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A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA−/CD45RO+ T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12‐myristate 13‐acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho‐PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL‐4 and minimal interferon gamma production (IFN‐γ), and lack of expression of transcriptional factor, T‐bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN‐γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias. [ABSTRACT FROM AUTHOR]

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4+ and CD8+ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4+ and CD8+ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially ‘corrected’ after birth.

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well-studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4- and 8-weeks of age. IETs increased in abundance over time and belonged to both γδ and αβ T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8α+ γδ T cells and CD4−CD8α+ αβ T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of γδ IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8α− γδ IETs, while CD2+CD8α+ γδ IET percentages were greater in the large intestine. Percentages of CD4−CD8α+ αβ IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4- to 8-weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs. [ABSTRACT FROM AUTHOR]

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation. [ABSTRACT FROM AUTHOR]

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo. Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8+ T cell responses in NOD.Cg-Rag1tm1Mom-Il2rγtm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8+ memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo. A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion. [ABSTRACT FROM AUTHOR]

Immunological programming of immune cells varies in response to changing environmental signals. This process is facilitated by modifiers that regulate the translational fate of mRNAs encoding various immune mediators, including cytokines and chemokines, which in turn determine the rapid activation, tolerance, and plasticity of the immune system. RNA-binding proteins (RBPs) recruited by the specific sequence elements in mRNA transcripts are one such modifier. These RBPs form RBP-RNA complexes known as Riboclusters. These riboclusters serve as RNA sorting machinery, where depending upon the composition of the ribocluster, control translation, degradation or storage of mRNA. Recent findings suggest that this regulation of mRNA homeostasis is critical for controlling the immune response. Here, we present the current knowledge of the ribocluster-mediated post-transcriptional regulation of immune mediators, and highlight recent findings regarding their implications for the pathogenesis of acute or chronic inflammatory diseases.

Immunological programing of immune cells varies in response to changing environmental signals. This process is facilitated by modifiers that regulate the translational fate of mRNAs encoding various immune mediators, including cytokines and chemokines, which in turn determine the rapid activation, tolerance, and plasticity of the immune system. RNA-binding proteins (RBPs) recruited by the specific sequence elements in mRNA transcripts are one such modifiers. These RBPs form RBP-RNA complexes known as "riboclusters." These riboclusters serve as RNA sorting machinery, where depending upon the composition of the ribocluster, translation, degradation, or storage of mRNA is controlled. Recent findings suggest that this regulation of mRNA homeostasis is critical for controlling the immune response. Here, we present the current knowledge of the ribocluster-mediated post-transcriptional regulation of immune mediators and highlight recent findings regarding their implications for the pathogenesis of acute or chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Intra-thymic injection is a powerful tool for adoptive transfer of cells, cellular tag reagents for tracking recent thymic emigrants (RTEs), or other substances directly into the thymus. The traditional approach developed decades ago requires an invasive surgery to open the thoracic cavity and visualize the thymus. Subsequently, a technique was developed requiring only a small skin incision needed to identify the precise injection site. Nevertheless, both techniques require surgical intervention, and this can lead to elevated animal stress levels and pain which necessitates analgesic medication administration. Here we describe a less invasive technique allowing in situ visualization and transfer of cell suspensions or substances into the thymus via an ultrasound-guided intra-thymic injection approach., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Abstract: Human hematopoietic stem cells giving rise to long term initiating cells in vitro are enriched in a CD34+ slow dividing fraction (SDF). Here, we tested reconstitution and multilineage differentiation of this CD34+ SDF in NOD/SCID mice. In the bone marrow a slightly higher percentage of human hematopoietic progenitors were recovered after the transfer of the SDF compared to the fast dividing fraction. Instead, T cell maturation in the rudimentary thymus and lymph node repopulation was only initiated by the SDF. The capacity of the SDF to differentiate and mature in the patients’ thymus could provide an advantage in immunocompetence recovery. [Copyright &y& Elsevier]

CXCL12 is an important CXC chemokine involved in numerous biological processes. We had previously demonstrated the synergistic participation of CXCL12 and IL-7 in the control of both survival and proliferation of CD34+ human thymic lymphoid progenitors. On this basis, we hypothesize a presumptive role for CXCL12 and its receptor, CXCR4, in the thymus involution. In this respect, in the current report we describe the expression of both molecules in the human thymus during aging. Our results demonstrate that, despite the profound alterations observed in the thymic epithelial microenvironment of aged thymuses, the proportions of different CD4/CD8 thymocyte subsets do not undergo significant variations. Remarkably, a strong CXCL12 expression was found in older thymuses, which appeared in the same locations as in younger thymuses: the subcapsulary and medullary areas. The proportions of CXCR4+ cells, most of them belonging to the CD3– compartment, showed no important variations in the older thymuses. However, within the CD34+ cell population, a significant reduction in the expression of CXCR4 molecules was observed. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4+ and CD8+ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4+ and CD8+ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially 'corrected' after birth. [ABSTRACT FROM AUTHOR]

Summary The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-γ ) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-γ release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression. [ABSTRACT FROM AUTHOR]

Most patients with primary immune deficiency suffer from recurrent infections; however, paradoxical autoimmune phenomena can also manifest. The aim of this study was to identify immunological markers of autoimmune phenomena associated with common variable immunodeficiency (CVID). The study included 33 adults with CVID divided into two groups: (1) those with noninfectious autoimmune complications (CVID-C (n = 24)) and (2) those with only infectious symptoms (CVID-OI (n = 9)). Flow cytometry of peripheral blood was performed and compared with systemic lupus erythematosus (SLE) patients (n = 17) and healthy controls (n = 20). We found that all lymphocytes were lower in CVID-C and SLE. NK cells were lowest in CVID-C. Th17 cells were significantly reduced in CVID-C and SLE. Tregs were significantly lower in CVID-C and SLE. Bregs did not significantly differ between any groups. Class-switched memory B cells were significantly lower in CVID-C and CVID-OI. Lastly, plasmablasts were significantly higher in SLE. Among the T cell subsets, CVID-C patients had lower naive and recent thymic emigrant CD4+ T cells. In conclusion, reduced Treg, Th17, and NK cells are features of CVID with autoimmune complications, and class-switched memory B cells can help distinguish patients with different causes of autoimmunity. Future studies are needed to confirm whether reductions of Treg, Th17, and NK cells might be a biomarker of more complicated CVID cases. [ABSTRACT FROM AUTHOR]

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases. [ABSTRACT FROM AUTHOR]

The murine heat stable antigen (HSA, mouse CD24) is a glycosyl phosphatidylinositol-anchored cell surface protein which is primarily expressed in immature but not mature cells of several hematopoletic lineages and in neuronal tissue. The function of HSA is not known but there is evidence in lymphocytes that it is involved in cell adhesion and in cell activation. We examined the effect of constitutive HSA expression on the maturation and on the function of B and T cells in transgenic mice. Transgenic HSA was strongly expressed throughout all stages of T cell maturation without changes in absolute cell number or proportions of subpopulations both in the thymus and in the periphery. The size of the B cell compartment was also unchanged. Thus, we conclude that in the T lineage the loss of HSA expression is not mandatory for maturation. On the functional level, two parameters of immune function were measured. When the ability to activate peripheral T cells expressing transgenic HSA was tested in a mixed lymphocyte reaction, no significant difference in the stimulation index and cytolytic activity was detected between transgenics and control littermates. However, when immunized with a T cell dependent antigen, transgenic mice showed 10-fold higher serum lgG1 titers. This suggests that the expression of transgenic HSA in cells normally negative for HSA (e.g. peripheral T cells or memory B cells) leads to a better stimulation of lymphocytes during a secondary antibody response. [ABSTRACT FROM PUBLISHER]

Antigen-specific cytotoxlc killer lymphocytes (CTLs) represent one of the major effector functions of the immune system. It is well established that, as a consequence of TCR recognition of the antigen-bearing target cell, resting T lymphocytes develop Into fully active antigen-specific CTLs. In contrast, natural killer (NK) cells are immediately tytic upon contact with an appropriate target cell. The tytic machinery of CTLs and NK cells Is thought to include the contents of their cytoplasmlc granules, in particular the pore-forming protein perforin. Here we report direct cytolytic activity by resting peripheral CD3CD8 T cells as a result of TCR-CD3 binding to the target cell; the murine OKT3 hybridoma (anti-human CD3) was used as a target. The cytotoxicity was more pronounced In the CD8CD45RO population, which contains ‘memory’ T cells, than In the reciprocal CD8CD45RA subset; CD8CD4 mature thymocytes were non-cytotoxlc. The cytolytic potential of these populations correlated with the presence or absence of perforin. The results demonstrate that the cytolytic machinery of T cells develops post-thymically and can be Immediately triggered by TCR-CD3 stimulation. [ABSTRACT FROM PUBLISHER]

Thymic B cells are a constituent of normal human thymic medulla. They are supposed to play a role in T cell maturation. Thymic B cells have been characterized morphologically and immunohistochemically at the light-microscopic level. Their ultrastructural appearance in vivo has not been demonstrated. Six normal infantile thymi were immunolabelled with the pan-B cell marker CD20 using a pre-embedding technique and viewed at the electron-microscopic level. Cells expressing CD20 had long cytoplasmic processes. They were all 'asteroid' in shape and in close contact with thymocytes. Also, their long cytoplasmic processes intermingled with cytoplasmic processes of cells that were presumed to be interdigitating reticulum cells (IDC) based on morphological criteria. Thymic B cells may act in concert with IDC during T cell maturation. [ABSTRACT FROM AUTHOR]

T cells play a crucial role in the adaptive immune system, and their maturation process is tightly regulated. Adenosine deaminase acting on RNA 1 (ADAR1) is the enzyme responsible for adenosine‐to‐inosine RNA editing in dsRNAs, and loss of ADAR1 activates the innate immune sensing response via melanoma differentiation‐associated protein 5 (MDA5), which interprets unedited dsRNA as non‐self. Although ADAR1 is highly expressed in the thymus, its role in the adaptive immune system, especially in T cells, remains elusive. Here, we demonstrate that T cell‐specific deletion of Adar1 in mice causes abnormal thymic T cell maturation including impaired negative selection and autoimmunity such as spontaneous colitis. This is caused by excessive expression of interferon‐stimulated genes, which reduces T cell receptor (TCR) signal transduction, due to a failure of RNA editing in ADAR1‐deficient thymocytes. Intriguingly, concurrent deletion of MDA5 restores thymocyte maturation and prevents colitis. These findings suggest that prevention of MDA5 sensing of endogenous dsRNA by ADAR1‐mediated RNA editing is required for preventing both innate immune responses and T cell‐mediated autoimmunity. Synopsis: ADAR1 is responsible for editing dsRNAs, and loss of ADAR1 activates the innate immune response. ADAR1‐mediated editing is essential for thymic T cell development, and for counteracting autoimmunity via MDA5‐sensing of unedited dsRNA. ADAR1‐mediated RNA editing is upregulated in a stage‐dependent manner during T cell maturation.T cell‐specific Adar1 KO mice exhibit spontaneous autoimmunity caused by aberrant T cell maturation.Type I interferon‐stimulated genes are upregulated by the activation of MDA5 in ADAR1‐deficient T cells.The concurrent deletion of MDA5 restores thymocyte maturation and prevents the recognition of unedited dsRNA as non‐self. ADAR1 is responsible for editing dsRNAs, and loss of ADAR1 activates the innate immune response. ADAR1‐mediated editing is essential for thymic T cell development, and for counteracting autoimmunity via MDA5‐sensing of unedited dsRNA. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14 + , CD169 + , and CD34 + cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4 + T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG + plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4 + T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo . Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8 + T cell responses in NOD.Cg-Rag1 tm1Mom -Il2rγ tm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8 + memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo . A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

An individual's immune function, susceptibility to infection, and response to immunosuppressive therapy are influenced in part by his/her T cell maturation state. Although childhood is the most dynamic period of immune maturation, scant information regarding the variability of T cell maturation in children with renal disease is available. In this study, we compared the T cell phenotype in children with renal failure (n=80) with that in healthy children (n=20) using multiparameter flow cytometry to detect markers of T cell maturation, exhaustion, and senescence known to influence immune function. We correlated data with the degree of renal failure (dialysis or nondialysis), prior immunosuppression use, and markers of inflammation (C-reactive protein and inflammatory cytokines) to assess the influence of these factors on T cell phenotype. Children with renal disease had highly variable and often markedly skewed maturation phenotypes, including CD4/CD8 ratio reversal, increased terminal effector differentiation in CD8 + T cells, reduction in the proportion of naïve T cells, evidence of T cell exhaustion and senescence, and variable loss of T cell CD28 expression. These findings were most significant in patients who had experienced major immune insults, particularly prior immunosuppressive drug exposure. In conclusion, children with renal disease have exceptional heterogeneity in the T cell repertoire. Cognizance of this heterogeneity might inform risk stratification with regard to the balance between infectious risk and response to immunosuppressive therapy, such as that required for autoimmune disease and transplantation., (Copyright © 2016 by the American Society of Nephrology.)

Objective: Characterized by chronic inflammation, dysfunction of exocrine glands, and systemic autoimmunity, primary Sjögren syndrome (pSS) is a common autoimmune disease in elderly women. Our study was performed to explore the potential involvement of microRNA (miRNA) in Chinese patients with pSS., Methods: Using microarrays, miRNA expression in peripheral blood mononuclear cells (PBMC) was profiled in 4 female patients with pSS and 3 healthy participants, followed by a large-scale study of 33 patients and 10 healthy individuals. Compared to the healthy participants, 202 miRNA were upregulated and 180 were downregulated in the patients with pSS. To confirm this finding, a set of regulated miRNA was further examined in a large patient group, using quantitative reverse transcriptase-PCR assays., Results: MiR-181a was the miRNA that most profoundly differed between patients with pSS and healthy individuals; however, similar miRNA-181a expression profiles were found in groups with different disease phenotypes. Together, these observations suggested that an elevated miRNA-181a level is a general phenomenon in Chinese patients with pSS., Conclusion: In addition to the elevated miR-181a levels, our study led to the speculation that elevated miR-181a levels in the PBMC of these patients compromise the maturation of B cells, enabling them to recognize and attack autoantigens and resulting in disease phenotypes. In addition to the regulation of human miRNA, many virus-derived miRNA were unexpectedly upregulated in the patients with pSS, suggesting that viral infection of PBMC plays a role in this disease.

Tacrolimus impairs allo- and viral-specific T cell responses. Belatacept, a costimulation-based alternative to tacrolimus, has emerged with a paradoxical picture of less complete control of alloimmunity with concomitant impaired viral immunity limited to viral-naïve patients. To reconcile these signatures, bulk population and purified memory and naïve lymphocytes from cytomegalovirus (CMV)-seropositive (n=10) and CMV-seronegative (n=10) volunteers were studied using flow cytometry, interrogating proliferation (carboxyfluorescein succinimidyl ester dilution) and function (intracellular cytokine staining) in response to alloantigens or CMV-pp-65 peptides. As anticipated, T cells from CMV-experienced, but not naïve, individuals responded to pp-65 with a small percentage of their repertoire (<2.5%) consisting predominantly of mature, polyfunctional (expressing interferon gamma, tumor necrosis factor alpha and IL-2) T effector memory cells. Both CMV naïve and experienced individuals responded similarly to alloantigen with a substantially larger percentage of the repertoire (up to 48.2%) containing proportionately fewer polyfunctional cells. Tacrolimus completely inhibited responses of CMV- and allo-specific T cells regardless of their maturation. However, belatacept's effects were decreasingly evident in increasingly matured cells, with minimal effect on viral-specific triple cytokine producers and CD28-negative allo-specific cells. These data indicate that belatacept's immunosuppressive effect, unlike tacrolimus's, wanes on progressively developed effector responses, and may explain the observed clinical effects of belatacept., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)