control group. Group II and III received a conditioning regimen of rMamu-IL-12 (10 and 20 microg/kg, respectively, subcutaneously [s.c.]) on days -2 and 0. Thereafter, group II received 2 microg of IL-12 per kg and group III received 10 microg/kg s.c. twice a week for 8 weeks. On day 0 all animals were infected with SIVmac251 intravenously. While all four group I animals and three of four group II animals died by 8 and 10 months post infection (p.i.), all four group III animals remained alive for >20 months p.i. The higher IL-12 dose led to lower plasma viral loads and markedly lower peripheral blood mononuclear cell and lymph node proviral DNA loads. During the acute viremia phase, the high-IL-12-dose monkeys showed an increase in CD3(-) CD8 alpha/alpha(+) and CD3(+) CD8 alpha/alpha(+) cells and, unlike the control and low-IL-12-dose animals, did not demonstrate an increase in CD4(+) CD45RA(+) CD62L(+) naive cells. The high-IL-12-dose animals also demonstrated that both CD8 alpha/alpha(+) and CD8 alpha/beta(+) cells produced antiviral factors early p.i., whereas only CD8 alpha/beta(+) cells retained this function late p.i. Long-term survival correlated with sustained high levels of SIV gag/pol and SIV env cytotoxic T lymphocytes and retention of high memory responses against nominal antigens. This is the first study to demonstrate the capacity of IL-12 to significantly protect macaques from SIV-induced disease, and it provides a useful model to more precisely identify correlates of virus-specific disease-protective responses.

Identification of protein kinases dysregulated in CD4(+) T cells in pathogenic versus apathogenic simian immunodeficiency virus infection.

Human immunodeficiency virus infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM) leads to a generalized loss of immune responses involving perturbations in T-cell receptor (TCR) signaling. In contrast, naturally SIV-infected sooty mangabeys (SM) remain asymptomatic and retain immune responses despite relatively high viral loads. However, SIV infection in both RM and SM led to similar decreases in TCR-induced Lck phosphorylation. In this study, a protein tyrosine kinase (PTK) differential display method was utilized to characterize the effects of in vivo SIV infection on key signaling molecules of the CD4(+) T-cell signaling pathways. The CD4(+) T cells from SIV-infected RM, but not SIV-infected SM, showed chronic downregulation of baseline expression of MLK3, PRK, and GSK3, and symptomatically SIV-infected RM showed similar downregulation of MKK3. In vitro TCR stimulation with or without CD28 costimulation of CD4(+) T cells did not lead to the enhancement of gene transcription of these PTKs. While the CD4(+) T cells from SIV-infected RM showed a significant increase of the baseline and anti-TCR-mediated ROR2 transcription, SIV infection in SM led to substantially decreased anti-TCR-stimulated ROR2 transcription. TCR stimulation of CD4(+) T cells from SIV-infected RM (but not SIV-infected SM) led to the repression of CaMKKbeta and the induction of gene transcription of MLK2. Studies of the function of these molecules in T-cell signaling may lead to the identification of potential targets for specific intervention, leading to the restoration of T-cell responses.

Failure to expand influenza and tetanus toxoid memory T cells in vitro correlates with disease course in SIV infected rhesus macaques.

Marked decreases in influenza (flu) and tetanus toxoid (T.T.) antigen specific CD8(+) and CD4(+) T cell memory responses were noted shortly after SIV infection in monkeys that go on to develop clinical disease within 18 months (normal progressor, NP) following SIV infection but not in monkeys that remain asymptomatic >3 years post SIV infection (long-term nonprogressor, LTNP). While PBMCs from NP and LTNP monkeys demonstrate both low and high avidity flu and T.T. specific CD8(+) and CD4(+)T cell immune responses prior to SIV infection, the PBMCs from NP but not LTNP fail to generate high avidity T cell responses post SIV infection. This failure to generate high avidity T cell responses in vitro correlated with increased apoptotic cell death in PBMC cultures from NP animals. Since high avidity antigen specific CTLs have been shown to be most efficient in eliminating viral infections, the present finding has important implications for the evaluation of the level of immune reconstitution following various modalities of therapy in HIV-1 infected patients.

Relative resistance in the development of T cell anergy in CD4+ T cells from simian immunodeficiency virus disease-resistant sooty mangabeys.

Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4(+) T cell responses. In contrast, CD4(+) T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4(+) T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4(+)T cells from humans and RM became anergized, surprisingly, CD4(+) T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4(+) T cells from SM since incubation of CD4(+) T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4(+) T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4(+) T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4(+) T cells from SM may contribute to the resistance of this species to SIV-induced disease.

Chronic immune stimulation accelerates SIV-induced disease progression.

The contribution of chronic immune stimulation on the progression of lentivirus-induced disease was evaluated in the SIVmac251 macaque model of AIDS. Following SIV inoculation, seroconversion and control of the acute viral replication phase, repeated immune stimulations with tetanus toxoid (TT), keyhole limpet hemocyanin (KLH) and allogeneic peripheral blood mononuclear cells (PBMC) were initiated in four monkeys. These animals showed a significant shortening of survival when compared with eight non-immune-stimulated control animals inoculated with the same route, dose and stock of SIVmac251 (median survival 9.5 months versus 17 months, P = 0.010). In addition, when the comparison was extended to another 22 control animals of different origin but inoculated by the same route with similar doses and stocks of SIVmac251, the difference in survival was still significant (9.5 versus 18 months, P = 0.003). This accelerated progression of symptomatic disease was not accompanied with significant increases in plasma viral loads, but suboptimal antibody responses to the immunizing antigens were noted, correlating with the length of survival. These findings may have implications for HIV-infected humans suffering from chronic infectious diseases.

Induction of long-term protective effects against heterologous challenge in SIVhu-infected macaques.

A group of three rhesus macaques were inoculated with SIV isolated from a human (SIVhu) accidentally exposed and infected with SIVsm. Extensive sequence analyses of SIVhu obtained from the human and macaques following infection indicated the presence of truncated nef. Not only did nef fail to repair itself in vivo postinfection (p.i.), but instead, further mutations added additional stop codons with increasing time p.i. Infection of these animals was associated with minimal acute viral replication, followed by undetectable plasma viral loads and only intermittent PCR detection up to 5 years p.i. The three SIVhu infected and three control monkeys were then challenged with the heterologous highly pathogenic SHIV89.6p. All three controls became infected and showed rapid declines in peripheral CD4(+) lymphocytes, disease, and death at 10 and 32 weeks p.i., respectively. In contrast, all three animals previously infected with SIVhu are healthy and exhibit stable CD4(+) lymphocyte levels and undetectable plasma viral loads at >20 months post-SHIV89. 6p challenge. Only transient, low levels of SHIV replication were noted in these animals. Whereas responses to SIVgag/pol were noted, no evidence for SIV/SHIV envelope cross-reactivity was detected by antibody or CTL analyses, suggesting that the protective immune mechanisms to the heterologous challenge isolate were most likely not directed to envelope but rather to other viral determinants.

Inverse correlation of telomerase activity/proliferation of CD4+ T lymphocytes and disease progression in simian immunodeficiency virus-infected nonhuman primates.

Both increased lymphocyte renewal with subsequent exhaustion of the immune system and impaired T-cell renewal have been put forth to account for CD4+ T-cell depletion and development of AIDS in HIV-1-infected humans and SIV-infected nonhuman primates. In the present study, telomeric terminal restriction fragment length and telomerase activity were used as measures of proliferative activity of T lymphocytes from three nonhuman primate species before and after being infected with SIV. In peripheral blood T cells, our data show both species and T-cell-subset-specific differences in proliferative activity accompanied by different patterns of disease progression. A significant postinfection increase in telomerase/proliferative activity in CD4+ T cells from seropositive sooty mangabeys and from normal progressor rhesus macaques was associated with asymptomatic infection or delayed disease progression, respectively, whereas a decrease in telomerase/proliferative activity detected in CD4+ T cells postinfection from SIVsmmPBj14-infected pigtailed macaques was associated with rapid CD4+ T-cell depletion and disease progression. The levels of telomerase activity observed in CD4+ T cells from peripheral blood closely parallelled those seen in CD4+ T cells in lymph node samples from selected animals. Our data suggest that an increase in proliferative activity of T lymphocytes in vivo may be associated with a favorable course of SIV infection in nonhuman primates.

A novel role for tumor necrosis factor-alpha in regulating susceptibility of activated CD4+ T cells from human and nonhuman primates for distinct coreceptor using lentiviruses.

Although CD4+ T-cell activation has long been shown to promote infection and replication of simian immunodeficiency virus (SIV) and HIV, recent studies have documented that not all activated CD4+ T cells from human and nonhuman primates are susceptible to infection with HIV/SIV, respectively. Activation of CD4+ T cells with anti-CD3 + anti-CD28 conjugated beads led to induction of a state of anti-viral resistance to infection with strains of viruses that primarily use CCR5 as a coreceptor. The studies reported herein were designed to address the mechanism by which anti-CD3 + anti-CD28-induced stimulation in turn induced antiviral resistance. Results of these studies show that the anti-viral resistance induced by activation of CD4+ T cells with anti-CD3 + anti-CD28 is primarily conferred by the synthesis of tumor necrosis factor-alpha (TNF-alpha), and highlight a unique regulatory role for TNF-alpha in regulating synthesis of MIP-1alpha, MIP-1beta, and regulated-on-activation normal T-expressed and secreted cells, which contributes to this state of antiviral resistance to R5-tropic strains of HIV/SIV. However, while TNF-alpha has a protective role in antiviral resistance of activated CD4+ T cells to R5-tropic viruses, it enhances CXCR4 expression of CD4+ T cells and mediates increased susceptibility to infection with X4-tropic strains of HIV and recombinant SIVs. The results of the studies reported herein also suggest that it is not the Th1 v/s Th2 cytokine profile but the mode of CD4+ T-cell activation that dictates the synthesis of distinct cytokines which regulate the expression of chemokines and chemokine receptors which in turn regulate and confer susceptibility/resistance to R5 v/s X4-tropic HIV and SIV.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Studies of CD40L expression by lymphoid cells from experimentally and naturally SIV-infected nonhuman primate species.

Dysfunction of T lymphocytes is well documented in HIV-1-infected individuals; however, the mechanisms responsible for the noted dysfunction are not well understood. CD40L is an important costimulatory molecule that helps initiate immune responses, and there is controversy regarding whether or not expression of CD40L is compromised in HIV-1-infected individuals. We have utilized the SIV infection of experimentally infected (disease-susceptible) and naturally infected (disease-resistant) nonhuman primates as animal models of human AIDS to address this issue. Little is known concerning the expression of CD40L in nonhuman primates. Studies were conducted to determine the frequency, density, phenotype, and kinetics of CD40L expression by in vitro activated peripheral blood mononuclear cells (PBMCs) from different species of uninfected and SIV-infected monkeys. Data obtained show marked differences in the density and phenotypic lineage that expresses CD40L in lymphoid cells from the three species examined. However, no detectable differences were noted in the frequency and density of CD40L expression by in vitro activated lymphoid cells from uninfected and SIV-infected disease-susceptible rhesus macaques and seropositive as compared to seronegative disease-resistant sooty mangabeys. These data suggest that phenotypic expression of CD40L is not compromised due to SIV infection.

Immune and hematopoietic parameters in HIV-1-infected chimpanzees during clinical progression toward AIDS.

Until recently, chimpanzees were considered susceptible to human immunodeficiency virus type 1 (HIV-1) infection, but refractory to disease induction based on the asymptomatic status of all experimentally infected chimpanzees after over 10 years postinfection (PI). However, a decline in peripheral CD4+ T cells was noted in one chimpanzee (C499) of the Yerkes cohort of HIV-1 infected apes, after 11 years PI concurrent with increasing plasma viral load. These clinical signs were followed by the occurrence of opportunistic infections, thrombocytopenia, and progressive anemia leading to euthanasia. A second chimpanzee (C455) was transfused with blood from C499 collected during the symptomatic stage. Shortly thereafter, this second animal showed a rapid decline in peripheral CD4+ T-cell levels and sustained high viral load. Hematological analyses showed a 50% decrease in CFU-GM for both apes during the symptomatic phase and a reduction of 40% and 73% of the total CFU despite normal levels of CD34+ cells in the bone marrow. Cryopreserved sequential PBMC samples from these two chimpanzees were analyzed for constitutive and PHA-P induced levels of cytokines and chemokines. Data show that whereas there were no detectable constitutive levels of mRNA coding for IL-2, 4, and 10, there appears to be a transient increase in IFN-gamma message level coincident with increased viremia and this IFN-gamma synthesis decreased with disease progression. PHA-induced cytokine mRNA analysis showed low or undetectable levels of IL-4 and IL-10 mRNA in all samples and a marked decrease in the levels of IL-2 shortly after HIV infection. In addition, there was also a gradual decrease in IFN-gamma mRNA with progression of disease. Of interest were the findings of high to normal levels of PHA-induced synthesis of the chemokines MIP-1alpha, MIP-1beta, and RANTES in samples during the asymptomatic and early symptomatic period, which also dramatically decreased at late stages of the disease. These data suggest important roles for IL-2, IFN-gamma, and the chemokines in the regulation of immune responses in HIV-1-infected chimpanzees.

Patterns of myocardial cell adhesion molecule expression in human endomyocardial biopsies after cardiac transplantation. Induced ICAM-1 and VCAM-1 related to implantation and rejection.

Conflicting patterns of myocardial cell adhesion molecule expression associated with cardiac rejection have emerged from numerous studies of randomly selected cardiac biopsies. We designed a prospective, longitudinal study which reports both qualitative and quantitative levels of myocardial ICAM-1, VCAM-1, E-selectin, and P-selectin expression in sequential human cardiac allograft biopsies. Intense ICAM-1 and VCAM-1 staining was found in all biopsies during the first three weeks after transplant and coincided with elevated serum levels of troponin T, a sensitive marker of ischemic myocyte injury. Baseline ICAM-1 and VCAM-1 expression returned within three to four weeks, as did serum troponin T levels in all patients who did not develop rejection. All 29 rejection episodes encountered were associated with intense ICAM-1 staining, while 24 of the 29 (83%) had intense VCAM-1 staining. Increased ELAM-1 and CD62 staining was only rarely observed. Persistence of increased ICAM-1 and VCAM-1 staining after treated rejection episodes predicted a recurrent rejection episode within two months (75% positive and 100% negative predictive value). Objective quantitative measurements by radioimmunoassay (RIA) confirmed these patterns of induced ICAM-1 and VCAM-1 expression. Thus, longitudinal monitoring of serial biopsies for myocardial ICAM-1 and VCAM-1 expression could be useful in the early detection of rejection episodes and monitoring the efficacy of immunosuppressive therapy.

Evidence for simian immunodeficiency virus-specific IgM and IgG response in peripheral blood mononuclear cells of serum enzyme-linked immunosorbent assay-negative nonhuman primates.

In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)-negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+ cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+ T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+ T cells that regulate SIV-specific Ig synthesis and show the detection of gag sequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.