Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Early antiretroviral therapy in SIV-infected rhesus macaques reveals a multiphasic, saturable dynamic accumulation of the rebound competent viral reservoir.

The rebound competent viral reservoir (RCVR)-virus that persists during antiretroviral treatment (ART) and can reignite systemic infection when treatment is stopped-is the primary barrier to eradicating HIV. We used time to initiation of ART during primary infection of rhesus macaques (RMs) after intravenous challenge with barcoded SIVmac239 as a means to elucidate the dynamics of RCVR establishment in groups of RMs by creating a multi-log range of pre-ART viral loads and then assessed viral time-to-rebound and reactivation rates resulting from the discontinuation of ART after one year. RMs started on ART on days 3, 4, 5, 6, 7, 9 or 12 post-infection showed a nearly 10-fold difference in pre-ART viral measurements for successive ART-initiation timepoints. Only 1 of 8 RMs initiating ART on days 3 and 4 rebounded after ART interruption despite measurable pre-ART plasma viremia. Rebounding plasma from the 1 rebounding RM contained only a single barcode lineage detected at day 50 post-ART. All RMs starting ART on days 5 and 6 rebounded between 14- and 50-days post-ART with 1-2 rebounding variants each. RMs starting ART on days 7, 9, and 12 had similar time-to-measurable plasma rebound kinetics despite multiple log differences in pre-ART plasma viral load (pVL), with all RMs rebounding between 7- and 16-days post-ART with 3-28 rebounding lineages. Calculated reactivation rates per pre-ART pVL were highest for RMs starting ART on days 5, 6, and 7 after which the rate of accumulation of the RCVR markedly decreased for RMs treated on days 9 and 12, consistent with multiphasic establishment and near saturation of the RCVR within 2 weeks post infection. Taken together, these data highlight the heterogeneity of the RCVR between RMs, the stochastic establishment of the very early RCVR, and the saturability of the RCVR prior to peak viral infection.

Timing of initiation of anti-retroviral therapy predicts post-treatment control of SIV replication.

One approach to 'functional cure' of HIV infection is to induce durable control of HIV replication after the interruption of antiretroviral therapy (ART). However, the major factors that determine the viral 'setpoint' level after treatment interruption are not well understood. Here we combine data on ART interruption following SIV infection for 124 total animals from 10 independent studies across 3 institutional cohorts to understand the dynamics and predictors of post-treatment viral control. We find that the timing of treatment initiation is an important determinant of both the peak and early setpoint viral levels after treatment interruption. During the first 3 weeks of infection, every day of delay in treatment initiation is associated with a 0.22 log10 copies/ml decrease in post-rebound peak and setpoint viral levels. However, delay in initiation of ART beyond 3 weeks of infection is associated with higher post-rebound setpoint viral levels. For animals treated beyond 3 weeks post-infection, viral load at ART initiation was the primary predictor of post-rebound setpoint viral levels. Potential alternative predictors of post-rebound setpoint viral loads including cell-associated DNA or RNA, time from treatment interruption to rebound, and pre-interruption CD8+ T cell responses were also examined in the studies where these data were available. This analysis suggests that optimal timing of treatment initiation may be an important determinant of post-treatment control of HIV.

Effects of Early Antiretroviral Therapy on the Composition and Diversity of the Fecal Microbiome of SIV-infected Rhesus Macaques (Macaca mulatta).

HIV-infected people develop reproducible disruptions in their gastrointestinal microbiota. Despite the suppression of HIV viremia via long-term antiretroviral therapy (ART), alterations still occur in gut microbial diversity and the commensal microbiota. Mounting evidence suggests these microbial changes lead to the development of gut dysbiosis-persistent inflammation that damages the gut mucosa-and correlate with various immune defects. In this study, we examined how early ART intervention influences microbial diversity in SIV-infected rhesus macaques. Using 16S rRNA sequencing, we defined the fecal microbiome in macaques given daily ART beginning on either 3 or 7 d after SIV infection (dpi) and characterized changes in composition, α diversity, and ß diversity from before infection through 112 dpi. The dominant phyla in the fecal samples before infection were Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria. After SIV infection and ART, the relative abundance of Firmicutes and Bacteroidetes did not change significantly. Significant reductions in α diversity occurred across time when ART was initiated at 3 dpi but not at 7 dpi. Principal coordinate analysis of samples revealed a divergence in ß diversity in both treatment groups after SIV infection, with significant differences depending on the timing of ART administration. These results indicate that although administration of ART at 3 or 7 dpi did not substantially alter fecal microbial composition, the timing of early ART measurably altered phylogenetic diversity.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA-treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription.

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 µg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

CD8+ T cells fail to limit SIV reactivation following ART withdrawal until after viral amplification.

To define the contribution of CD8+ T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8+ T cell depletion using a rhesusized anti-CD8ß monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8+ T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA+ cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8+ T cell-depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8+ T cell-depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8+ T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell.

The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti-IL-15- and control-treated groups in both cohorts. However, RM treated with anti-IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

In Vivo Validation of the Viral Barcoding of Simian Immunodeficiency Virus SIVmac239 and the Development of New Barcoded SIV and Subtype B and C Simian-Human Immunodeficiency Viruses.

Genetically barcoded viral populations are powerful tools for evaluating the overall viral population structure as well as assessing the dynamics and evolution of individual lineages in vivo over time. Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone simian immunodeficiency virus (SIV) SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences were within a 34-base barcode that could be tracked using next-generation deep sequencing. Here, we assessed the population size, distribution, and authenticity of individual viral clonotypes within this synthetic swarm using samples from 120 rhesus macaques infected intravenously. The number of replicating barcodes in plasma correlated with the infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of the inoculum size. Overall, 97% of detectable clonotypes in the viral stock were identified in the plasma of at least one infected animal. Additionally, we prepared a second-generation barcoded SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and an additional barcoded stock with suboptimal nucleotides corrected (SIVmac239Opt5M). We also generated four barcoded stocks from subtype B and C simian-human immunodeficiency virus (SHIV) clones. These new SHIV clones may be particularly valuable models to evaluate Env-targeting approaches to study viral transmission or viral reservoir clearance. Overall, this work further establishes the reliability of the barcoded virus approach and highlights the feasibility of adapting this technique to other viral clones.IMPORTANCE We recently developed and published a description of a barcoded simian immunodeficiency virus that has a short random sequence inserted directly into the viral genome. This allows for the tracking of individual viral lineages with high fidelity and ultradeep sensitivity. This virus was used to infect 120 rhesus macaques, and we report here the analysis of the barcodes of these animals during primary infection. We found that the vast majority of barcodes were functional in vivo We then expanded the barcoding approach in a second-generation SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and a barcoded stock of SIVmac239Opt5M whose sequence had 5 changes from the wild-type SIVmac239 sequence. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing a human immunodeficiency virus (HIV) type 1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.

The Landscape of Persistent Viral Genomes in ART-Treated SIV, SHIV, and HIV-2 Infections.

Evaluation of HIV cure strategies is complicated by defective proviruses that persist in ART-treated patients but are irrelevant to cure. Non-human primates (NHP) are essential for testing cure strategies. However, the persisting proviral landscape in ART-treated NHPs is uncharacterized. Here, we describe viral genomes persisting in ART-treated, simian immunodeficiency virus (SIV)-infected NHPs, simian-human immunodeficiency virus (SHIV)-infected NHPs, and humans infected with HIV-2, an SIV-related virus. The landscapes of persisting SIV, SHIV, and HIV-2 genomes are also dominated by defective sequences. However, there was a significantly higher fraction of intact SIV proviral genomes compared to ART-treated HIV-1 or HIV-2 infected humans. Compared to humans with HIV-1, SIV-infected NHPs had more hypermutated genomes, a relative paucity of clonal SIV sequences, and a lower frequency of deleted genomes. Finally, we report an assay for measuring intact SIV genomes which may have value in cure research.

Lymph nodes as barriers to T-cell rejuvenation in aging mice and nonhuman primates.

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T-cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T-cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T-cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA-treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.

Early antiretroviral therapy limits SIV reservoir establishment to delay or prevent post-treatment viral rebound.

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4-9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years.

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

OBJECTIVE: To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells. DESIGN: Case report. METHODS: Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing. RESULTS: HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells. CONCLUSION: Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

Effect of Anti-IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques.

IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other γ-chain (γc) cytokines has not been fully defined in primates. In this article, we address this question by determining the effect of IL-15 inhibition with a rhesusized anti-IL-15 mAb on T and NK cell dynamics in rhesus macaques. Strikingly, anti-IL-15 treatment resulted in rapid depletion of NK cells and both CD4(+) and CD8(+) effector memory T cells (TEM) in blood and tissues, with little to no effect on naive or central memory T cells. Importantly, whereas depletion of NK cells was nearly complete and maintained as long as anti-IL-15 treatment was given, TEM depletion was countered by the onset of massive TEM proliferation, which almost completely restored circulating TEM numbers. Tissue TEM, however, remained significantly reduced, and most TEM maintained very high turnover throughout anti-IL-15 treatment. In the presence of IL-15 inhibition, TEM became increasingly more sensitive to IL-7 stimulation in vivo, and transcriptional analysis of TEM in IL-15-inhibited monkeys revealed engagement of the JAK/STAT signaling pathway, suggesting alternative γc cytokine signaling may support TEM homeostasis in the absence of IL-15. Thus, IL-15 plays a major role in peripheral maintenance of NK cells and TEM However, whereas most NK cell populations collapse in the absence of IL-15, TEM can be maintained in the face of IL-15 inhibition by the activity of other homeostatic regulators, most likely IL-7.

Effect of IL-7 Therapy on Naive and Memory T Cell Homeostasis in Aged Rhesus Macaques.

Aging is associated with gradual deterioration of adaptive immune function, a hallmark of which is the profound loss of naive T cells (TN) associated with decline in thymic output and export of new cells into the peripheral T cell pool. Because the lymphotropic cytokine IL-7 plays crucial roles in both development of TN in the thymus and TN homeostasis in the periphery, we sought to determine the extent to which therapeutic administration of IL-7 could reverse TN deficiency in aging rhesus macaques (RM), either by enhancement of the demonstrably reduced thymopoiesis or by peripheral TN expansion. Our results indicate that treatment of both adult (8-15 y) and old (>20 y) RM with recombinant simian IL-7 (rsIL-7) results in only transient increases in peripheral CD4(+) and CD8(+) TN numbers with no long-term benefit, even with repeated therapy. This transient effect was due to peripheral TN expansion and not enhanced thymic function, and appeared to be limited by induction of IL-7 nonresponsiveness. However, rsIL-7 therapy had a more promising effect on the central memory T cell (TCM) population (both CD4(+) and CD8(+)) in adult and old RM, doubling the numbers of these cells in circulation and maintaining this larger population long term. IL-7 therapy did not reduce TCR diversity of the memory T cell compartment, suggesting that rsIL-7-induced expansion was symmetrical. Thus, although rsIL-7 failed to counter age-associated TN loss, the ability of this therapy to expand clonotypically diverse CD4(+) and CD8(+) TCM populations might potentially improve adaptive immune responsiveness in the elderly.