Comprehensive early and lasting loss of memory CD8 T cells and functional memory during acute and persistent viral infections

Viral infections have been shown to induce lymphopenias that lower memory CD8 T cell frequencies, and they also have been shown to cause a permanent loss of memory cells specific to previously encountered pathogens. In this study, the patterns and significance of virus-induced memory CD8 T cell depletion were examined in mice immune to heterologous (Pichinde, vesicular stomatitis, vaccinia) viruses and subsequently challenged with acute or persistent lymphocytic choriomeningitis virus infections. Memory CD8 T cell loss was comprehensive and occurred in both lymphoid and peripheral tissues of the immune host. The impact of the loss of memory T cells was reflected by in vivo cytotoxicity assays, which showed decreased clearance of epitope-expressing targets. Memory CD8 T cell loss occurred very early (day 2) after infection, and was thereafter sustained, consistent more with an active deletion model than with a competition model. Cross-reactive T cells, in contrast, increased in number, but memory cells were reduced whether or not there was competition from cross-reactive T cells. Memory T cell loss was more profound during persistent infection than after acute infection. Adoptive transfer studies showed that, unlike the resolved acute infection, in which the reduced memory frequencies became stable, memory T cell loss was a continuously ongoing process during persistent infection. This study therefore links an early virus-induced lymphopenia to a subsequent long-term loss of CD8 T cell memory and offers a new mechanism for immune deficiency during persistent viral infections.     

Daidzein prevents the increase in CD4+CD28null T cells and B lymphopoesis in ovariectomized mice: a key mechanism for anti-osteoclastogenic effect

Estrogen deficiency leads to an upregulation of TNF-α producing T cells and B-lymphopoesis which augments osteoclastogenesis. Estrogen deficiency also increases the population of premature senescent CD4⁺CD28null T cells which secrete a higher amount of TNF-α thus leading to enhanced osteoclastogenesis. Isoflavonoids like daidzein and genistein are found mostly in soybeans, legumes, and peas. These share structural similarity with 17β-stradiol (E2) and have osteoprotective role. This study explores the effect of daidzein (Daid) on the proliferation of TNF-α producing T cells, premature senescent T cells and B cell lymphopoesis under estrogen deficient conditions. For this study adult Balb/c mice were treated with Daid at 10 mg/kg body weight dose by oral gavage daily post ovariectomy (Ovx). After six weeks animals were autopsied and bone marrow and spleen cells were collected for FACS analysis. Blood serum was collected for ELISA. It was observed that Ovx mice treated with Daid for six weeks show reduction in Ovx induced expansion of CD4⁺ T cells in bone marrow and spleen when analysed by flow cytometry. Estrogen deficiency led to increased prevalence of TNF-α secreting CD4⁺CD28null T cells, however, treatment with Daid increased the percentage of CD4⁺CD28⁺ T cells. Co-culture of CD4⁺CD28null T cells and bone marrow resulted in enhanced osteoclastogenesis as evident by increased tartarate resistant acid phosphatase (TRAP) expression, an osteoclast marker. However, treatment with Daid resulted in reduced osteoclastogenesis in CD4⁺CD28null T cells and bone marrow cell co-culture. Daid also regulated B lymphopoesis and decreased mRNA levels of RANKL in B220⁺ cells. Taken together, we propose that one of the mechanisms by which Daid prevents bone loss is by reversing the detrimental immune changes as a result of estrogen deficiency.     

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28⁻CD57⁺CD8⁺ T cells between the groups. However, the frequency of Tim-3⁺CD8⁺ and Tim-3⁺CD4⁺ exhausted T cells, but not PD-1⁺ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1⁺CD8⁺ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3⁺CD8⁺ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

Modulation of CD28 expression: distinct regulatory pathways during activation and replicative senescence.

The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site alpha and beta, within the CD28 minimal promoter. Both alpha- and beta-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site beta- but not site alpha-binding activities. In contrast, T cell activation induces a parallel decline in both site alpha- and beta-binding activities. CD4+ and CD8+ T cells differ in their beta-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack alpha- and beta-bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells.     

Control of memory CD8+ T cell differentiation by CD80/CD86-CD28 costimulation and restoration by IL-2 during the recall response

Memory CD8+ T cell responses have been considered to be independent of CD80/CD86-CD28 costimulation. However, recall responses are often severely blunted in CD28-/- mice. Whether this impairment represents a requirement for CD28 costimulation for proper memory CD8+ T cell development or a requirement during the recall response is unknown. Furthermore, how CD28 costimulation affects the phenotype and function of memory CD8+ T cells has not been characterized in detail. In this study, we investigate these questions by studying the role of the CD28 costimulatory pathway in memory CD8+ T cell responses to acute and persistent DNA virus infections. Memory CD8+ T cells against vaccinia virus (VV) infection which develop without CD28 costimulation exhibit lower expression of differentiation markers CD27 and CD122 (IL-15Rbeta). These memory CD8+ T cells also fail to produce IL-2. Our data indicate that for an optimal recall response, CD28 costimulation is required both for T cell priming and also during the recall response. Similar requirements were observed for memory CD8+ T cell responses during persistent infection with murine gammaherpesvirus 68 (MHV-68) infection, indicating CD28 may play the same role in both acute and persistent infections. Finally, we show deficits in the recall response are restored by IL-2 signaling during recall, but not during priming. The data presented show that CD28 costimulation not only controls the magnitude of the primary response but also affects development of memory CD8+ T cells and is required during the recall response in addition to initial T cell priming.     

CD7 and CD28 are required for murine CD4+CD25+ regulatory T cell homeostasis and prevention of thyroiditis

CD7 and CD28 are T cell Ig superfamily molecules that share common signaling mechanisms. To determine roles CD7 and CD28 might play in peripheral lymphocyte development and function, we have generated CD7/CD28-double-deficient mice. CD7- and CD28-single-deficient and CD7/CD28-double-deficient mice had normal levels of CD4 and CD8-single-positive T cells in thymus and spleen. However, CD28-deficient mice had decreased CD4+CD25+ T cells in spleen compared with wild-type mice, and CD7/CD28-double-deficient mice had decreased numbers of CD4+CD25+ T cells in both thymus and spleen compared with both wild-type and CD28-deficient mice. Functional studies demonstrated that CD4+CD25+ T cells from CD28-deficient and CD7/CD28-double-deficient mice could mediate suppression of CD3 mAb activation of CD4+CD25- wild-type T cells, but were less potent than wild-type CD4+CD25+ T regulatory cells. Thyroiditis developed in aged CD7/CD28-double-deficient mice (>1 year) that was not seen in age-matched control mice or single CD7- or CD28-deficient mice, thus suggesting in vivo loss of T regulatory cells allowed for the development of spontaneous thyroiditis. Taken together, these data demonstrated collaborative roles for both CD7 and CD28 in determination of number and function of CD4+CD25+ T regulatory cells in the thymus and peripheral immune sites and in the development of spontaneous thyroiditis.     

Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4(+)C25(+) Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4(+)CD25(-) T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4(+)CD25(-) T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.     

Major features of immunesenescence,including reduced thymic output,are ameliorated by high levels of physical activity in adulthood

It is widely accepted that aging is accompanied by remodelling of the immune system including thymic atrophy and increased frequency of senescent T cells, leading to immune compromise. However, physical activity, which influences immunity but declines dramatically with age, is not considered in this literature. We assessed immune profiles in 125 adults (55–79 years) who had maintained a high level of physical activity (cycling) for much of their adult lives, 75 age‐matched older adults and 55 young adults not involved in regular exercise. The frequency of naïve T cells and recent thymic emigrants (RTE) were both higher in cyclists compared with inactive elders, and RTE frequency in cyclists was no different to young adults. Compared with their less active counterparts, the cyclists had significantly higher serum levels of the thymoprotective cytokine IL‐7 and lower IL‐6, which promotes thymic atrophy. Cyclists also showed additional evidence of reduced immunesenescence, namely lower Th17 polarization and higher B regulatory cell frequency than inactive elders. Physical activity did not protect against all aspects of immunesenescence: CD28^?veCD57^+ve senescent CD8 T‐cell frequency did not differ between cyclists and inactive elders. We conclude that many features of immunesenescence may be driven by reduced physical activity with age.     

IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.     

Pathogenic features of CD4(+)CD28(-) T cells in immune disorders

Aging of the immune system contributes to the increased morbidity and mortality of the elderly population and may occur prematurely in patients with immune disorders. One of the main characteristics of immunosenescence is the expansion of CD4(+)CD28(-) T cells in the blood. These cells are effector memory T cells with cytotoxic capacity, and have been recently described to have pathogenic potential in a variety of immune disorders. Interestingly, CD4(+)CD28(-) T cells have now been found to infiltrate target tissues of patients with multiple sclerosis, rheumatoid arthritis, myopathies, acute coronary syndromes, and other immune-related diseases. In this review, we discuss potential factors and mechanisms that may induce the expansion of these cells, as well as their putative pathogenic mechanisms in immune disorders.     

The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28

CD28 plays crucial costimulatory roles in T cell proliferation, cytokine production, and germinal center response. Mice that are deficient in the inducible costimulator (ICOS) also have defects in cytokine production and germinal center response. Because the full induction of ICOS in activated T cells depends on CD28 signal, the T cell costimulatory capacity of ICOS in the absence of CD28 has remained unclear. We have clarified this issue by comparing humoral immune responses in wild-type, CD28 knockout (CD28 KO), and CD28-ICOS double-knockout (DKO) mice. DKO mice had profound defects in Ab responses against environmental Ags, T-dependent protein Ags, and vesicular stomatitis virus that extended far beyond those observed in CD28 KO mice. However, DKO mice mounted normal Ab responses against a T-independent Ag, indicating that B cell function itself was normal. Restimulated CD4(+) DKO T cells that had been primed in vivo showed decreased proliferation and reduced IL-4 and IL-10 production compared with restimulated CD4(+) T cells from CD28 KO mice. Thus, in the absence of CD28, ICOS assumes the major T cell costimulatory role for humoral immune responses. Importantly, CD28-mediated ICOS up-regulation is not essential for ICOS function in vivo.     

Foxp3+ regulatory T cells control persistence of viral CNS infection

We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4(+) CD25(+) Foxp3(+) Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8(+) T cells predominantly recognising the H-2D(b)-presented viral hemagglutinin epitope MV-H(22-30) (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8(+) effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.     

Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

肿瘤患者CD8+T细胞中CD28表达的研究进展

CD28与B7结合形成的共刺激信号是T细胞激活的第二信号,肿瘤患者CD8^＋T细胞上CD28分子在肿瘤免疫中发挥着重要作用。人体抗肿瘤免疫主要由CD8^＋T细胞介导,根据CD28的表达与否可将CD8^＋T细胞分为细胞毒T细胞（CD8^＋CD28^＋,CTL）和抑制性T细胞（CD8^＋C28^-,Ts）。CTL是体内杀伤肿瘤细胞的主要功能性细胞之一,当该细胞与肿瘤接触时,通过共刺激信号而被激活,发挥其对肿瘤细胞的杀伤作用;Ts在机体的免疫耐受中发挥作用。现就肿瘤患者CD8^＋T细胞上CD28的表达作一综述。     

Four functionally distinct populations of human effector-memory CD8+ T lymphocytes

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.     

Generation and growth of CD28nullCD8+ memory T cells mediated by IL-15 and its induced cytokines

Accumulation of CD28(null)CD8+ T cells and the defects of these cells in response to antigenic stimulation are the hallmarks of age-associated decline of T cell function. However, the mechanism of these age-associated changes is not fully understood. In this study, we report an analysis of the growth of human CD28(null) and CD28+CD8+ memory T cells in response to homeostatic cytokine IL-15 in vitro. We showed that 1) there was no proliferative defect of CD28(null)CD8+ memory T cells in response to IL-15 compared with their CD28+ counterparts; 2) stable loss of CD28 expression occurred in those actively dividing CD28+CD8+ memory T cells responding to IL-15; 3) the loss of CD28 was in part mediated by TNF-alpha that was induced by IL-15; and 4) CCL4 (MIP-1beta), also induced by IL-15, had a significant inhibitory effect on the growth of CD28(null) cells, which in turn down-regulated their expression of CCL4 receptor CCR5. Together, these findings demonstrate that CD28(null)CD8+ memory T cells proliferate normally in response to IL-15 and that IL-15 and its induced cytokines regulate the generation and growth of CD28(null)CD8+ T cells, suggesting a possible role of IL-15 in the increase in CD28(null)CD8+ T cells that occurs with aging.     

Loss of CD127 expression links immune activation and CD4(+) T cell loss in HIV infection

Although chronic immune activation correlates with CD4(+) T cell loss in HIV infection, an understanding of the factors mediating T cell depletion remains incomplete. We propose that reduced expression of CD127 (IL-7 receptor alpha chain, IL-7Ralpha), induced by immune activation, contributes to CD4(+) T cell loss in HIV infection. In particular, loss of CD127 on central memory CD4(+) T cells (T(CM)) severely restrains the regenerative capacity of the memory component of the immune system, resulting in a limited ability to control T cell homeostasis. Studies from both pathogenic and controlled HIV infection indicate that the containment of immune activation and preservation of CD127 expression are critical to the stability of CD4(+) T cells in infection. A better understanding of the factors regulating CD127 expression in HIV disease, particularly on T(CM) cells, might unveil new approaches exploiting the IL-7/IL-7R receptor pathway to restore T cell homeostasis and promote immune reconstitution in HIV infection.