Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation

The differentiation of human CD4⁺ T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel‐based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC‐MS/MS is required to provide a reference dataset for proteome‐based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 ( http://proteomecentral.proteomexchange.org/dataset/PXD001066 ).     

Signal transduction via the T cell antigen receptor in naïve and effector/memory T cells

T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.     

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/<Emphasis Type="Italic">neu</Emphasis> peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

Virtual memory cells make a major contribution to the response of aged influenza-naïve mice to influenza virus infection

Background

A diverse repertoire of naïve T cells is thought to be essential for a robust response to new infections. However, a key aspect of aging of the T cell compartment is a decline in numbers and diversity of peripheral naïve T cells. We have hypothesized that the age-related decline in naïve T cells forces the immune system to respond to new infections using cross-reactive memory T cells generated to previous infections that dominate the aged peripheral T cell repertoire.

Results

Here we confirm that the CD8 T cell response of aged, influenza-naïve mice to primary infection with influenza virus is dominated by T cells that derive from the memory T cell pool. These cells exhibit the phenotypic characteristics of virtual memory cells rather than true memory cells. Furthermore, we find that the repertoire of responding CD8 T cells is constrained compared with that of young mice, and differs significantly between individual aged mice. After infection, these virtual memory CD8 T cells effectively develop into granzyme-producing effector cells, and clear virus with kinetics comparable to naïve CD8 T cells from young mice.

Conclusions

The response of aged, influenza-naive mice to a new influenza infection is mediated largely by memory CD8 T cells. However, unexpectedly, they have the phenotype of VM cells. In response to de novo influenza virus infection, the VM cells develop into granzyme-producing effector cells and clear virus with comparable kinetics to young CD8 T cells.

     

Differential effects of age,cytomegalovirus-seropositivity and end-stage renal disease (ESRD) on circulating T lymphocyte subsets

The age- and cytomegalovirus (CMV)-seropositivity-related changes in subsets and differentiation of circulating T cells were investigated in end-stage renal disease (ESRD) patients (n = 139) and age-matched healthy individuals. The results show that CMV-seropositivity is associated with expansion of both CD4⁺ and CD8⁺ memory T cells which is already observed in young healthy individuals. In addition, CMV-seropositive healthy individuals have a more differentiated memory T cell profile. Only CMV-seropositive healthy individuals showed an age-dependent decrease in CD4⁺ naïve T cells. The age-related decrease in the number of CD8⁺ naïve T cells was CMV-independent. In contrast, all ESRD patients showed a profound naïve T-cell lymphopenia at every decade. CMV-seropositivity aggravated the contraction of CD4⁺ naïve T cells and increased the number of differentiated CD4⁺ and CD8⁺ memory T cells. In conclusion, CMV-seropositivity markedly alters the homeostasis of circulating T cells in healthy individuals and aggravates the T cell dysregulation observed in ESRD patients.     

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach

This study compares the total liver proteome of inbred alcohol‐preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three‐step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2‐DE. Scanned gels of two sample groups, alcohol‐exposed iP and alcohol‐naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC‐MS/MS. Twenty‐four individual rats, 12 alcohol‐naïve, and 12 alcohol‐exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid β‐oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.     

Strong homeostatic TCR signals induce formation of self‐tolerant virtual memory CD8 T cells

Virtual memory T cells are foreign antigen‐inexperienced T cells that have acquired memory‐like phenotype and constitute 10–20% of all peripheral CD8⁺ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen‐experienced memory T cells are incompletely understood. By analyzing T‐cell receptor repertoires and using retrogenic monoclonal T‐cell populations, we demonstrate that the virtual memory T‐cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self‐reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T‐cell compartment via modulating the self‐reactivity of individual T cells. Although virtual memory T cells descend from the highly self‐reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self‐reactivity in polyclonal T cells for the generation of functional T‐cell diversity.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.     

Age-related differences in polyfunctional T cell responses

Background

A reduced number of naïve T cells along with an accumulation of differentiated cell types in aging have been described but little is known about the polyfunctionality of the T cell responses. In this study we compared the individual and polyfunctional expression of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 by T cell subsets, including the newly described stem cell like memory T cells (T_SCM), in response to stimulation with superantigen staphylococcal enterotoxin B (SEB) in older (median age 80, n?=?23) versus younger (median age 27; n?=?23) adults.

Results

Older age was associated with a markedly lower frequency of CD8+ naïve T cells (11% vs. 47%; p?<?0.0001) and an expansion in memory T cell subsets including central memory (p?<?0.05), effector memory and effector T cells (p?<?0.001 for both). There was also a decline in CD4+ naïve T cells in older subjects (33% vs. 45%; p?=?0.02). There were no differences in frequencies or polyfunctional profiles of T_SCM between groups. CD8+ naïve cells in the older group had increased expression of all functional parameters measured compared to the younger subjects and exhibited greater polyfunctionality (p?=?0.04). CD4+ naïve T cells in the older group also showed greater polyfunctionality with a TNF-α and IL-2 predominance (p?=?0.005). CD8+ effector memory and effector T cells exhibited increased polyfunctionality in the older group compared with younger (p?=?0.01 and p?=?0.003).

Conclusions

These data suggest that aging does not have a negative effect on polyfunctionality and therefore this is likely not a major contributor to the immunesenescence described with aging.

     

α-Fetoprotein Influence on the Conversion of Naïve T-Helpers into Memory T-Cell Effector Subpopulations

The effect of native α-fetoprotein (AFP) on the conversion of naïve T-helpers into central memory T-cells (TCM) and effector subpopulations of the preterminally differentiated (TEM) and terminally differentiated (TEMRA) memory T-cells was studied. AFP was found to prevent the conversion of naïve T-helpers into effector subpopulations of memory T cells (TEM and TEMRA) while reducing the total production of IL-4 and IFN-γ by the studied cell populations. The data reveal a new role of AFP in the immune tolerance formation during pregnancy.     

Immunologic considerations for generating memory CD8 T cells through vaccination

Following infection or vaccination, naïve CD8 T cells that receive the appropriate integration of antigenic, co‐stimulatory and inflammatory signals undergo a programmed series of biological changes that ultimately results in the generation of memory cells. Memory CD8 T cells, in contrast to naïve cells, more effectively limit or prevent pathogen re‐infection because of both qualitative and quantitative changes that occur following their induction. Unlike vaccination strategies aimed at generating antibody production, the ability to generate protective memory CD8 T cells has proven more complicated and problematic. However, recent experimental results have revealed important principles regarding the molecular and genetic basis for memory CD8 T cell formation, as well as identified ways to manipulate their development through vaccination, resulting in potential new avenues to enhance protective immunity.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

Thymic output and functionality of the IL-7/IL-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life

During aging, the thymus undergoes a marked involution that is responsible for profound changes in the T‐cell compartment. To investigate the capacity of the thymus to produce new cells at the limit of human lifespan, we analyzed some basic mechanisms responsible for the renewal and maintenance of peripheral T lymphocytes in 44 centenarians. Thymic functionality was analyzed by the quantification of cells presenting the T‐cell receptor rearrangement excision circles (TREC). A new method based upon real‐time PCR was used, and we found that most centenarians (84%) had undetectable levels of TREC+ cells. Six‐color cytofluorimetric analysis revealed that centenarians had an extremely low number of naïve T cells; central memory and effector memory T cells were greatly increased, while terminally differentiated cells were as numerous as in young (aged 20–45) or middle‐aged (aged 58–62) donors. Interleukin (IL)‐7 and IL‐7 receptor α‐chain (CD127) levels were the same at all ages, as shown by ELISA, flow cytometry and real‐time PCR. However, IL‐7 plasma levels were higher in centenarian females than males. The presence of TREC+ cells and of very few naïve T lymphocytes suggests that in centenarians such cells could either derive from residues of thymic lymphopoietic islets, or even represent long‐living lymphocytes that have not yet encountered their antigen. IL‐7 could be one of the components responsible, among others, for the higher probability of reaching extreme ages typical of females.     

Early-life atomic-bomb irradiation accelerates immunological aging and elevates immune-related intracellular reactive oxygen species

Reactive oxygen species (ROS) play an important role in immune responses; however, their excessive production and accumulation increases the risk of inflammation-related diseases. Although irradiation is known to accelerate immunological aging, the underlying mechanism is still unclear. To determine the possible involvement of ROS in this mechanism, we examined 10,023 samples obtained from 3752 atomic-bomb survivors in Hiroshima and Nagasaki, who participated in repeated biennial examinations from 2008 to 2016, for the effects of aging and radiation exposure on intracellular ROS (H₂O₂ and O₂^•−) levels, percentages of T-cell subsets, and the effects of radiation exposure on the relationship between cell percentages and intracellular ROS levels in T-cell subsets. The cell percentages and intracellular ROS levels in T-cell subsets were measured using flow cytometry, with both fluorescently labeled antibodies and the fluorescent reagents, carboxy-DCFDA and hydroethidine. The percentages of naïve CD4⁺ and CD8⁺ T cells decreased with increasing age and radiation dose, while the intracellular O₂^•− levels in central and effector memory CD8⁺ T cells increased. Additionally, when divided into three groups based on the percentages of naïve CD4⁺ T cells, intracellular O₂^•− levels of central and effector memory CD8⁺ T cells were significantly elevated with the lowest radiation dose group in the naïve CD4⁺ T cells. Thus, the radiation exposure-induced decrease in the naïve CD4⁺ T cell pool size may reflect decreased immune function, resulting in increased intracellular ROS levels in central and effector memory CD8⁺ T cells, and increased intracellular oxidative stress.     

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis‐infected and culture filtrate protein‐treated macrophages

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC‐MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis‐infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP‐treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis‐infected cells. The exosomes from CFP‐treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.     

Plasmodium vivax infection induces expansion of activated naïve/memory T cells and differentiation into a central memory profile

Immunity to malaria is widely believed to wane in the absence of reinfection, but direct evidence for the presence or absence of durable immunological memory to malaria is limited. Here, we characterized the profile of circulating naïve and memory (including central and effector) CD4⁺ T cells responses of individuals naturally infected by Plasmodium vivax. In the current study, we demonstrated that acute P. vivax infection induces a significant increase in the absolute number of both naïve and memory cells, which were responsible for the production of anti-inflammatory (IL-10) and pro-inflammatory (IFN-γ) cytokines. Finally, we described the profile of memory cell subtypes (T_CM-CD45RO^highCCR7⁺ and T_EM-CD45RO^highCCR7⁻), as well as the pattern of cell migration based on CD62L selectin expression, demonstrating that P. vivax-infected donors presented with a predominantly central memory cell profile. Our results indicate that the expansion of both naïve and memory T cells, responsible for the production of both pro-inflammatory and regulatory cytokines, which might also contribute to the modulation of immune responses during P. vivax infection.