Cutting edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells

IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate. Presently, we showed that CXCR1(+) cells were predominantly found among CD8(+) T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8(+) T cells. Indeed, human CMV-specific CD8(+) T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8(+) T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor. The results of a chemotaxis assay showed that the migration of CXCR1(+)CD8(+) T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8(+) T cells.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.     

CXCR3 in T cell function

CXCR3 is a chemokine receptor that is highly expressed on effector T cells and plays an important role in T cell trafficking and function. CXCR3 is rapidly induced on naïve cells following activation and preferentially remains highly expressed on Th1-type CD4⁺ T cells and effector CD8⁺ T cells. CXCR3 is activated by three interferon-inducible ligands CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC). Early studies demonstrated a role for CXCR3 in the trafficking of Th1 and CD8 T cells to peripheral sites of Th1-type inflammation and the establishment of a Th1 amplification loop mediated by IFNγ and the IFNγ-inducible CXCR3 ligands. More recent studies have also suggested that CXCR3 plays a role in the migration of T cells in the microenvironment of the peripheral tissue and lymphoid compartment, facilitating the interaction of T cells with antigen presenting cells leading to the generation of effector and memory cells.     

CXCR3 is induced early on the pathway of CD4+ T cell differentiation and bridges central and peripheral functions

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs, or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4(+) T cells. Using CD4(+) T cells from cord blood, we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu, although on resting cells, expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils, CXCR3(+)CD4(+) T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4(+) T cells by comparing its pattern of expression with that of CCR5, another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5, supporting a model of sequential induction of chemokine receptors during CD4(+) T cell differentiation. Taken together, our data show that CXCR3 can be expressed at all stages of CD4(+) T cell activation and differentiation, bridging central function in lymphoid organs and effector function in peripheral tissues.     

Decreased CXCR3+ CD8 T cells in advanced human immunodeficiency virus infection suggest that a homing defect contributes to cytotoxic T-lymphocyte dysfunction

To exert their cytotoxic function, cytotoxic T-lymphocytes (CTL) must be recruited into infected lymphoid tissue where the majority of human immunodeficiency virus (HIV) replication occurs. Normally, effector T cells exit lymph nodes (LNs) and home to peripheral sites of infection. How HIV-specific CTL migrate into lymphoid tissue from which they are normally excluded is unknown. We investigated which chemokines and receptors mediate this reverse homing and whether impairment of this homing could contribute to CTL dysfunction as HIV infection progresses. Analysis of CTL chemokine receptor expression in the blood and LNs of untreated HIV-infected individuals with stable, chronic infection or advanced disease demonstrated that LNs were enriched for CXCR3(+) CD8 T cells in all subjects, suggesting a key role for this receptor in CTL homing to infected lymphoid tissue. Compared to subjects with chronic infection, however, subjects with advanced disease had fewer CXCR3(+) CD8 T cells in blood and LNs. CXCR3 expression on bulk and HIV-specific CD8 T cells correlated positively with CD4 count and negatively with viral load. In advanced infection, there was an accumulation of HIV-specific CD8 T cells at the effector memory stage; however, decreased numbers of CXCR3(+) CD8 T cells were seen across all maturation subsets. Plasma CXCL9 and CXCL10 were elevated in both infected groups in comparison to the levels in uninfected controls, whereas lower mRNA levels of CXCR3 ligands and CD8 in LNs were seen in advanced infection. These data suggest that both CXCR3(+) CD8 T cells and LN CXCR3 ligands decrease as HIV infection progresses, resulting in reduced homing of CTL into LNs and contributing to immune dysfunction.     

CXCR3 ligands: redundant, collaborative and antagonistic functions

CXCR3 is a chemokine receptor that is rapidly induced on na?ve T cells following activation, and preferentially remains highly expressed on type-1 helper (Th1)-type CD4(+) T cells, effector CD8(+) T cells and innate-type lymphocytes, such as natural killer (NK) and NKT cells. CXCR3 is activated by three interferon (IFN)-γ-inducible ligands CXCL9 (monokine induced by gamma-interferon), CXCL10 (interferon-induced protein-10) and CXCL11 (interferon-inducible T-cell alpha chemoattractant). Although some studies have revealed that these ligands have redundant functions in vivo, other studies have demonstrated that the three CXCR3 ligands can also collaborate and even compete with each other. Differential regulation of the three ligands at specific times in defined anatomically restricted locations in vivo likely participates in the fine control of T-cell trafficking over the course of an immune response. Among the differences in regulation, CXCL10 is induced by a variety of innate stimuli that induce IFN-α/β as well as the adaptive immune cell cytokine IFN-γ, whereas CXCL9 induction is restricted to IFN-γ. In this review, we will discuss how the balance, timing and pattern of CXCR3 ligand expression appears to regulate the generation of effector T cells in the lymphoid compartment and subsequent migration into peripheral sites of Th1-type inflammation in which the CXCR3 ligands also then regulate the interactions and migratory behavior of effector T cells in an inflamed peripheral tissue.     

The Function of the Chemokine Receptor CXCR6 in the T Cell Response of Mice against Listeria monocytogenes

The chemokine receptor CXCR6 is expressed on different T cell subsets and up-regulated following T cell activation. CXCR6 has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16 on liver sinusoidal endothelial cells. Here, we analyzed the role of CXCR6 in CD8⁺ T cell responses to infection of mice with Listeria monocytogenes. CD8⁺ T cells responding to listerial antigens acquired high expression levels of CXCR6. However, deficiency of mice in CXCR6 did not impair control of the L. monocytogenes infection. CXCR6-deficient mice were able to generate listeria-specific CD4⁺ and CD8⁺ T cell responses and showed accumulation of T cells in the infected liver. In transfer assays, we detected reduced accumulation of listeria-specific CXCR6-deficient CD8⁺ T cells in the liver at early time points post infection. Though, CXCR6 was dispensable at later time points of the CD8⁺ T cell response. When transferred CD8⁺ T cells were followed for extended time periods, we observed a decline in CXCR6-deficient CD8⁺ T cells. The manifestation of this cell loss depended on the tissue analyzed. In conclusion, our results demonstrate that CXCR6 is not required for the formation of a T cell response to L. monocytogenes and for the accumulation of T cells in the infected liver but CXCR6 appears to influence long-term survival and tissue distribution of activated cells.     

Down-modulation of CXCR3 surface expression and function in CD8+ T cells from cutaneous T cell lymphoma patients

Viruses can escape destruction by the immune system by exploitation of the chemokine-chemokine receptor system. It is less established whether human cancers can adopt similar strategies to evade immunologic control. In this study, we show that advanced cutaneous T cell lymphoma (CTCL) is associated with selective and efficient inactivation of CXCR3-dependent T cell migration. Our studies demonstrate that this alteration is at least in part due to CXCR3 down-regulation in vivo by elevated serum levels of CXCR3 ligands. The T cell population most affected by this down-regulatory mechanism are CD8+ cytotoxic effector T cells. In CTCL patients, cytotoxic effector T cells have strongly reduced surface CXCR3 expression, accumulate in peripheral blood, but are virtually absent from CTCL tumor lesions, indicating an inability to extravasate into lymphoma tissue. CTCL-associated inactivation of effector cell recruitment may be a paradigmatic example of a new type of immune escape mechanisms shielding the neoplasm from a tumoricidal attack.     

CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system

IFN-gamma-inducible protein 10/CXCL10 is a chemokine associated with type 1 T cell responses, regulating the migration of activated T cells through binding to the CXCR3 receptor. Expression of both CXCL10 and CXCR3 are observed during immunopathological diseases of the CNS, and this receptor/ligand pair is thought to play a central role in regulating T cell-mediated inflammation in this organ site. In this report, we investigated the role of CXCL10 in regulating CD8(+) T cell-mediated inflammation in the virus-infected brain. This was done through analysis of CXCL10-deficient mice infected intracerebrally with lymphocytic choriomeningitis virus, which in normal immunocompetent mice induces a fatal CD8(+) T cell-mediated meningoencephalitis. We found that a normal antiviral CD8(+) T cell response was generated in CXCL10-deficient mice, and that lack of CXCL10 had no influence on the accumulation of mononuclear cells in the cerebrospinal fluid. However, analysis of the susceptibility of CXCL10-deficient mice to lymphocytic choriomeningitis virus-induced meningitis revealed that these mice just like CXCR3-deficient mice were partially resistant to this disease, whereas wild-type mice invariably died. Furthermore, despite marked up-regulation of the two remaining CXCR3 ligands: CXCL9 and 11, we found a reduced accumulation of CD8(+) T cells in the brain parenchyma around the time point when wild-type mice succumb as a result of CD8(+) T cell-mediated inflammation. Thus, taken together these results indicate a central role for CXCL10 in regulating the accumulation of effector T cells at sites of CNS inflammation, with no apparent compensatory effect of other CXCR3 ligands.     

Immunogenic,but Not Steady-State,Antigen Presentation Permits Regulatory T-Cells To Control CD8+ T-Cell Effector Differentiation by IL-2 Modulation

Absorption of IL-2 is one proposed mechanism of CD4+CD25+FoxP3+ regulatory T cell (Treg) suppression. Direct in vivo experimental evidence for this has recently been obtained. While modulation of IL-2 bioavailability controls CD8+ T-cell effector differentiation under strongly immunogenic conditions it is not known whether Treg modulate CD8+ T cell responses through this mechanism under steady-state conditions. Here we assess this using a mouse model in which dendritic cells (DC) are manipulated to present cognate antigen to CD8+ T cells either in the steady-state or after activation. Our observations show that Treg exert a check on expansion and effector differentiation of CD8+ T cells under strongly immunogenic conditions associated with TLR ligand activation of DC, and this is mediated by limiting IL-2 availability. In contrast, when DC remain unactivated, depletion of Treg has little apparent effect on effector differentiation or IL-2 homeostasis. We conclude that while modulation of IL-2 homeostasis is an important mechanism through which Treg control CD8+ effector differentiation under immunogenic conditions, this mechanism plays little role in modulating CD8+ T-cell differentiation under steady-state conditions.     

Opposing effects of CXCR3 and CCR5 deficiency on CD8+ T cell-mediated inflammation in the central nervous system of virus-infected mice

T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice were generated and infected intracerebrally with noncytolytic lymphocytic choriomeningitis virus. Because these chemokine receptors are mostly expressed by overlapping subsets of activated CD8+ T cells, it was expected that absence of both receptors would synergistically impair effector T cell invasion and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CXCR3/CCR5-deficient mice, more CD8+ T cells were found in the parenchyma of double-deficient mice when these were analyzed around the time when the difference in clinical outcome becomes manifest. Taken together, these results indicate that while CXCR3 plays an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response.     

CXCR1+CD4+ T cells in human allergic disease

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-gamma expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.     

CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells

CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Valpha24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with alpha-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX(3)CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.     

CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system

The chemokine receptor CXCR3 promotes the trafficking of activated T and NK cells in response to three ligands, CXCL9, CXCL10, and CXCL11. Although these chemokines are produced in the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), their role in the pathogenesis of CNS autoimmunity is unresolved. We examined the function of CXCR3 signaling in EAE using mice that were deficient for CXCR3 (CXCR3(-/-)). The time to onset and peak disease severity were similar for CXCR3(-/-) and wild-type (WT) animals; however, CXCR3(-/-) mice had more severe chronic disease with increased demyelination and axonal damage. The inflammatory lesions in WT mice consisted of well-demarcated perivascular mononuclear cell infiltrates, mainly in the spinal cord and cerebellum. In CXCR3(-/-) mice, these lesions were more widespread throughout the CNS and were diffused and poorly organized, with T cells and highly activated microglia/macrophages scattered throughout the white matter. Although the number of CD4(+) and CD8(+) T cells infiltrating the CNS were similar in CXCR3(-/-) and WT mice, Foxp3(+) regulatory T cells were significantly reduced in number and dispersed in CXCR3(-/-) mice. The expression of various chemokine and cytokine genes in the CNS was similar in CXCR3(-/-) and WT mice. The genes for the CXCR3 ligands were expressed predominantly in and/or immediately surrounding the mononuclear cell infiltrates. We conclude that in EAE, CXCR3 signaling constrains T cells to the perivascular space in the CNS and augments regulatory T cell recruitment and effector T cell interaction, thus limiting autoimmune-mediated tissue damage.     

Tissue-Protective Effects of NKG2A in Immune-Mediated Clearance of Virus Infection

Virus infection triggers a CD8⁺ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8⁺ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1^b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8⁺ T cells. We have previously shown that Qa1^b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8⁺ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8⁺ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8⁺ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8⁺ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8⁺ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.