CD4 SP Research Articles

MiR-9-1 Controls Osteoblastic Regulation of Lymphopoiesis

The highly conserved MicroRNA-9 (miR-9) family consists of three members, miR-9-1, miR-9-2 and miR-9-3. These three microRNAs are encoded by unique genes located on different chromosomes, the transcripts of which are processed into the same mature miR-9 sequence. MiR-9 is known to play a crucial role in brain development and neurogenesis, its involvement in hematopoiesis has remained uncertain. To explore this, we investigated the role of miR-9-1 in controlling hematopoiesis. Our findings revealed that miR-9-1 and miR-9-2 but not miR-9-3 were expressed in all hematopoietic cells, with higher levels observed in HSCs, MPPs, and CLPs. In order to understand the impact of miR-9 on hematopoiesis, we employed CRISPR/Cas9-mediated genome editing to create miR-9-1-deficient mice. Deletion of miR-9-1 resulted in reduced mature miR-9 expression, leading to 43% of the mice exhibiting retarded growth and postweaning lethality by 12 weeks of age. The growth-retarded miR-9-1-deficient mice experienced severe lymphopenia, primarily affecting B and T cell development. Further analysis revealed that the populations of pro/pre- and immature B cells in bone marrow (BM) were dramatically reduced in growth-retarded miR-9-1-deficient relative to wild-type control mice. The populations of peripheral T1, T2, FO and MZ B cells were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. In addition, the numbers of DN, DP, and SP thymocytes and CD4 + and CD8 + T cells in the spleen were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. Consistent with the specific impairment of lymphocyte (B and T cell) development and maturation, the growth-retarded miR-9-1-deficient mice exhibited a reduction of hematopoietic progenitors including ST-HSCs, MPPs, CMPs and especially CLPs, but had normal numbers of GMPs and MEPs. Interestingly, transplantation of miR-9-1-deficient BM cells into wild-type recipients resulted in normal lymphopoiesis, indicating that the lymphopenia was not due to hematopoietic cell autonomous effects but was caused by alterations in the BM stromal microenvironment. We then focused on the effect of miR-9-1 deficiency on the BM niche, particularly the mesenchymal stem cell (MSC) and osteoblastic (OB) niches. The numbers of MSCs in BM stromal cells were comparable between miR-9-1-deficient and wild type mice, whereas the number of osteoblastic cells (OBs) was significantly reduced in the BM stromal cells from growth-retarded miR-9-1-deficient relative to wild-type mice. In agreement with the reduction of OBs in miR-9-1-deficient mice, the mutant MSCs failed to differentiate into OBs. Notably, miR-9-1 deficiency did not influence the differentiation of osteoclasts and adipocytes, suggesting a specific impairment in osteoblastic bone formation. To elucidate the molecular mechanism underlying miR-9-1's regulation of osteoblastic differentiation, we conducted high-throughput RNA sequencing analysis on miR-9-1-deficient MSCs and OBs. The results revealed reduced expression of master transcription factors involved in osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Additionally, we identified Follistatin (Fst) as a direct target of miR-9-1. MiR-9-1 deficiency led to increased Fst levels, which, in turn, inhibited BMP signaling in MSCs and reduced IL-7 and IGF-1. In summary, our studies demonstrate that miR-9-1 plays a pivotal role in controlling osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

Systemic CD4 cytotoxic T cells improve protection against PRRSV-1 transplacental infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major swine pathogens causing reproductive failure in sows. Although modified-live virus (MLV) vaccines are available, only partial protection against heterologous strains is produced, thus vaccinated sows can be infected and cause transplacental infection. The immune effector mechanisms involved are largely unknown. The present study investigated the role of cytotoxic lymphocytes, including cytotoxic T cells (CTL), NKT, and NK cells, from blood in preventing PRRSV-1 transplacental infection in vaccinated primiparous sows (two doses vaccinated). Sows from a PRRSV-1 unstable farm were bled just before the last month of gestation (critical period for transplacental infection), then followed to determine whether sows delivered PRRSV-1-infected (n=8) or healthy (n=10) piglets. After that, functions of CTL, NKT, and NK cells in the two groups of sows were compared. No difference was found through cell surface staining. But upon in vitro re-stimulation with the circulating field virus, sows that delivered healthy piglets displayed a higher frequency of virus-specific CD107a+ IFN-γ-producing T cells, which accumulated in the CD4+ compartment including CD4 single-positive (CD4 SP) and CD4/CD8α double-positive (CD4/CD8α DP) subsets. The same group of sows also harbored a higher proportion of CD107a+ TNF-α-producing T cells that predominantly accumulated in CD4/CD8α double-negative (CD4/CD8α DN) subset. Consistently, CD4 SP and CD4/CD8α DN T cells from sows delivering healthy piglets had a higher virus-specific proliferative response. Additionally, in sows that delivered PRRSV-1-infected piglets, a positive correlation of virus-specific IFN-γ response with average Ct values of umbilical cords of newborn piglets per litter was observed. Our data strongly suggest that CTL responses correlate with protection against PRRSV-1 transplacental infection, being executed by CD4 T cells (IFN-γ related) and/or CD4/CD8α DN T cells (TNF-α related).

VSIG4(+) peritoneal macrophages induce apoptosis of double-positive thymocyte via the secretion of TNF-\u03b1 in a CLP-induced sepsis model resulting in thymic atrophy

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(−) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(−) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(−) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

Global dynamics of stage-specific transcription factor binding during thymocyte development

In vertebrates, multiple transcription factors (TFs) bind to gene regulatory elements (promoters, enhancers, and silencers) to execute developmental expression changes. ChIP experiments are often used to identify where TFs bind to regulatory elements in the genome, but the requirement of TF-specific antibodies hampers analyses of tens of TFs at multiple loci. Here we tested whether TF binding predictions using ATAC-seq can be used to infer the identity of TFs that bind to functionally validated enhancers of the Cd4, Cd8, and Gata3 genes in thymocytes. We performed ATAC-seq at four distinct stages of development in mouse thymus, probing the chromatin accessibility landscape in double negative (DN), double positive (DP), CD4 single positive (SP4) and CD8 SP (SP8) thymocytes. Integration of chromatin accessibility with TF motifs genome-wide allowed us to infer stage-specific occupied TF binding sites within known and potentially novel regulatory elements. Our results provide genome-wide stage-specific T cell open chromatin profiles, and allow the identification of candidate TFs that drive thymocyte differentiation at each developmental stage.

Desert hedgehog knockout mice show a reduced ratio of CD4+:CD8+ in thymic developing T cells

<p class="abstract"><strong>Background:</strong> Thymic differentiation is important and determines the strength of adaptive immunity in mammals in their later days in life. There are many factors that have been found to influence the development of T lymphocytes in the thymus and these include the effect of hedgehog signalling family of proteins. Immunologists and other basic science researchers have established the role of Indian hedgehog and sonic hedgehog in thymocytes development in the recent past, but the role of hedgehog is not well known. The aim of this study was to determine the influence of Desert hedgehog in CD4:CD8 ratio in developing thymocytes of mice.</p><p class="abstract"><strong>Methods:</strong> Smashed thymocytes from mice deficient of Desert hedgehog and those with an intact gene coding for this protein were prepared in a cell suspension using standard procedurs. The cell suspensions were stained using fluorochrome-labelled monoclonal anti: CD4-PE, CD8-TRI, CD3-FITC, CD5-FITC, CD44-PE and CD25-FITC (e-Bioscience). Samples were analyzed using a three-color flow cytometry. The flow cytometry-generated data was analyzed using flowjo (Treestar, USA). Statistical significance of the findings was determined using paired t-test and reported at p<0.05. </p><p class="abstract"><strong>Results:</strong> There was a general upward trend on CD4+CD8+ double positive thymocytes in Desert hedgehog mice relative to WT controls. An analysis of CD4:8 ratio revealed a reduced ratio in Dhh KO mice compared to that of WT controls attributable to the finding that there might have been a preferential differentiation of DP CD4+CD8+ to SP CD4+ in Dhh knockout mice as demonstrated by percentage of thymic subsets. The results of this study were not statistically significant and this was blamed on the fewer number of animals used in the study.</p><p class="abstract"><strong>Conclusions:</strong> Dhh might have a role arresting the DP cell subjects from differentiating preferentially to CD4+ T cells. To get statistically significant findings, these experiments could be repeated with a larger animal sample.</p>

SHP1 alters TCR signal strength and metabolism

Abstract Recently, we have shown thymocytes lacking SHP1 have decreased cell numbers post selection for both CD4 SP and CD8 SP. This thymocyte selection defect led to decreases in tetramer positive precursor numbers in the periphery. Specifically we analyzed CD8 T cells for LCMV GP33–41:Kd and CD4 T cells for MOG35–55:IAb. Immunization with LCMV Armstrong revealed about 50% reduction of antigen specific T cells, both CD4+ and CD8+. Even though SHP1 deficient T cells have reduced numbers, they exhibit superior proliferation as observed by cell trace violet coupled with increased death. Though SHP1 has many implications in T cell signaling, a novel aspect is impacting metabolism as the SHP1 deficient T cells display increased metabolic activity. Naïve SHP1 deficient CD8 T cells exhibit increased mitochondrial respiration. During expansion, these T cells maintain increased mitochondrial respiration compared to wildtype controls. Comparatively, lack of SHP1 also causes an increase in glucose uptake throughout expansion. Altogether, SHP1 appears to impact TCR mediated signal threshold which leads to increased T cell metabolism in peripheral tissues.

Canine peripheral blood CD4+CD8+ double-positive T cell subpopulations exhibit distinct T cell phenotypes and effector functions

Canine peripheral blood CD4+CD8α+ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4+ but also from CD8+ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4dimCD8αbright, CD4brightCD8αbright, and the CD4brightCD8αdim subsets. Previously we could show that total canine peripheral CD4+CD8α+ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4dimCD8αbright subpopulation has the lowest and the CD4brightCD8αbright subset the highest frequency of CD25+ cells pointing to the CD4brightCD8αbright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4brightCD8αbright subset contains the highest frequencies of effector-memory Tcells (TEM). Upon in vitro stimulation, the CD4brightCD8αbright and CD4dimCD8αbright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4dimCD8αbright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4dimCD8αbright subset, while Foxp3+ regulatory Tcells were detected in the CD4brightCD8αdim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4+ and CD8+ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.

Defining the role of CCR8 in promoting thymocyte development and thymic epithelial homeostasis

Abstract Generation of functional and self-tolerant T cells in the thymus is crucial to preventing autoimmune diseases. Medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present diverse auto-antigens to developing thymocytes; strong TCR reactivity in response to self-antigens causes thymocytes to undergo negative selection, eliminating autoreactive cells from the repertoire. Chemokine receptors expressed on the surface of developing thymocytes have been shown to promote accumulation of post-positive selection thymocytes in the medulla, where they may encounter mTECs and DCs presenting auto-antigens. CCR8 is highly expressed by post-positive selection CD4 thymocytes while its ligands, CCL1 and CCL8 are expressed by mTECs. Thus, CCR8 signaling may promote medullary entry of post-positive selection thymocytes and efficient interactions with mTECs and enforcing central tolerance. Our preliminary data indicate that CCR8−/− thymi have a reduced medullary area, implicating impaired thymocyte:mTEC interactions that impacts normal proliferation and differentiation of mTECs. Furthermore, 2-photon imaging reveals that CCR8 deficiency results in a significant increase in the path straightness of CD4 SP thymocytes, which could impact the efficiency of interactions with medullary stromal cells. Strikingly, CCR8 deficiency results in increased cellularity of CD4SP OTII TCR transgenic thymocytes in a wild-type host, suggesting CCR8 is critical for efficient clonal deletion to endogenous auto-antigens. Thus, our preliminary data indicate that CCR8 contributes to the migratory properties and stromal interactions of post-positive selection thymocytes that promote efficient negative selection.

Histone deacetylase 3 is necessary for T cell maturation (HEM2P.238)

Abstract Recent thymic emigrants (RTEs) are newly generated T cells that need to undergo post-thymic maturation in the periphery. The mechanism of T cell maturation remains unclear. Histone deacetylase 3 (HDAC3) controls gene regulation, chromatin accessibility and genomic stability. However, the function of HDAC3 in T cell has yet to be elucidated. As we previously demonstrated that HDAC3 is a binding partner to NKAP, we hypothesize that HDAC3, like NKAP, may regulate T cell maturation. We generated CD4-cre HDAC3 cKO mice, which have similar numbers of DN, DP, CD4 SP and CD8 SP thymocytes as compared to WT mice; however, the peripheral numbers of CD4+ and CD8+ T cells are dramatically decreased in CD4-cre HDAC3 cKO mice. When we examined Rag1-GFP CD4-cre HDAC3 cKO mice, the majority of peripheral naïve T cells are Rag1-GFP+ RTEs indicating a block in T cell maturation. There is no defect in the positive selection, negative selection, migration, or homeostasis in CD4-cre HDAC3 cKO mice. However, expression of the complement inhibitor CD55, which is induced during maturation, is severely decreased. In the periphery, similar to NKAP-deficient T cells, HDAC3-deficient T cells are targeted for elimination by complement. In addition, HDAC3-deficient T cells have decreased sialic acid modifications on the cell surface, mainly due to α2-3, and α2-8 linkages, that recruit natural IgM to initiate the classical complement pathway. Thus, HDAC3 is required for T cell maturation.

HDAC3 is required for thymocyte positive selection (HEM2P.236)

Abstract In order to generate functional peripheral T cells, proper gene regulation during T cell development is critical. Development involves both the activation and repression of genes as cells progress through each developmental checkpoint. Histone acetylation/deacetylation is one mechanism regulating gene expression. Acetylation by histone acetyl transferases (HATs) allows for gene expression and deacetylation by histone deacetylase (HDAC) enzymes promote gene silencing, and their role in regulating T cell development is not clear. In this study, our lab focused on HDAC3. We examined the role HDAC3 plays during T cell development by utilizing a CD2-Cre-mediated loss of HDAC3 (CD2icre HDAC3-cKO), which deletes early in thymocyte development. When HDAC3 is absent, there is a developmental block during positive selection, leading to the production of very few CD4 and CD8 SP thymocytes leading to a severe defect in peripheral T cell numbers. During positive selection, HDAC3-deficient DP thymocytes fail to down-regulate RORγt as they progress to the SP stage. Consistent with the failure to down-regulate RORγt, thymocytes post-positive selection express high levels of Bcl-xl and fail to express Bcl-2, which is required for their survival. Moreover, HDAC3-deficient thymocytes show increased apoptosis. These results suggest that HDAC3 is required for the down-regulation of RORγt during positive selection.

Fine-tuning of T-cell development by the CD3γ di-leucine-based TCR-sorting motif.

The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulation is abolished in thymocytes from CD3γLLAA mice with a mutated CD3γ diL motif, and that CD3γLLAA mice have reduced numbers of thymocytes compared with aged-matched wild-type mice. We found that early thymocyte development at the β-selection checkpoint is impaired resulting in reduced numbers of double negative (DN) 4 cells in CD3γLLAA mice. This was not caused by reduced proliferation but most probably by increased down-regulation of the antiapoptotic molecule Bcl-2 causing enhanced apoptosis during the transition from the DN3 to the DN4 stage. In contrast, proliferation of immature CD8 single positive (ISP) thymocytes was increased resulting in normal numbers of ISP in CD3γLLAA mice. Despite the normal numbers of ISP, CD3γLLAA mice had reduced numbers of double positive and SP thymocytes indicating that the CD3γ diL motif also affected later stages of T-cell development. In accordance, we found that positive and negative selection, differentiation toward CD4 and CD8 SP T cells and the development of nonconventional T cells were affected in CD3γLLAA mice. In conclusion, our study identifies an important role of the CD3γ diL motif in T-cell development most probably mediated by its fine-tuning of pre-TCR and TCR expression, down-regulation and signaling.

Human T-Cell Development and Thymic Egress: An Infectious Disease Perspective.

Emigration of mature naïve CD4 SP T cells from the human thymus to the periphery is not fully understood, although elucidation of the mechanisms that govern egress of T cells is crucial to understanding both basic immunology and the immune response in diseases such as HIV infection. Recent work has brought to light the requirement for sphingosine-1-phosphate (S1P) and its receptors in a variety of fields including mature naïve T-cell egress from the thymus of mice. We are examining the expression and function of this novel requisite T-cell egress receptor within the human thymus, characterizing changes observed in the expression and function of this receptor in infectious diseases. To perform this work, we use a variety of humanized murine models reviewed in this article. Future work in the field of T-cell egress, especially as it pertains to S1P receptors, should advance the fields of basic T-cell immunology and immunopathology and open new avenues for exploration into novel therapeutics.

Thymus-deriving natural regulatory T cell generation in vitro: Role of the source of activation signals

In this research we have examined different sources of activation signals in order to optimize culture conditions for in vitro generation of thymus-deriving natural regulatory T cells (nTregs). We have established a novel model using JAWS II dendritic cell line of immature phenotype and compared it to commonly used methods for the generation of Tregs from peripheral lymphoid organs or blood T cells. In our model the first activation signal is provided by anti-CD3 monoclonal antibodies while the second is delivered by costimulatory molecules expressed on JAWS II cells. The presence of JAWS II cells co-cultured in vitro with unsorted thymocytes directly isolated from the thymus gland creates environment favoring SP CD4+ differentiation, provides the apoptotic cells clearance, maintains the survival of thymocytes and facilitate nTreg generation. Moreover the usage of immature dendritic cells stimuli enables to conduct research on agents affecting nTreg survival, proliferation and development in conditions of cell-to-cell contact of undifferentiated thymocytes with dendritic cells.

Cutting edge: Antigen-specific thymocyte feedback regulates homeostatic thymic conventional dendritic cell maturation.

Thymic dendritic cells (DC) mediate self-tolerance by presenting self-peptides to and depleting autoreactive thymocytes. Despite a significant role in negative selection, the events regulating thymic DC maturation and function under steady-state conditions are poorly understood. We report that cross-talk with thymocytes regulates thymic conventional DC (cDC) numbers, phenotype, and function. In mice lacking TCR-expressing thymocytes, thymic cDC were reduced and exhibited a less mature phenotype. Furthermore, thymic cDC in TCR-transgenic mice lacking cognate Ag expression in the thymus were also immature; notably, however, thymic cDC maturation was re-established by an Ag-specific cognate interaction with CD4+ or CD8+ single-positive thymocytes (SP). Blockade of CD40L during Ag-specific interactions with CD4 SP, but not CD8 SP, limited the effect on cDC maturation. Together, these novel findings demonstrate that homeostatic maturation and function of thymic cDC are regulated by feedback delivered by CD4 SP and CD8 SP via distinct mechanisms during a cognate Ag-specific interaction.

The age-related loss of FoxN1 and associated thymic involution leads to increased susceptibility to autoimmunity by impairing negative selection rather than influencing the generation of Tregs. (HEM4P.235)

Abstract The thymus maintains immunotolerance to self-antigen by deleting self-reactive T cells and generating nTregs. The thymus involutes with age, which is driven by the loss of FoxN1. Thymic involution is thought to be linked with increased susceptibility to autoimmune disease. However, how the age-related loss of FoxN1 induced thymic involution impacts autoimmunity remains unclear. We used a FoxN1 cKO mouse model that mimics natural thymic involution. We found impaired negative selection in the FoxN1 cKO thymus, evidenced by an increase in the frequency of CD4 and CD8 SP thymocytes and a decrease in Aire+ mTECs. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they may be self-reactive T cells. Additionally, we found that nTreg frequency was increased in the thymus, but not in the spleen. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells into young Rag2-/- mice. We found that the young periphery was able to reverse Treg accumulation. Additionally, the adoptive transfer led to an increase in salivary infiltration independent of peripheral age. We conclude that loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, the age-related accumulation of Tregs depends on the age of the microenvironment in which they stay.

Increased Cell Surface Free Thiols Identify Effector CD8+ T Cells Undergoing T Cell Receptor Stimulation

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8+ T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8+ T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8+ T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8+ T cells. Finally, CSFT levels on effector CD8+ T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8+ T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.

The impact of short CYLD in medullary thymic epithelial cell development (P1425)

Abstract CYLD is a deubiquitinating enzyme and acts as a negative regulator of NF-κB signaling. To analyse the function of CYLD in vivo we used CYLDex7/8 mice, which are characterized by loss of the full-length transcript and overexpression of a short splice variant of cyld (sCYLD). In these mice thymic and peripheral CD4 and CD8 T cells were reduced compared to controls. Hence, we were interested to investigate the contribution of CYLD in positive and negative selection in the thymus in vivo. For this purpose we crossed the HY-TCR transgene (HYtg) to CYLDex7/8 mice. The analysis of CYLDex7/8 HYtg males revealed an increase in CD4+CD8+ DP as well as in CD8 SP thymocytes, suggesting a less pronounced negative selection in CYLD mutant mice compared to HYtg control mice. Since medullary thymic epithelial cells (mTECs) play an essential role in the negative selection process of thymocytes, the levels of mTECs in the medullary compartment were examined by histology and FACS analysis. We observed reduced numbers of mTECs, combined with decreased expression levels of the mTEC markers UEA-1, keratin-5, claudin-3 and claudin-4. Interestingly, specifically immature mTECs were significantly reduced compared to controls, suggesting a developmental block in the transition of immature to mature mTECs. Taken together, these results display a crucial role of sCYLD in the development and maturation of mTECs.

Interchangeability of Themis1 and Themis2 in Thymocyte Development Reveals Two Related Proteins with Conserved Molecular Function

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.

The acute effects of sub-lethal gamma irradiation on the thymus and T cells were directly affected by age, sex and radiation exposure dose (115.8)

Abstract To study the effects of A-bomb radiation on immunosenescence, we developed a mouse model to evaluate the effects of sub-lethal gamma irradiation, varying exposure dose and analysis time points in males and females. The current data report the effects on 2 and 6-month old mice exposed at 0, 0.5, 1, 2, or 4 Gray (Gy), analyzed 1.5 months after irradiation. We saw a dose dependent reduction of DN1 thymocytes, especially of DN1a,b subsets. Although DN cells were reduced, the DP subset was increased, with a dose dependent restoration of total thymocyte numbers at all but the highest exposures. Significantly, there was a dose dependent increase of CD4 and decrease of CD8 SP cells, resulting in an increased CD4/CD8 ratio. Thymic NK cell numbers were increased at low doses, but decreased at the 4Gy dose. In males, total peripheral T cell numbers showed a dose-dependent decline while thymocyte numbers showed the opposite effect. In contrast, peripheral T cells showed the same increased CD4/CD8 ratio as thymocytes. Newly produced thymocytes contributed more to restoration of peripheral T cells at low doses, but peripheral homeostasis appeared to have more effect at 4Gy. Thymic epithelial cells (TECs) were also affected, with an increase of total TEC number at low doses followed by a dose dependent reduction at higher doses. These results suggest that age, sex and exposure dose all affect the restoration of the immune system during early recovery period after sub-lethal irradiation.