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.     

Generating functional CD8+ T cell memory response under transient CD4+ T cell deficiency: implications for vaccination of immunocompromised individuals

Studies based on either MHC class II-knockout or CD4+ T cell-depleted murine models have demonstrated a critical role for CD4+ T cells in the generation of CD8+ T cell memory. However, it is difficult to extend these findings to immunocompromised humans where a complete loss of CD4+ T cells is rarely observed. Here, we have developed a model setting, which allows studies on the generation of CD8+ T cell memory responses in a transient CD4+ T cell-deficient setting similar to that seen in immunocompromised patients. Immunisation with an adenoviral vaccine under transient helpless or help-deficient conditions showed varying degrees of impact on the priming of CD8+ T cell responses. Antigen-specific T cells generated under normal CD4+ T cell help and transient help-deficient conditions showed similar effector phenotype and were capable of proliferation upon secondary antigen encounter. Most importantly, in spite of CD4+ T cell deficiency, the long-term CD8+ T cell memory response remained functionally stable and showed comparable cytotoxic effector function as seen in CD8+ T cells generated with normal CD4+ T cell numbers. These findings provide evidence that in spite of partially impaired activation of a primary CD8+ T cell response, a fully functional and stable memory CTL response can be induced under conditions of severe transient CD4+ T cell deficiency.     

A distinct role for ICOS-mediated co-stimulatory signaling in CD4+ and CD8+ T cell subsets

While the ligand of inducible co-stimulator (ICOS), B7 homologous protein, is widely expressed in somatic cells, B7-1 and B7-2 expression is mainly limited to lymphoid organs. Thus, the activation of T cells through ICOS without a CD28-mediated signal may occur in physiological situations. In order to gain a better understanding of the role of the ICOS co-stimulatory signal in immune responses, we studied the cellular response of T cells to beads coated with anti-ICOS or anti-CD28, plus sub-optimal anti-CD3 mAb. We demonstrate that while CD28 ligation induced expansion of both CD4+ and CD8+ populations, ICOS ligation only resulted in the expansion of CD8+ T cells, and induced apoptosis in the CD4+ T cell population. It was found that IL-2 is critically required for CD8+ T cell expansion triggered by ICOS ligation, whereas it had only a limited effect on the expansion of CD4+ T cells. This distinct reactivity of CD4+ and CD8+ T cell populations to exogenous IL-2 strongly correlates with the expression level of IL-2 receptor beta-chain, CD122, on T cells. Furthermore, we defined a small but distinct population of memory phenotype CD4+ T cells that constitutively express ICOS. Interestingly, while naive CD4+ T cells were unable to produce IL-2, ICOS-expressing T cells produced a substantial amount of IL-2 by stimulation with anti-ICOS/CD3 beads, suggesting that IL-2, which is indispensable for CD8+ T cell expansion, is produced by this ICOS-expressing T cell population. These results provide evidence indicating that the ICOS co-stimulatory signal plays a distinct role in the development of CD4+ and CD8+ T cell-mediated immune responses.     

CD4+ T-cell help amplifies innate signals for primary CD8+ T-cell immunity

CD8⁺ T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8⁺ T-cell immunity have been shown to depend on CD4⁺ T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4⁺ T cells, various dendritic cell subsets, and CD8⁺ T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8⁺ T-cell expansion and differentiation. This model facilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity.     

Split peripheral tolerance: CD40 ligation blocks tolerance induction for CD8 T cells but not for CD4 T cells in response to intestinal antigens

The role of antigen-presenting cells in the balance between immunity and tolerance to intestinal antigens remains poorly understood. In this study, we examined whether CD40 ligation affects the induction of CD4 and CD8 T cell tolerance in response to intestinal antigens. We show that an agonistic anti-CD40 mAb treatment did not block the induction of OVA-specific CD4 T cell tolerance, whereas this approach enabled strong priming of OVA-specific cytotoxic T cells (CTL), preventing CTL tolerance to intestinal antigen. Such CTL priming was independent of CD4 T cell help but required B7 costimulation. Co-administration of anti-CD40 mAb increased the synthesis of IL-2 and the expression of CD25 by CD8 T cells, but neither IL-2 production nor CD25 expression by CD4 T cells was enhanced by anti-CD40 mAb. However, neutralization of TGF-beta together with addition of agonistic anti-CD40 mAb was able to reverse CD4 T cell tolerance. These findings suggest that the induction of tolerance versus immunity against intestinal antigens is determined by the status of the antigen-presenting cells and that signals via CD40 differently regulate the outcome of CD4 and CD8 T cells in vivo.     

The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity

Control of virus infections and eradication of tumors usually involves the lytic activity of CD8+ cytotoxic T lymphocytes (CTLs). The induction of effective CTL immunity relies on several factors, one of the most important of which is CD4+ T cell help. Numerous studies have demonstrated the dependence of CTL priming on the presence of CD4+ T cells, but until recently little was known of the mechanisms regulating this process. Based on reports that CD4+ and CD8+ T cells must recognize antigen on the same antigen-presenting cell (APC), help was originally thought to be provided through the delivery of short-range, CD4+ T cell-secreted cytokines. However, the results of subsequent studies favor an alternative mechanism, whereby CD4+ T cells modify the APC, converting it into a stimulatory cell for CD8+ T cell priming. It is important that CD40 and its ligand, CD40L, have been implicated in the provision of this help and, in particular, the generation of long-lasting CTL memory.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

CD27 costimulation contributes substantially to the expansion of functional memory CD8+ T cells after peptide immunization

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long‐lived memory cells. The costimulatory receptor, CD27, is essential for optimal T‐cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8⁺ T‐cell reactivation remains controversial. We have used OVA and anti‐CD40 to establish a memory CD8⁺ T‐cell population and report here that their secondary expansion, driven by peptide and anti‐CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8⁺ T‐cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector‐ and central‐memory CD8⁺ T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8⁺ T‐cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

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.     

Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival

Intraperitoneal peptide injection of TCR-transgenic mide or expression of antigen in hepatocytes leads to an accumulation in the liver of specific apoptotic CD8+ T cells expressing activation markers. To determine whether liver cells are capable of directly activating naive CD8+ T cells, we have studied the ability of purified hepatocytes to activate TCR-transgenic CD8+ T cells in vitro. We show that hepatocytes which do not express CD80 and CD86 co-stimulatory molecules are able to induce activation and effective proliferation of specific naive CD8+ T cells in the absence of exogenously added cytokines, a property only shared by professional antigen-presenting cells (APC). Specific T cell proliferation induced by hepatocytes was comparable in magnitude to that seen in response to dendritic cells and was independent of CD4+ T cell help or bystander professional APC co-stimulation. During the first 3 days, the same number of divisions was observed in co-cultures of CD8+ T cells with either hepatocytes or splenocytes. Both APC populations induced expression of early T cell activation markers and specific cytotoxic T lymphocyte (CTL) activity. However, in contrast to T cells activated by splenocytes, T cells activated by hepatocytes lost their cytolytic function after 3 days of co-culture. This correlated with death of activated T cells, suggesting that despite efficient activation, proliferation and transient CTL function, T cells activated by hepatocytes did not survive. Death could be prevented by adding antigen-expressing splenocytes or exogenous IL-2 to the co-culture, indicating that hepatocytes are not involved in direct killing of CD8+ T cells but rather fail to promote survival. Dying cells acquired a CD8low TCRlow B220+ phenotype similar to the one described for apoptotic intrahepatic T cells, suggesting an alternative model to account for the origin of these cells in the liver. The importance of these findings for the understanding of peripheral tolerance and the ability of liver grafts to be accepted is discussed.     

IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL

Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i) higher IFN-gamma secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL.     

Concurrent interaction of DCs with CD4+ and CD8+ T cells improves secondary CTL expansion: It takes three to tango

T‐cell help is essential for CTL‐memory formation. Nevertheless, it is unclear whether the continuous presence of CD4⁺ T‐helper (Th) cells is required during dendritic cell (DC)/CD8⁺ T‐cell encounters, or whether a DC will remember the helper signal after the Th cell has departed. This question is relevant for the design of therapeutic cancer vaccines. Therefore, we investigated how human DCs need to interact with CD4⁺ T cells to mediate efficient repetitive CTL expansion in vitro. We established an autologous antigen‐specific in vitro system with monocyte‐derived DCs, as these are primarily used for cancer vaccination. Contrary to common belief, a sequential interaction of licensed DCs with CD8⁺ T cells barely improved CTL expansion. In sharp contrast, simultaneous encounter of Th cells and CTLs with the same DC during the first in vitro encounter is a prerequisite for optimal subsequent CTL expansion in our in vitro system. These data suggest that, in contrast to DC maturation, the activation of DCs by Th cells, which is necessary for optimal CTL stimulation, is transient. This knowledge has significant implications for the design of new and more effective DC‐based vaccination strategies. Furthermore, our in vitro system could be a valuable tool for preclinical immunotherapeutical studies.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo.

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

Clonal restriction of the expansion of antigen-specific CD8+ memory T cells by transforming growth factor-{beta}

Recent evidence showed that transforming growth factor-beta (TGF-beta) regulates the global expansion of CD8+ T cells, which are CD44hi, a marker for memory cells. However, it is not clear whether this regulatory mechanism also applies to the antigen-specific CD8+ memory cells. By using a murine mixed lymphocyte culture (MLC) model, we examined the effect of TGF-beta on antigen-specific CD8+ memory cells [cytotoxic T lymphocyte (CTL)]. We found that the secondary CTL response in CD8+ memory cells from untreated MLC was not affected by TGF-beta but augmented by interleukin (IL)-2, whereas the CD8+ memory cells from TGF-beta-pretreated MLC (MLC-TGF-beta) failed to mount a significant, secondary CTL response, even when IL-2 was added. In exploring this dichotomy, in combination with flow cytometry analysis, we found that prolonged exposure to TGF-beta reduces the CTL activity in CD8+ memory cells. The increase by IL-2 and the reduction by TGF-beta of the CTL responses were clonal-specific. TGF-beta did not affect the CTL response to a third-party antigen or polyclonal T cell activation. Experiments performed with transgenic 2C cells gave similar results. Cell-cycle study performed with adoptive transfer of the cell tracker-labeled MLC cells revealed that the in vivo expansion of CD8+ memory cells from MLC-TGF-beta was restricted severely, and the restriction was clonal-specific, thus offering direct evidence to show that TGF-beta induces clonal restriction of CD8+ memory cell expansion.     

CD8+ suppressor-mediated regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.     

Priming of CD8+ T cell responses by pathogens typically depends on CD70-mediated interactions with dendritic cells

The CD27/CD70-interaction has been shown to provide a costimulatory and survival signal for T cells in vitro and in vivo. Recently, CD70 expression by DC was found to be important for the priming of CD8+ T cells. We show here that blocking CD70 interactions has a significant impact on priming of CD8+ T cell responses by vaccinia virus (VV), Listeria monocytogenes and vesicular stomatitis virus (VSV) in mice. However, the priming of specific CD8+ T cells upon infection with lymphocytic choriomeningitis virus (LCMV) was only marginally reduced by CD70-blockade. Blocking of CD70 prevented CD8+ T cell priming in DIETER mice, a model in which presentation of LCMV-derived epitopes can be induced selectively in dendritic cells (DC). In contrast, CD70-CD27 interactions were not important for the priming of VSV-specific CD4+ T cells or class switch of neutralizing antibodies. As we show that priming of CD8+ T cells by the pathogens used here is dependent on antigen presentation by DC and that infection results in up-regulation of CD70 on DC, we conclude that CD70 expression on DC plays an important role in the priming of CD8+ T cells by pathogens. Moreover, the lack of CD70 cannot be completely compensated for by other costimulatory molecules.     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.