Chronic intravenous injections of antigen induce and maintain tolerance in T cell receptor-transgenic mice

Antigen-specific T cell tolerance can be induced by systemic injection of high-dose antigen. In particular, a single intravenous (i.v.) injection of influenza virus hemagglutinin peptide in HNT-TCR transgenic mice induces T cell tolerance through thymocyte apoptosis as well as anergy and deletion of peripheral CD4⁺ T cells. We now show that this tolerance is reversed after 8 weeks probably due to the short in vivo half-life of the peptide. Since durable tolerance is required for this strategy to be of therapeutic value, we tested whether weekly i.v. injections of peptide (up to 12 weeks) could maintain the CD4⁺ T cell tolerance. Each injection induces a profound deletion of thymocytes, although their level recovers before the next injection. Therefore, during the treatment period, the thymus undergoes cycles of contraction/expansion. In the periphery, the number of CD4⁺ T cells is stably decreased and the persisting CD4⁺ T cells are hyporeactive both in vitro and in vivo. This tolerance is essentially peripheral since comparable results were obtained in thymectomized HNT-TCR mice injected weekly. Our data show that stable antigen-specific tolerance can be induced by repeated i.v. injections of antigen. These findings might have implications for the treatment of T cell-mediated autoimmune diseases.     

Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8⁺ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4^?/? CD8⁺ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4‐mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory‐like CD8⁺ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4^?/? CD8⁺ T cells was impaired, whereas their number eventually reached a level similar to that of wild‐type CD8⁺ T cells. Conversely, memory‐like Irf4^?/? CD8⁺ T cells underwent homeostatic proliferation in a manner similar to that of wild‐type memory CD8⁺ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8⁺ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8⁺ T cells, whereas the maintenance of memory CD8⁺ T cells is IRF4‐independent.     

Activation of caspase-3-like enzymes in non-apoptotic T cells

The activation of the caspase family of cysteine proteases is a key step in the implementation of apoptotic cell death leading to further downstream effects such as DNA fragmentation. In cultured tumor cells, caspase activity appears only when cells are undergoing apoptosis. Here we show that human and murine T lymphocytes acquire high intracellular activities of cell death-specific caspases upon activation by mitogens and IL-2 without evidence that apoptosis is proceeding. The highest activity is seen when cells are mitogen activated for 3 days. On a per cell basis, caspase activity in activated T cells is much higher than in tumor cells induced to undergo apoptosis. In the presence of exogenously added IL-2 cells stay alive and maintain a high level of caspase activity while IL-2 withdrawal results in cell death and decline of caspase activity. Caspase activity can also be measured in extracts from spleen and lymph nodes from mice injected with superantigen. While in tumor cell lines caspase activity correlates with cleavage of poly(ADP)-ribose polymerase (PARP) and DNA fragmentation, in activated T cells cleavage products of cellular PARP can be detected whereas DNA fragmenting activity appears only upon IL-2 withdrawal which coincides with cell death. These data show that caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. Furthermore, they provide a molecular correlate for the high susceptibility of activated T cells for apoptosis.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

Ligation of CD5 on resting B cells,but not on resting T cells,results in apoptosis

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5⁺ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5⁺ B cells were more sensitive to IgM-induced apoptosis than CD5⁻ B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.     

Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on T cell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vβ8.2⁺ T cells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vβ8.2⁺ T cell population in the CNS to 26.2 % compared to 7.4 % in saline-treated rats and 7.6 % in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vβ8.2⁺ population in the popliteal lymph node draining the inoculation site (1.4 %) or in the spleen (1.6 %) above that occurring in saline-treated rats (1.6 % and 1.1 %, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP_{72 – 89}, was decreased in the CNS but not in the popliteal lymphnode by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vβ8.2⁺ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vβ8.2⁺ T cells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of auto reactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.     

Efficient induction of cytotoxic CD8+ T cells against exogenous proteins: establishment and characterization of a T cell line specific for the membrane protein ActA of Listeria monocytogenes

The property of listeriolysin (LLO) to introduce soluble passenger proteins into the cytosol of antigen-presenting cells allows the induction of CD8⁺ cytotoxic T cells against such antigens. To overcome the potential problem of presentation of the immunodominant epitope LLO91-99 by H-2K^d, a variant LLO92A was established in which Tyr 92 was replaced by Ala. Immunization of BALB/c mice with purified LLO92A failed to stimulate cytotoxic T cells specific for either the epitope LLO91-99 or for any other LLO-derived peptide. Injection of mixtures of purified LLO92A and soluble nucleoprotein (NP) of influenza virus into mice resulted in a strong cytotoxic T cell response exclusively directed against NP. The LLO92A variant was successfully used to generate, propagate and characterize a CD8⁺ T cell line specific for the membrane-bound virulence factor ActA of Listeria monocytogenes. Interestingly, wild-type ActA bound to the surface of live L. monocytogenes was not presented by MHC class I molecules to the CD8⁺ T cell line.     

Hyaluronan‐binding by CD44 reduces the memory potential of activated murine CD8 T cells

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44^?/? and CD44^+/+ OT‐I CD8 T cells were adoptively transferred into mice challenged with Listeria‐OVA, there was a slight increase in the percentage of CD44^+/+ cells at the effector site. However, CD44^+/+ cells were out‐competed by CD44^?/? cells after the contraction phase in the lymphoid tissues, and the CD44^?/? cells preferentially formed more memory cells. The hyaluronan‐binding CD44^+/+ CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non‐binding CD44^+/+ and CD44^?/? OT‐I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.     

Direct and indirect T cell priming by dendritic cell vaccines

The mechanisms by which dendritic cell (DC) vaccines prime host T cells in vivo was analyzed. Mice were immunized with syngeneic bone marrow-derived DC and as surrogate antigen β-galactosidase (β-gal) was used. DC either pulsed with peptide, loaded with β-gal antigen or gene-modified induced β-gal-specific cytotoxic T lymphocytes (CTL) and moderate rejection of an in vivo challenge with β-gal expressing tumors. In addition, β-gal-specific CTL lysed the syngeneic DC that were used as vaccines. Using SCID mice reconstituted with F1 lymphocytes, direct priming by gene-modified DC vaccines was demonstrated by the presence of β-gal-specific CTL of the haplotype exclusively expressed by DC while indirect priming by host antigen-presenting cells (APC) was shown by the detection of CTL of the haplo type exclusively present on host APC and absent on DC vaccines. Since DC immunization in syngeneic mice was associated with an increase in NK1.1⁺/Ly49C⁻ cells and detectable lysis of DC in vitro by lymphokine-activated killer cells, DC vaccines appear to interact with host natural killer cells as well as with antigen-specific T cells. These effector cells in turn may lyse DC vaccines thereby leading to the release of antigens that can be taken up by host APC.     

Interrupting CD28 costimulation before antigen rechallenge affects CD8+ T‐cell expansion and effector functions during secondary response in mice

The role of CD28‐mediated costimulation in secondary CD8⁺ T‐cell responses remains controversial. Here, we have used two tools — blocking mouse anti‐mouse CD28‐specific antibodies and inducible CD28‐deleting mice — to obtain definitive answers in mice infected with ovalbumin‐secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN‐γ production during the secondary immune response. In contrast, cell‐intrinsic deletion of CD28 in transferred TCR‐transgenic CD8⁺ T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation‐impaired CD8⁺ T cells respond to CD28‐dependent help from their environment by enhanced functional differentiation. Finally, we report that cell‐intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long‐term memory. Thus, if given sufficient time, the progeny of primed CD8⁺ T cells adapt to the absence of this costimulator.     

β-Galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a β-galactoside-binding protein (βGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8⁺ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8⁺ T cells. LGALS1 expression could be induced in CD4⁺ and CD8⁺ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric βGBP was released by activated CD8⁺ T cells and in vitro experiments further showed that recombinant βGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8⁺ T cells. Thus, these data indicate a role of βGBP as an autocrine negative growth factor for CD8⁺ T cells.     

An ongoing immune response to HIV envelope gp120 in human CD4-transgenic mice contributes to T cell decline upon intravenous administration of gp120

The mechanisms accounting for T cell depletion in AIDS patients are not yet fully understood, nor are the roles of host factors in HIV pathogenesis. We show here that an ongoing humoral immune response to HIV gp120 can sensitize non-infected cells towards apoptosis. Thus, i.v. injection of 1 μg recombinant(r) gp120 into gp120-immunized human CD4-transgenic mice (huCD4 Tg), which express huCD4 on both T and B cells, results in T and B cell depletion in peripheral blood and lymphoid tissues. On day 6 after a bolus injection of gp120, the numbers of peripheral T cells and B cells in gp120-immunized huCD4 Tg decreased sevenfold and two- to threefold, respectively. Annexin V staining revealed a higher percentage of early apoptotic cells on day 1 of gp120 i.v. injection from gp120-primed huCD4 Tg spleens compared to gp120-primed controls. Boosting the primed huCD4 Tg mice with soluble gp120 and hen egg-white lysozyme led to lower secondary titers to both antigens than found in controls. Furthermore, splenocytes from gp120-pretreated immunized huCD4 Tg had a lower level of stimulation in response to anti-CD3 treatment. These in vivo results are consistent with in vitro data demonstrating that cross-linking CD4 on splenocytes of huCD4 Tg by rgp120_SF2 and anti-gp120 not only sensitizes T cells for apoptosis, but also induces apoptosis per se, and suggest that anti-gp120 responsiveness can contribute to T cell depletion in AIDS.     

Generating long‐lived CD8+ T‐cell memory: Insights from epigenetic programs

T‐cell‐based immunological memory has the potential to provide the host with life‐long protection against pathogen reexposure and thus offers tremendous promise for the design of vaccines targeting chronic infections or cancer. In order to exploit this potential in the design of new vaccines, it is necessary to understand how and when memory T cells acquire their poised effector potential, and moreover, how they maintain these properties during homeostatic proliferation. To gain insight into the persistent nature of memory T‐cell functions, investigators have turned their attention to epigenetic mechanisms. Recent efforts have revealed that many of the properties acquired among memory T cells are coupled to stable changes in DNA methylation and histone modifications. Furthermore, it has recently been reported that the delineating features among memory T cells subsets are also linked to distinct epigenetic events, such as permissive and repressive histone modifications and DNA methylation programs, providing exciting new hypotheses regarding their cellular ancestry. Here, we review recent studies focused on epigenetic programs acquired during effector and memory T‐cell differentiation and discuss how these data may shed new light on the developmental path for generating long‐lived CD8⁺ T‐cell memory.     

CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8⁺ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8⁺ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1⁺ CD8⁺ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8⁺ T cells.     

Mini-review CD4 T cells are required for CD8 T cell memory generation

Whereas the role of CD4 T cells in B cell memory generation is well established and unequivocal, the role that CD4 T cells play in CD8 responses was until recently far more elusive and controversial. A series of recent reports, however, have re-assessed the role of CD4 help on CD8 responses and have given rise to surprisingly unambiguous conclusions. While studying very different systems, they demonstrated that CD4 T cells are absolutely required for the generation of bona fide CD8 memory cells; the reports allow, for the first time, strong analogies to be made between B and CD8 memory cell generation. These data invite us to drastically change our idea of CD4 help on CD8 responses because they show that the old dichotomy - Th-dependent versus Th-independent CD8 responses - is no longer accurate.     

AMPK: A metabolic switch for CD8+ T‐cell memory

Adenosine monophosphate‐activated protein kinase (AMPK) is a serine/threonine kinase and is crucial for cellular energy homeostasis. The exact role of AMPK during memory CD8⁺ T‐cell differentiation, a process that changes from the metabolically active state of effector T cells to one of quiescence in memory cells is not well understood; however, a report by Cantrell and colleagues [Eur. J. Immunol. 2013. 43: 889‐896] in this issue of the European Journal of Immunology shows that AMPK, by sensing glucose stress, is an important upstream molecule of mammalian target of rapamycin (mTOR) complex 1 for memory CD8⁺ T‐cell differentiation. This study provides new insights into how AMPK monitors energy stress to control effector and memory CD8⁺ T‐cell formation as discussed in this Commentary.     

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.     

Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment

Memory T cells can be classified as central memory (T(CM), CD45RA(neg)CCR7(+)), effector memory (T(EM), CD45RA(neg)CCR7(neg)), and terminally differentiated cells (T(TD), CD45RA(+)CCR7(neg)) with different homing and effector capacities. In 101 healthy subjects aged from 5 to 96 years, distinct dynamics were evidenced between circulating CD4(+) and CD8(+) T cell populations. Naive CD4(+) and CD8(+) T cells decreased linearly with age, CD8(+) twice more rapidly. Memory cells outnumbered naive cells on average at 37.4 in the CD4(+) and 29.5 years of age in the CD8(+) pool. CD4(+) T(CM) and T(EM) cells were positively correlated and increased linearly at a similar rate with age, while CD4(+) T(TD) remained rare. CD8(+) T(EM) and T(TD) accumulated linearly with age, while T(CM) increased only slightly, and each memory subset was negatively correlated to the two others. Almost all CD8(+) T(TD) and some CD8(+) T(EM) had lost CD28 expression. Despite different dynamics, each individual CD4(+) naive and memory subset was correlated to the synonymous CD8(+) subset. Half of the subjects aged 65 years or older were characterized by extremely reduced CD8(+) naive and increased CD8(+) T(TD) cell counts, which could indicate an acceleration of the decay of the immune system from this age onward.