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.     

Aged regulatory T cells protect from autoimmune inflammation despite reduced STAT3 activation and decreased constraint of IL-17 producing T cells

Regulatory T cells (Tregs) are specialized CD4⁺ T lymphocytes helping defend against autoimmunity and inflammation. Although age is associated with increased inflammation and autoimmunity, few reports address age effects of immune regulation or auto‐aggressive T cells. We show here that young and aged naïve CD4⁺ T cells are equivalently auto‐aggressive in vivo in T cell‐driven autoimmune colitis. Young and aged CD4⁺ Tregs equally suppressed age‐matched T cell proliferation in vitro and controlled clinical and pathologic T cell‐driven autoimmune colitis, suggesting equivalent regulatory function. However, whereas young and aged CD4⁺ Tregs suppressed interferon (IFN)‐γ⁺ T cells equivalently in this model, aged CD4⁺ Tregs unexpectedly failed to restrain interleukin (IL)‐17⁺ T cells. Nonetheless, young and aged CD4⁺ Tregs equally restrained IL‐17⁺ T cells in vivo during acute inflammation, suggesting a chronic inflammation‐related defect in aged CD4⁺ Tregs. In support, aged Tregs expressed reduced STAT3 activation, a defect associated with poor IL‐17‐producing T cell restraint. Aged naïve mice had markedly increased programmed death (PD)‐1⁺ T cells, but these exhibited no significant auto‐aggressive or regulatory functions in T cell‐driven colitis. Young CD8⁺ CD122^? T cells induce autoimmune bone marrow failure, but we show that aged CD8⁺ CD122^? T cells do not. These data demonstrate no apparent age‐related increase in auto‐aggressive T cell behavior, but disclose previously unrecognized functional defects in aged CD4⁺ Tregs during chronic inflammation. IL‐17 can be inflammatory and contributes to certain autoimmune disorders. Reduced aged Treg function during chronic inflammation and reduced IL‐17 restraint could contribute to age‐related inflammation or autoimmunity.     

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.     

Age‐associated alterations in CD8α+ dendritic cells impair CD8 T‐cell expansion in response to an intracellular bacterium

Age‐associated decline in immunity to infection has been documented across multiple pathogens, yet the relative contributions of the aged priming environment and of lymphocyte‐intrinsic defects remain unclear. To address the impact of the aging environment on T‐cell priming, adult naïve OT‐I TCR transgenic CD8 T cells, specific for the H‐2K^b‐restricted immunodominant OVA_257‐264 epitope, were transferred into adult or old recipient mice infected with the recombinant intracellular bacterium Listeria monocytogenes carrying the chicken ovalbumin protein (Lm‐OVA). We consistently found that adult OT‐I CD8 expansion was reduced in aged recipient mice, and this correlated with numeric, phenotypic, and functional defects selectively affecting CD8α+ dendritic cells (DC). Following Lm‐OVA infection, aged mice failed to accumulate CD8α+ DC in the spleen, and these cells expressed much lower levels of critical costimulatory molecules in the first three days following infection. Further, aged CD8α+ DC showed impaired uptake of the bacteria at very early time points following infection. Treatment of aged mice with Flt3 ligand (Flt3L) improved the number of DC present in the spleen prior to Lm‐OVA infection, and improved, but did not reconstitute, OT‐I expansion to Lm‐OVA infection. These results suggest that age‐associated changes in antigen uptake, pathogen sensing, and/or antigen presentation contribute to impaired adaptive immune responses to microbial pathogens with aging.     

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.     

Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4⁺ and CD8⁺ T cells, microarray analysis was performed pre‐ and post‐TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T‐cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4⁺ and CD8⁺ naïve T cells. Evaluation of these differences provides in‐depth insight into potential functional and phenotypical differences among these populations.     

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-β, IL-10 and IFN-γ by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-γ is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-β only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-β induced IL-10 production. IL-10 produced in naïve subjects when TGF-β is neutralized or in LCL patients did not modify the IFN-γ production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-β is neutralized clearly showed that they are memory CD45RA⁻ CD8⁺ T cells. In LCL patients, IL-10 producing cells are both CD45RA⁻ CD4 and CD8⁺ T cells. The role of these IL-10 producing CD8⁺ T cells in the development of the diseases should be carefully evaluated.     

Microparticle delivery of Interleukin-7 to boost T-cell proliferation and survival

In HIV infections, homoeostasis of T cells is dysregulated such that there is a depletion of CD4⁺ T cells and a progressive loss of naïve CD4⁺ and CD8⁺ T cells. Methodologies that can improve the function of some or all of these cells will likely enhance immune responsiveness in HIV infection. Interleukin‐7 (IL‐7) is a cytokine that has been shown to be critical in homeostatic expansion of naïve CD8⁺ and CD4⁺ cells in lymphopenic hosts, as well as regulating effector T cell to memory T‐cell transition and memory T‐cell homeostasis. In animal studies and clinical trials, repeated injections of IL‐7 are used to boost both CD4⁺ and CD8⁺ cell counts. Daily injections, however, are painful, inconvenient, and provide a frequent route for pathogen entry. We developed a poly (D ,L ‐lactide‐co‐glycolide; PLGA) microparticle controlled release system to administer IL‐7 in which a single injection of microparticles can provide therapeutic delivery of IL‐7. IL‐7 encapsulated PLGA microparticles were first synthesized using a water/organic/water double emulsion method, release from the particles was then optimized using in vitro release studies and therapeutic effectiveness was finally studied in animal studies. These PLGA microparticles showed effective delivery of IL‐7 for 1 week in vitro. These results were translated to in vivo delivery as well, which was followed for 9 days. Controlled release of IL‐7 in mice demonstrated biological activity in both CD4⁺ and CD8⁺ T cells in mice, which was consistent with previously reported results using daily injections. Biotechnol. Bioeng. 2012; 109:1835–1843. © 2012 Wiley Periodicals, Inc.     

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T_CM) and effector (=T_EM) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL).In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7^?CD45RA^?CD8⁺ T_EM cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T_EM cells was significantly increased. T_EM phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result.Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T_EM cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.     

B7-H1 signaling is integrated during CD8+ T cell priming and restrains effector differentiation

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cells is integrated during priming of naïve CD8⁺ T cells and functions to limit the differentiation of effector T cell responses. CD8⁺ T cells primed by B7-H1-deficient dendritic cells exhibit increased production of IFN-γ, enhanced target cell killing, and improved anti-tumor activity. Additionally, enhanced memory populations arise from CD8⁺ T cells primed by B7-H1-deficient dendritic cells. Based on these findings, we suggest that early blockade of B7-H1 signaling should be investigated as a strategy to improve dendritic cell-based anti-tumor immunotherapy.     

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.     

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.

     

Compartment resolved reference proteome map from highly purified naïve,activated, effector,and memory CD8+ murine immune cells

Differentiation of CD8⁺ T lymphocytes into effector and memory cells is key for an adequate immune response and relies on complex interplay of pathways that convey signals from the cell surface to the nucleus. In this study, we investigated the proteome of four cytotoxic T‐cell subtypes; naïve, recently activated effector, effector, and memory cells. Cells were fractionated into membrane, cytosol, soluble nuclear, chromatin‐bound, and cytoskeletal compartments. Following LC‐MS/MS analysis, identified peptides were analyzed via MaxQuant. Compartment fractionation and gel‐LC‐MS separation resulted in 2399 proteins identified in total. Comparison between the different subsets resulted in 146 significantly regulated proteins for naïve and effector cells, followed by 116 for activated, and 55 for memory cells. Besides Granzyme B signaling (for activated and/ or effector cells vs. naïve cells), the most prominent changes occurred in the TCA cycle and aspartate degradation. These changes suggest that correct balancing of metabolism is key for differentiation processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001065 ( http://proteomecentral.proteomexchange.org/dataset/PXD001065 ).     

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.     

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 ).     

Systemic analyses of immunophenotypes of peripheral T cells in non-segmental vitiligo: implication of defective natural killer T cells

Although it is widely believed that non‐segmental vitiligo (NSV) results from the autoimmune destruction of melanocytes, a clear understanding of defects in immune tolerance, which mediate this uncontrolled self‐reactivity, is still lacking. In the present study, we systemically evaluated circulating regulatory T (Treg) cells, including CD4⁺CD25⁺FoxP3⁺ Treg cells and invariant natural killer T (iNKT) cells, as well as naïve and memory CD4⁺ and CD8⁺ T cells and their cytokine production, in a cohort of 43 progressive NSV patients with race‐, gender‐, and age‐matched healthy controls. We found that the general immunophenotypes of CD4⁺ and CD8⁺ T cells and the percentage of CD4⁺CD25⁺FoxP3⁺ Tregs were comparable between NSV and healthy controls. However, percentages of peripheral iNKT cells were significantly decreased in NSV patients compared to that in healthy controls. Our data confirm the previous notion that the percentage of peripheral CD4⁺CD25⁺FoxP3⁺ Tregs remains unaltered in NSV and suggests the involvement of defective iNKT cells in the pathogenesis of NSV.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.