Safety and Immunogenicity After a Three-Dose SARS-CoV-2 Vaccine Schedule in Allogeneic Stem Cell Transplant Recipients.

In allogeneic stem cell transplant (Allo-SCT) recipients, the cell-mediated and humoral immunogenicity of the 3-dose SARS-CoV-2 vaccination schedule has not been investigated in prospective studies. In a prospective cohort, we recruited 122 Allo-SCT recipients since August 2021, when Ontario began offering a 3-dose vaccine schedule for Allo-SCT recipients. We determined humoral and cell-mediated immunity and adverse effects of the 3-dose SARS-COV-2 vaccination schedule in Allo-SCT recipients. In immunogenicity analysis (n = 95), the median (interquartile range [IQR]) antibody titer against the receptor-binding domain (RBD) of the spike (S) protein after the third dose (10,358.0 U/mL [IQR = 673.9-31,753.0]) was significantly higher than that after the first (10.2 U/mL [IQR = 0.6-37.0]) and the second doses (125.6 U/mL [IQR = 2.8-1251.0]) (P < .0001). The haploidentical donor status was an independent risk factor (adjusted odds ratio = 7.67, 95% confidence interval [CI], 1.86-31.60) for suboptimal antibody response (anti-RBD < 100 U/mL). S-specific CD4+ and CD8+ T-cell responses were measured in a subset of Allo-SCT recipients (n = 20) by flow cytometry. Most developed antigen-specific CD4+ (55%-80%) and CD8+ T-cells (80%) after 2 doses of vaccine. Frequencies of CD4+ polyfunctional (P = .020) and IL-2 monofunctional (P = .013) T-cells significantly increased after the third dose. Twenty-three episodes (23/301 doses [7.6%]) of new-onset or worsening pre-existing graft-versus-host disease (GVHD) occurred, including 4 episodes after the third dose. We observed 4 relapses (3.27%). Seven patients developed SARS-CoV-2 infection despite vaccination, although none required hospitalization. In conclusion, the 3-dose SARS-CoV-2 vaccine schedule provided immunity associated with a low risk of GVHD and other adverse effects. This prospective cohort showed that the third dose of SARS-CoV-2 vaccine in allogeneic stem cell transplant recipients promoted better humoral and cellar immune responses than after the initial series without increasing the risk of GVHD or severe adverse effects.

Sirtuin 2-mediated deacetylation of cyclin-dependent kinase 9 promotes STAT1 signaling in type I interferon responses.

Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.

IFN-γ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5.

It is well established that activation of the transcription factor signal transducer and activator of transcription 1 (STAT1) is required for the interferon-γ (IFN-γ)-mediated antiviral response. Here, we found that IFN-γ receptor stimulation also activated Unc-51-like kinase 1 (ULK1), an initiator of Beclin-1-mediated autophagy. Furthermore, the interaction between ULK1 and the mitogen-activated protein kinase kinase kinase MLK3 (mixed lineage kinase 3) was necessary for MLK3 phosphorylation and downstream activation of the kinase ERK5. This autophagy-independent activity of ULK1 promoted the transcription of key antiviral IFN-stimulated genes (ISGs) and was essential for IFN-γ-dependent antiviral effects. These findings define a previously unknown IFN-γ pathway that appears to be a key element of the antiviral response.

Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus.

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038µM and 0.016µM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination (87%). They exhibited lower IC50 (0.98-6.2µM) compared with FPV (36.8µM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25µM). When dosed >50µM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.

Interferon γ (IFNγ) Signaling via Mechanistic Target of Rapamycin Complex 2 (mTORC2) and Regulatory Effects in the Generation of Type II Interferon Biological Responses.

We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses.

Small Molecule Agonists for the Type I Interferon Receptor: An In Silico Approach.

Type I interferons (IFNs) exhibit broad-spectrum antiviral activity, with potential utility against emerging acute virus infections that pose a threat to global health. Recombinant IFN-αs that have been approved for clinical use require cold storage and are administered through intramuscular or subcutaneous injection, features that are problematic for global distribution, storage, and administration. Cognizant that the biological potency of an IFN-α subtype is determined by its binding affinity to the type I IFN receptor, IFNAR, we identified a panel of small molecule nonpeptide compounds using an in silico screening strategy that incorporated specific structural features of amino acids in the receptor-binding domains of the most potent IFN-α, IFN alfacon-1. Hit compounds were selected based on ease of synthesis and formulation properties. In preliminary biological assays, we provide evidence that these compounds exhibit antiviral activity. This proof-of-concept study validates the strategy of in silico design and development for IFN mimetics.

Central role of ULK1 in type I interferon signaling.

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.

Regulatory effects of SKAR in interferon α signaling and its role in the generation of type I IFN responses.

We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.

Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.

We provide evidence that type I IFN-induced STAT activation is diminished in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2. Our studies show that transient or stable knockdown of Rictor or Sin1 results in defects in activation of elements of the STAT pathway and reduced STAT-DNA binding complexes. This leads to decreased expression of several IFN-inducible genes that mediate important biological functions. Our studies also demonstrate that Rictor and Sin1 play essential roles in the generation of the suppressive effects of IFNα on malignant erythroid precursors from patients with myeloproliferative neoplasms. Altogether, these findings provide evidence for critical functions for Rictor/Sin1 complexes in type I IFN signaling and the generation of type I IFN antineoplastic responses.

Small molecule mimetics of an interferon-α receptor interacting domain.

Small molecules that mimic IFN-α epitopes that interact with the cell surface receptor, IFNAR, would be useful therapeutics. One such 8-amino acid region in IFN-α2, designated IRRP-1, was used to derive 11 chemical compounds that belong to 5 distinct chemotypes, containing the molecular features represented by the key residues Leu30, Arg33, and Asp35 in IRRP-1. Three of these compounds exhibited potential mimicry to IRRP-1 and, in cell based assays, as predicted, effectively inhibited IFNAR activation by IFN-α. Of these, compound 3 did not display cell toxicity and reduced IFN-α-inducible STAT1 phosphorylation and STAT-DNA binding. Based on physicochemical properties' analyses, our data suggest that moieties with acidic pKa on the small molecule may be a necessary element for mimicking the carboxyl group of Asp35 in IRRP-1. Our data confirm the relevance of this strategy of molecular mimicry of ligand-receptor interaction domains of protein partners for small molecule drug discovery.

Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.

Role of Schlafen 2 (SLFN2) in the generation of interferon alpha-induced growth inhibitory responses.

The precise STAT-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I interferons (IFNs) remain unknown. We provide evidence that Type I IFNs regulate expression of Schlafens (SLFNs), a group of genes involved in the control of cell cycle progression and growth inhibitory responses. Using cells with targeted disruption of different STAT proteins and/or the p38 MAP kinase, we demonstrate that the IFN-dependent expression of distinct Schlafen genes is differentially regulated by STAT complexes and the p38 MAP kinase pathway. We also provide evidence for a key functional role of a member of the SLFN family, SLFN2, in the induction of the growth-suppressive effects of IFNs. This is shown in studies demonstrating that knockdown of SLFN2 enhances hematopoietic progenitor colony formation and reverses the growth-suppressive effects of IFNalpha on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form more colonies in soft agar, implicating this protein in the regulation of anchorage-independent growth. Altogether, our data implicate SLFN2 as a negative regulator of the metastatic and growth potential of malignant cells and strongly suggest a role for the SLFN family of proteins in the generation of the antiproliferative effects of Type I IFNs.

Activation of protein kinase C{eta} by type I interferons.

Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKCeta does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFNalpha on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKCeta as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis.

Dual regulatory roles of phosphatidylinositol 3-kinase in IFN signaling.

PI3K is activated by the type I and II IFN receptors, but its precise role in the generation of IFN responses is not well understood. In the present study we used embryonic fibroblasts from mice with targeted disruption of the genes encoding for both the p85alpha and p85beta regulatory subunits of PI3'-kinase (p85alpha(-/-)beta(-/-)) to precisely define the role of PI3K in the control of IFN-induced biological responses. Our data demonstrate that PI3K plays dual regulatory roles in the induction of IFN responses by controlling both IFN-alpha- and IFN-gamma-dependent transcriptional regulation of IFN-sensitive genes and simultaneously regulating the subsequent initiation of mRNA translation for such genes. These processes include the Isg15, Cxcl10, and/or Irf7 genes, whose functions are important in the generation of the biological effects of IFNs. Consistent with this, the induction of IFN antiviral responses is defective in double p85alpha/p85beta knockout cells. Thus, integration of signals via PI3K is a critical event during engagement of the IFN receptors that complements both the transcriptional activity of Jak-STAT pathways and controls initiation of mRNA translation.

De novo design of nonpeptidic compounds targeting the interactions between interferon-alpha and its cognate cell surface receptor.

Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus erythematosus, respectively. Specific sequences on the surface of IFN, IFN receptor recognition peptides (IRRPs) mediate the binding and signal transduction when IFN interacts with IFNAR. Structural features of two such IRRPs, IRRP-1 and IRRP-3, were used as templates to design small molecule mimetics. In silico screening was used to identify the molecular structural features mimicking their surface characteristics. A set of 26 compounds were synthesized and their ability to interfere with IFN-IFNAR interactions was investigated. Two compounds exhibited antagonist activity, specifically, blocking IFN-inducible Stat phosphorylation Stat complex-DNA binding. Design principles revealed here pave the way toward a novel series of small molecules as antagonists for IFN-IFNAR interactions.

Role of the Akt pathway in mRNA translation of interferon-stimulated genes.

Multiple signaling pathways are engaged by the type I and II IFN receptors, but their specific roles and possible coordination in the generation of IFN-mediated biological responses remain unknown. We provide evidence that activation of Akt kinases is required for IFN-inducible engagement of the mTOR/p70 S6 kinase pathway. Our data establish that Akt activity is essential for up-regulation of key IFN-alpha- and IFN-gamma-inducible proteins, which have important functional consequences in the induction of IFN responses. Such effects of the Akt pathway are unrelated to regulatory activities on IFN-dependent STAT phosphorylation/activation or transcriptional regulation. By contrast, they reflect regulatory activities on mRNA translation via direct control of the mTOR pathway. In studies using Akt1 and Akt2 double knockout cells, we found that the absence of Akt kinases results in dramatic reduction in IFN-induced antiviral responses, establishing a critical role of the Akt pathway in IFN signaling. Thus, activation of the Akt pathway by the IFN receptors complements the function of IFN-activated JAK-STAT pathways, by allowing mRNA translation of IFN-stimulated genes and, ultimately, the induction of the biological effects of IFNs.

Suppression of interferon (IFN)-inducible genes and IFN-mediated functional responses in BCR-ABL-expressing cells.

The interferons (IFNs) are cytokines that play key roles in host defense against viral infections and immune surveillance against cancer. We report that BCR-ABL transformation of hematopoietic cells results in suppression of IFN-dependent responses, including transcription of IFN-inducible genes and generation of IFN-mediated antiviral effects. BCR-ABL transformation suppresses expression of several IFN-regulated genes containing IFN-sensitive response element (ISRE) or GAS elements in their promoters, including Isg15, Irf1, Irf9, and Ifit2 (interferon-induced protein with tetratricopeptide repeats 2). Suppression of transcription of ISRE-containing genes is also seen in cells expressing various BCR-ABL kinase domain mutants, including T315I, H396P, Y253F, and E255K, but not kinase-defective BCR-ABL. Such effects are associated with impaired IFN-dependent phosphorylation of Stat1 on Tyr(701) and Stat3 on Tyr(705) and defective binding of Stat complexes to ISRE or GAS elements. Beyond suppression of Stat activities, BCR-ABL inhibits IFN-inducible phosphorylation/activation of the p38 MAPK, suggesting a dual mechanism by which this abnormal fusion protein blocks IFN transcriptional responses. The inhibitory activities of BCR-ABL ultimately result in impaired IFNalpha-mediated protection against encephalomyocarditis virus infection and reversal of IFN-dependent growth suppression. Altogether, our data provide evidence for a novel mechanism by which BCR-ABL impairs host defenses and promotes malignant transformation, involving dual suppression of IFN-activated signaling pathways.

Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling.

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNalpha or IFNgamma results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNalpha- or IFNgamma-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1(-/-)) knockout MEFs, as compared with wild-type 4E-BP1(+/+) MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2(-/-)) or hamartin (TSC1(-/-)) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.

CCL5-CCR5-mediated apoptosis in T cells: Requirement for glycosaminoglycan binding and CCL5 aggregation.

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.