Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

Dynamics of Cytokine/Chemokine Responses in Intestinal CD4+ and CD8+ T Cells during Acute Simian Immunodeficiency Virus Infection

Loss of intestinal CD4⁺ T cells was associated with decreased production of several T-helper 1 (T_H1) and T_H2 cytokines and increased production of interleukin 17 (IL-17), gamma interferon (IFN-γ), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD8⁺ T cells 21 days after simian immunodeficiency virus (SIV) infection in rhesus macaques. Shifting of mucosal T_H1 to T_H2 or T-cytotoxic 1 (T_C1) to T_C2 cytokine profiles was not evident. Additionally, both CD4⁺ and CD8⁺ T cells showed upregulation of macrophage migration inhibition factor (MIF) and basic fibroblast growth factor (FGF-basic) cytokines that have been linked to HIV disease progression.     

Targeting the Shift from M1 to M2 Macrophages in Experimental Autoimmune Encephalomyelitis Mice Treated with Fasudil

We observed the therapeutic effect of Fasudil and explored its mechanisms in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Fasudil, a selective Rho kinase (ROCK) inhibitor, was injected intraperitoneally at 40 mg/kg/d in early and late stages of EAE induction. Fasudil ameliorated the clinical severity of EAE at different stages, and decreased the expression of ROCK-II in spleen, accompanied by an improvement in demyelination and inhibition of inflammatory cells. Fasudil mainly inhibited CD4⁺IL-17⁺ T cells in early treatment, but also elevated CD4⁺IL-10⁺ regulatory T cells and IL-10 production in late treatment. The treatment of Fasudil shifted inflammatory M1 to anti-inflammatory M2 macrophages in both early and late treatment, being shown by inhibiting CD16/32, iNOS, IL-12, TLR4 and CD40 and increasing CD206, Arg-1, IL-10 and CD14 in spleen. By using Western blot and immunohistochemistry, iNOS and Arg-1, as two most specific markers for M1 and M2, was inhibited or induced in splenic macrophages and spinal cords of EAE mice treated with Fasudil. In vitro experiments also indicate that Fasudil shifts M1 to M2 phenotype, which does not require the participation or auxiliary of other cells. The polarization of M2 macrophages was associated with the decrease of inflammatory cytokine IL-1β, TNF-α and MCP-1. These results demonstrate that Fasudil has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

M2-like macrophages polarized by Foxp3− Treg-of-B cells ameliorate imiquimod-induced psoriasis

Our group have demonstrated that splenic B cells contributed to the CD4⁺CD25⁻ naive T cells conversion into CD4⁺CD25⁺Foxp3⁻ regulatory T cells without adding appended cytokines, named Treg-of-B cells which were potent suppressors of adaptive immunity. We like to investigate whether Treg-of-B cells could promote alternatively activated macrophage (M2 macrophages) polarization and alleviate inflammatory disease, psoriasis. In this study, we co-cultured the bone marrow-derived macrophages (BMDMs) with Treg-of-B cells under LPS/IFN-γ stimulation and analyzed the M2-associated gene and protein using qPCR, western blotting, and immunofluorescence staining. We also examined the therapeutic effect of Treg-of-B cell-induced M2 macrophage for skin inflammation using imiquimod (IMQ)-induced psoriatic mouse model. Our results showed that BMDMs co-cultured with Treg-of-B cells upregulated typical M2-associated molecules, including Arg-1, IL-10, Pdcd1lg2, MGL-1, IL-4, YM1/2 and CD206. In an inflammatory environment, TNF-α and IL-6 production by macrophages co-cultured with Treg-of-B cells was decreased significantly. The molecular mechanism revealed that Treg-of-B cells promoted M2 macrophage polarization via STAT6 activation in a cell contact-dependent manner. Moreover, the treatment with Treg-of-B cell-induced M2 macrophages attenuated the clinical manifestations of psoriasis, such as scaling, erythema and thickening in the IMQ-induced psoriatic mouse model. T cell activation in draining lymph nodes was decreased in the Treg-of-B cell-induced M2 macrophage group after IMQ application. In conclusion, our findings suggested that Foxp3⁻ Treg-of-B cells could induce alternatively activated M2 macrophages through STAT6 activation, providing a cell-based therapeutic strategy for psoriasis.     

The Effects of Co-transplantation of Olfactory Ensheathing Cells and Schwann Cells on Local Inflammation Environment in the Contused Spinal Cord of Rats

Inflammatory response following spinal cord injury (SCI) is important in regulation of the repair process. Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are important donor cells for repairing SCI in different animal models. However, synergistic or complementary effects of co-transplantation of both cells for this purpose have not been extensively investigated. In the present study, we investigated the effects of co-transplantation of OECs and SCs on expression of pro- or anti-inflammatory factor and polarization of macrophages in the injured spinal cord of rats. Mixed cell suspensions containing OECs and SCs were transplanted into the injured site at 7 days after contusion at the vertebral T10 level. Compared with the DMEM, SC, or OEC group, the co-transplantation group had a more extensive distribution of the grafted cells and significantly reduced number of astrocytes, microglia/macrophage infiltration, and expression of chemokines (CCL2 and CCL3) at the injured site. The co-transplantation group also significantly increased arginase⁺/CD206⁺ macrophages (IL-4) and decreased iNOS⁺/CD16/32⁺ macrophages (IFN-γ), which was followed by higher IL-10 and IL-13 and lower IL-6 and TNF-α in their expression levels, a smaller cystic cavity area, and improved motor functions. These results indicate that OEC and SC co-transplantation could promote the shift of the macrophage phenotype from M(IFN-γ) to M(IL-4), reduce inflammatory cell infiltration in the injured site, and regulate inflammatory factors and chemokine expression, which provide a better immune environment for SCI repair.     

CD8+ T cell/adipocyte inflammatory cross talk and ensuing M1 macrophage polarization are reduced by fish-oil-derived n-3 polyunsaturated fatty acids,in part by a TNF-α-dependent mechanism

Obese visceral adipose tissue (AT) inflammation is driven by adipokine-mediated cross talk between CD8⁺ T cells and adipocytes, a process mitigated by long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) but underlying mechanisms and ensuing effects on macrophage polarization status are unknown. Using an in vitro co-culture model that recapitulates the degree of CD8⁺ T cell infiltration reported in obese AT, 3T3-L1 adipocytes were co-cultured for 24 h with purified splenic CD8⁺ T cells from C57Bl/6 mice consuming either a 10% w/w safflower oil (control, CON) or 7% w/w safflower oil + 3% w/w fish oil (FO) diet for 4 weeks (n=8–10/diet). Co-cultured cells were in direct contact or in a contact-independent condition separated by a Transwell permeable membrane and stimulated with lipopolysaccharide (10 ng/ml) to mimic in vivo obese endotoxin levels. In contact-dependent co-cultures, FO reduced inflammatory (IL-6, TNFα, IFN-γ) and macrophage chemotactic (CCL2, CCL7, CCL3) mRNA expression and/or secreted protein, NF-κB p65 activation, ROS accumulation, NLRP3 inflammasome priming (Nlrp3, Il1β mRNA) and activation (caspase-1 activity) compared to CON (P<.05). The anti-inflammatory action of FO was reproduced by the addition of a TNF-α neutralizing antibody (1 μg/ml) to CON co-cultures (CON/anti-TNF-α), albeit to a lesser degree. Conditioned media from FO and CON/anti-TNF-α co-cultures, in turn, reduced RAW 264.7 macrophage mRNA expression of M1 polarization markers (iNos, Cd11c, Ccr2) and associated inflammatory cytokines (Il6, Tnfα, Il1β) compared to CON. These data suggest that inflammatory CD8⁺ T cell/adipocyte cross talk is partially attributable to TNF-α signaling, which can be mitigated by LC n-3 PUFA.     

Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment

 Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4⁺ and CD8⁺ T cells expressing certain families of T cell receptor (TCR) variable-region β (V_β) chain. T cells respond with profound cytokine production and induction of cytotoxicity. Repeated injections, however, cause deletion and anergy of both CD4⁺ and CD8⁺ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V_β11⁺ as well as reduced cytokine levels in serum upon challenge with SEA. Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4⁺ and CD8⁺ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing down-regulation of endogenous IL-2 production in vivo. Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor α, interferon γ and IL-6, on a cellular level. These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy. Received: 29 August 1996 / Accepted: 16 January 1997     

Human Cytomegalovirus Interleukin-10 Polarizes Monocytes toward a Deactivated M2c Phenotype To Repress Host Immune Responses

Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14⁺ monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4⁺ T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4⁺ T cell responses at sites of infection.     

Suppression of murine experimental autoimmune encephalomyelitis by interleukin-2 receptor targeted fusion toxin, DAB389IL-2

Previously we have shown that DAB₃₈₉IL-2, a recombinant fusion toxin targeting IL-2R bearing cells, suppressed disease in the rat experimental autoimmune encephalomyelitis (EAE) model of acute multiple sclerosis (MS). Our present study demonstrates that DAB₃₈₉IL-2 can also effectively suppress acute (A)-EAE, relapsing (R)-EAE and chronic (C)-EAE in mouse demyelinating models. DAB₃₈₉IL-2 significantly suppressed mitogenic proliferation of spleen cells while mutant fusion proteins DA_glu53B₃₈₉IL-2 and DAB₃₈₉IL-2_8-10 did not. EAE was successfully suppressed when DAB₃₈₉IL-2 was administered in various regimens between days 1 and 15 post immunization in all three models. CD4⁺IL-2R⁺ cells were reduced in the spleen but not in the lymph nodes of DAB₃₈₉IL-2-treated mice during A-EAE while the number of CD8⁺ cells was unchanged. DAB₃₈₉IL-2 also significantly reduced the number of CD4⁺, CD8⁺, CD25⁺, TCRγδ⁺ phenotype and CD11b⁺ macrophages/microglia within spinal cord lesions. These data strongly suggest that DAB₃₈₉IL-2 specifically targeted myelin protein-activated CD4⁺ T cells and strengthens the argument for the use of DAB₃₈₉IL-2 in treatment strategies for MS.     

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity,which is suppressed by regulatory T cells in breast cancer

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8⁺ T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8⁺ T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8⁺ T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8⁺ T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8⁺ T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8⁺ T cells but could significantly enhance IL-2-mediated promotion of CD8⁺ T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8⁺ T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4⁺CD25⁺ regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8⁺ T cells, an effect suppressible by CD4⁺CD25⁺ Treg cells.     

4-1BB Signaling Enhances Primary and Secondary Population Expansion of CD8+ T Cells by Maximizing Autocrine IL-2/IL-2 Receptor Signaling

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is primarily expressed on activated T cells and is known to enhance proliferation of T cells, prevent activation-induced cell death, and promote memory formation of CD8⁺ T cells. In particular, it is well acknowledged that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells rather than CD4⁺ T cells, but the underlying mechanism remains unclear. Here we found that 4-1BB triggering markedly increased IL-2Rα (CD25) and IL-2 expressions of CD8+ T cells but minimally for CD4⁺ T cells. Proliferation of CD8⁺ T cells was moderately enhanced by direct 4-1BB triggering in the absence of signaling through IL-2Rα/IL-2 interactions, but further promoted in the presence of IL-2Rα/IL-2 interactions. Among the TNFRSF members including OX40, GITR, CD30, and CD27, 4-1BB was superior in the ability to induce IL-2Rα expression on CD8+ T cells. When the primary and secondary expansions of CD8⁺ T cells in vivo were examined by adoptively transferring OVA-specific CD8⁺ T cells along with the treatment with agonistic anti-4-1BB and/or antagonistic anti-CD25 F(ab’)₂ mAb, 4-1BB triggering enhanced both primary and secondary expansion of CD8⁺ T cells in vivo, and the 4-1BB effects were moderately suppressed in primary expansion while completely abolished in secondary expansion of OVA-specific CD8⁺ T cells by blocking IL-2Rα. These results suggest that 4-1BB-mediated increases of IL-2Rα and IL-2 prolong the effects of transient TCR- and 4-1BB-mediated signaling in CD8⁺ T cells, and that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells through the amplification of autocrine IL-2/IL-2R signaling loop.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of human antigen-specific memory CD8<Superscript>+</Superscript> T cells

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8⁺ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8⁺ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8⁺ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8⁺ T cells. The lytic capacity of memory CTLs toward CEA⁺ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8⁺ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8⁺ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8⁺ T cells.     

IL-32γ induces chemotaxis of activated T cells via dendritic cell-derived CCL5

Interleukin (IL)-32 has been associated with a variety of inflammatory diseases including rheumatoid arthritis, vasculitis and Crohn’s disease. We have previously reported that IL-32γ, the IL-32 isoform with the highest biological activity, could act as an immune modulator through regulation of dendritic cell (DC) functions in immune responses. Cell locomotion is crucial for induction of an effective immune response. In this study, we investigated the effect and underlying mechanisms of IL-32γ on recruitment of T cells. IL-32γ upregulated the expression of several chemokines including CCL2, CCL4, and CCL5 in the DCs. In particular, IL-32γ significantly increased CCL5 expression in a dose-dependent manner. Treatment with JNK and NF-κB inhibitors suppressed IL-32γ-induced CCL5 expression in DCs, indicating that IL-32γ induced CCL5 production through the JNK and NF-κB pathways. Furthermore, supernatants from IL-32γ-treated DCs showed chemotactic activities controlling migration of activated CD4⁺ and CD8⁺ T cells, and these activities were suppressed by addition of neutralizing anti-CCL5 antibody. These results show that IL-32γ effectively promotes migration of activated T cells via CCL5 production in DCs. The chemotactic potential of IL-32γ may explain the pro-inflammatory effects of IL-32 and the pathologic role of IL-32 in immune disorders such as rheumatoid arthritis.