Chronic myelogenous leukemia maintains specific CD8+ T cells through IL‐7 signaling

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells. The disease progresses after several years from an initial chronic phase to a blast phase. Leukemia‐specific T cells are regularly detected in CML patients and may be involved in the immunological control of the disease. Here, we analyzed the role of leukemia‐specific CD8⁺ T cells in CML disease control and the mechanism that maintains CD8⁺ T‐cell immunosurveillance in a retroviral‐induced murine CML model. To study antigen‐specific immune responses, the glycoprotein of the lymphocytic choriomeningitis virus was used as model leukemia antigen. Leukemia‐specific CTL activity was detectable in vivo in CML mice and depletion of CD8⁺ T cells rapidly led to disease progression. CML‐specific CTL were characterized by the expression of the IL‐7 receptor α‐chain. In addition, leukemia cells produced IL‐7 that was crucial for the maintenance of leukemia‐specific CTL and for disease control. Therefore, CML cells maintain the specific CD8⁺ T‐cell‐mediated immune control by IL‐7 secretion. This results in prolonged control of disease and probably contributes to the characteristic chronic phase of the disease.     

CD28 days later: Resurrecting costimulation for CD8+ memory T cells

Rapid activation and proliferative expansion of specific CD8⁺ memory T (CD8⁺T_M) cells upon antigen re‐encounter is a critical component of the adaptive immune response that confers enhanced immune protection. In this context, however, the requirements for costimulation in general, and CD28 signaling in particular, remain incompletely defined. In the current issue of the European Journal of Immunology, Fröhlich et al. [Eur. J. Immunol. 2016. 46: 1644‐1655] provide definitive evidence that optimal elaboration of CD8⁺T_M‐cell recall responses is indeed contingent on CD28 expressed by these cells. Here, we discuss the “CD28 costimulation paradigm” in its historical context and highlight some of the unresolved complexities pertaining to CD28‐dependent interactions that shape CD8⁺ T‐cell phenotypes, functionalities, and recall reactivity.     

HIV-specific CD8 T cells express low levels of IL-7Ralpha: implications for HIV-specific T cell memory

Chronic infections in mice can result in defects in memory CD8 T cell properties including low expression of the IL-7Ralpha (CD127). To determine whether defects in memory CD8 T cell formation exist during human chronic infections and to what extent these defects may be allele- or epitope-specific, we compared influenza (Flu), vaccinia (VV) and EBV-specific CD8 T cells to HIV-specific CD8 T cells, using a panel of 13 HIV tetramers. Compared to Flu, VV or EBV, HIV tetramer+ CD8 T cells expressed significantly lower levels of CD127, and this reduction was pervasive across all epitopes and alleles tested and over a wide range of viral loads and CD4 counts. These results indicate impaired HIV-specific memory CD8 T cell differentiation, regardless of level of control of viremia, epitopes targeted or restricting HLA alleles.     

Circulating memory CD8+ T cells are limited in forming CD103+ tissue-resident memory T cells at mucosal sites after reinfection

Tissue-resident memory CD8⁺ T cells (T_RM) localize to barrier tissues and mediate local protection against reinvading pathogens. Circulating central memory (T_CM) and effector memory CD8⁺ T cells (T_EM) also contribute to tissue recall responses, but their potential to form mucosal T_RM remains unclear. Here, we employed adoptive transfer and lymphocytic choriomeningitis virus reinfection models to specifically assess secondary responses of T_CM and T_EM at mucosal sites. Donor T_CM and T_EM exhibited robust systemic recall responses, but only limited accumulation in the small intestine, consistent with reduced expression of tissue-homing and -retention molecules. Murine and human circulating memory T cells also exhibited limited CD103 upregulation following TGF-β stimulation. Upon pathogen clearance, T_CM and T_EM readily gave rise to secondary T_EM. T_CM also formed secondary central memory in lymphoid tissues and T_RM in internal tissues, for example, the liver. Both T_CM and T_EM failed to substantially contribute to resident mucosal memory in the small intestine, while activated intestinal T_RM, but not liver T_RM, efficiently reformed CD103⁺ T_RM. Our findings demonstrate that circulating T_CM and T_EM are limited in generating mucosal T_RM upon reinfection. This may pose important implications on cell therapy and vaccination strategies employing memory CD8⁺ T cells for protection at mucosal sites.     

Frontline: An in-depth evaluation of the production of IL-2 by antigen-specific CD8 T cells in vivo

IL-2 is an important cytokine that is capable of inducing both proliferation and apoptosis of activated T cells. CD4 T cells are thought to be the major producers of IL-2, but CD8 T cells also produce copious amounts of this cytokine. However, our current understanding regarding the kinetics of IL-2 production by antigen-specific CD8 T cells, and the proportion of these cells that produce IL-2 in vivo, is extremely limited. We now demonstrate that virus-specific CD8 T cells initiate IL-2 production by 6 h post-infection and prior to cell division in vivo. Interestingly, peak levels of IL-2 production were achieved very early during the response and prior to the proliferative peak. We also show -- using transgenic mice expressing herpes simplex virus-1 thymidine kinase under the control of the IL-2 promoter -- that, unlike what has been reported for antigen-specific CD4 T cells, the majority of antigen-specific CD8 T cells produce IL-2 during primary as well as secondary immune responses in vivo.     

IL-15-independent antiviral function of primary and memory CD8+ T cells

Memory CD8+ T cells are comprised of CD122hi IL-15-dependent and CD122lo IL-15-independent subsets. Induction and retention of IL-15-independent memory CD8+ T cells was assessed in IL-15-/- and wild-type (wt) mice immunized with recombinant vaccinia virus (rVV) or Sindbis virus (rSIN) vectors expressing the identical foreign epitope. Both vectors induced epitope-specific CD8+ T cell expansion and function, independent of IL-15. Similar kinetics of rVV clearance confirmed effective CD8+ T cell function in IL-15-/- mice. CD44hi CD122hi CD8+ T cells, mainly of the CD62L-/lo phenotype, increased more dramatically and declined more rapidly in IL-15-/- mice, independent of the vector. Rapid IL-15-independent memory CD8+ T cell expansion following challenge of immune mice compensated for the limited memory CD8+ populations in IL-15-/- mice. However, despite expansion and expression of potent effector function, viral clearance was delayed in the absence of IL-15, coinciding with a rapid loss in cytolytic function.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

Critical role for IL-21 in both primary and memory anti-viral CD8+ T-cell responses

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.     

Defect of CD8^＋ Memory T Cells Developed in Absence of IL-12 Priming for Secondary Expansion

IL-12 priming plays an important role in stimulation of CD8^＋ effector T cells and development of CD8^＋ memory T （Tm） cells. However, the functional alteration of CD8^＋ Tm cells developed in the absence of IL-12 priming is elusive. In this study, we investigated the capacity of secondary expansion of CD8~ Tm cells developed from transgenic OT I CD8^＋ T cells. The latter cells were in vitro and in vivo stimulated by ovalbumin （OVA）-pulsed dendritic cells [DCovA and （IL-12^-/-）DCovA] derived from wild-type C57BL/6 and IL-12 gene knockout mice, respectively. We demonstrated that IL-12 priming is important not only in CD8^＋ T cell clonal expansion, but also in generation of CD8^＋ Tm cells with the capacity of secondary expansion upon antigen re-encounter. However, IL-12 signaling is not involved in CD8^＋ Tm cell survival and recall responses. Therefore, this study provides useful information for vaccine design and development. Cellular ＆ Molecular Immunology.     

Proliferation and interleukin 5 production by CD8hi CD57+ T cells

CD8hi CD57+ T cells have previously been described as effector memory T cells with minimal expansion capacity and high susceptibility to activation-induced cell death. In contrast, we demonstrate here that CD8hi CD57+ T cells are capable of rapid expansion using multiple techniques including [(3)H]thymidine uptake, flow cytometric bead-based enumeration and standard haemocytometer counting. Previous reports can be explained by marked inhibition of activation-induced expansion and increased 7-amino-actinomycin D uptake by CD8hi CD57+ T cells following treatment with CFSE, a dye previously used to measure their proliferation, combined with specific media requirements for the growth of this cell subset. The ability of CD8hi CD57+ T cells to further differentiate is highlighted by a distinct cytokine profile late after activation that includes the unexpected release of high levels of interleukin 5. These data indicate that CD8hi CD57+ T cells should not be considered as "end-stage" effector T cells incapable of proliferation, but represent a highly differentiated subset capable of rapid division and exhibiting novel functions separate from their previously described cytotoxic and IFN-gamma responses.     

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.     

CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART)

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL-7R alpha chain) by memory CD8 T lymphocytes in HIV-infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four-colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127(+) cells among memory CD8 lymphocytes that resulted in a higher CD127(-) CD45RA(-)CD27(+) CD8 T cell count in HIV-infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti-retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down-regulation of CD127 by interleukin (IL)-7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL-7 confirmed that IL-7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV-infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL-7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.     

人类CD8+记忆T细胞体外扩增方法的研究

 目的： 研究体外扩增人类CD8+记忆T细胞的新方法,为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段。方法：将anti-CD3抗体、anti-CD28抗体、CD70、白细胞介素（IL）-2、IL-7和IL-15进行排列组合,设计出63种刺激方式,对体外分离得到的正常人外周血CD8+ T细胞进行体外扩增;培养14 d后进行细胞计数,并检测CD8+ T细胞的纯度以及CD8+中枢记忆T细胞（TCM）和CD8+效应记忆T细胞（TEM）所占的比例,进而计算出CD8+ T细胞、CD8+ TCM和CD8+ TEM的体外扩增倍数,从而确定理想的刺激方法。结果：体外扩增CD8+ T细胞、CD8+ TCM和CD8+ TEM的理想刺激方式均为anti-CD3抗体、IL-2和IL-7三者的组合;该刺激方式使3种细胞在培养14 d后分别扩增了13.19、13.28和15.27倍。结论：Anti-CD3抗体、IL-2和IL-7三者的组合,是刺激人类CD8+记忆T细胞体外扩增的相对理想方法。     

Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization

Transplantation tolerance induced by neonatal injection of semi-allogeneic spleen cells is associated with a pathological syndrome caused by T helper type 2 (Th2) differentiation of donor-specific CD4(+) T lymphocytes. We have shown previously that this Th2-biased response is inhibited by host CD8(+) T cells. Herein, we demonstrate that upon neonatal immunization with (A/J × BALB/c)F(1) spleen cells, BALB/c mice expand a population of CD8(+) T cells expressing both CD25 and forkhead box P3 (FoxP3) markers. In this setting, CD8(+) CD25(+) T cells predominantly produce interferon (IFN)-γ and interleukin (IL)-10 and are efficient in controlling IL-4, IL-5 and IL-13 production by donor-specific CD4(+) T cells in vitro. CD8(+) FoxP3(-) T cells are single producers of IFN-γ or IL-10, whereas CD8(+) FoxP3(+) T cells are double producers of IFN-γ and IL-10. We further demonstrate that IFN-γ and IL-10 are two major cytokines produced by CD8(+) T cells involved in the in vivo regulation of Th2-type pathology. In this setting, we conclude that neonatal alloimmunization induces the expansion of several regulatory CD8(+) T cells which may control Th2 activities via IFN-γ and IL-10.     

Enhancement of proliferation and downregulation of TRAIL expression on CD8+ T cells by IL-21

Mounting evidence indicates that the cytokine IL-21 can significantly enhance the survival of CD8(+) T lymphocytes. Given that CD4(+) T lymphocytes constitute the main cellular source for IL-21 in vivo, it is tempting to speculate a direct role in mediating the "help" provided by these CD4(+) T cells to the CD8 response. A new report in this issue of the European Journal of Immunology advances this notion by showing that CD8(+) T cells lacking the IL-21 receptor phenocopy those primed in the absence of CD4(+) T cells (the so-called "helpless" CD8(+) T cells) in their induction of the pro-apoptotic factor TRAIL. This finding helps to define the role of IL-21 in the CD8 response, and raises new questions relevant for achieving a broader understanding of this multifunctional cytokine.     

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells

Low CD8⁺ T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8⁺ T lymphocyte numbers. The A‐A‐T haplotype was associated with a severe clinical expression of HH and low CD8⁺ T lymphocyte numbers, while the G‐G‐G haplotype was associated with a milder clinical expression of HH and high CD8⁺ T lymphocyte numbers. As CD8⁺ T lymphocytes are a very heterogeneous population, in this study we analysed the CD8⁺ subpopulations of naive, central memory (T_CM) and effector memory (T_EM), and further subsets of CD8⁺ T_EM cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8⁺ T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T_EM cells and the T_EM CD27^‐CD28^‐ subset no effect of age was observed in HH [R² = 0·001, not significant (n.s.) and R² = 0·01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R² = 0·09, P = 0·017 and R² = 0·22, P = 0·0005, respectively). Interestingly, patients homozygous for the A‐A‐T haplotype have lower numbers of CD8⁺ T_EM cells due especially to lower numbers of T_EM CD27^‐CD28^‐ (0·206 ± 0·119 and 0·066 ± 0·067 × 10⁶ cells/ml, respectively) than patients carrying the G‐G‐G haplotype (0·358 ± 0·195 and 0·246 ± 0·202 × 10⁶ cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8⁺ T cells into the most mature phenotype.     

Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infections

IL-2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down-regulating T cell responses by inducing activation-induced cell death as well as regulatory T cells. The in vivo analysis of IL-2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL-2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL-2R-competent and IL-2R-deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL-2R signaling during acute/resolved viral infection. In marked contrast, IL-2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus-specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL-2R signaling is either essential or largely dispensable for induction and maintenance of virus-specific CD8+ T cell responses.     

TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 T cells

Generation and maintenance of antigen-specific effector and memory T cells are central events in immune responses against infections. We show that TNF receptor-associated factor 2 (TRAF2) maintains a survival signaling axis in effector and memory CD8 T cells required for immune responses against infections. This signaling axis involves activation of Tpl2 and its downstream kinase ERK by NF-κB-inducing kinase (NIK) and degradation of the proapoptotic factor Bim. NIK mediates Tpl2 activation by stimulating the phosphorylation and degradation of the Tpl2 inhibitor p105. Interestingly, while NIK is required for Tpl2-ERK signaling under normal conditions, uncontrolled NIK activation due to loss of its negative regulator, TRAF2, causes constitutive degradation of p105 and Tpl2, leading to severe defects in ERK activation and effector/memory CD8 T cell survival. Thus, TRAF2 controls a previously unappreciated signaling axis mediating effector/memory CD8 T cell survival and protective immunity.