Appearance Of CD4 Research Articles

Abstract PR02: Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients

Abstract About 30% of DLBCLs occur in patients over 75 years old. Due to comorbidities, this potentially curable disease is treated with a reduced dose chemoimmunotherapy regimen, associated with worse response rates and outcomes than the standard approach. Our data indicates that even those able to tolerate full-dose treatments have comparatively worse outcomes. We found that DLBCLs in older patients are more frequently of the ABC-DLBCL subgroup, and their microenvironment (TME) has infiltration of inflammatory and immunosuppressed cells, both characteristics independently associated with chemoimmunotherapy resistance. These features may imply we are facing different biologically diseases. To investigate this, we developed aged DLBCL murine models by implanting three murine lymphoma cells (harboring Setd2/BCL2, Ezh2/BCL2 and Tp53 mutations) in the spleen of young (3-4 months) and aged (21-23 months) mice and monitoring tumor growth by in vivo imaging. We also performed an integrative characterization of the TME from young and aged mice by single cell RNA-sequencing (coding and BCR/TCR), immunophenotyping (flow cytometry) and (MS)-based proteomics. For these two different murine DLBCL cell lines utilized, aged mice had higher tumor burden and showed significantly worse survival than younger mice. In addition, DLBCL cells isolated from aged mice displayed higher clonality and the activation of pro-inflammatory transcriptional programs than DLBCL cells isolated from younger mice. Regarding non-malignant TME B cells, CD11c+ T-bet+ Zeb2+ age-associated B cells (AABs) were significantly more abundant in aged v young mice. AABs displayed a secretory phenotype and transcriptional up-regulation of genes associated with pro-inflammatory programs such as IL-6, Stat3 and Nfatc1. TME T cells showed profound changes including increased infiltration of Foxp3+ Tregs and Pd1+ Cd8+ T cells, as well as the appearance of Cd8+ Gzk+ aged-associated T cells (ATT). Aged mice presented alterations in the myeloid compartment with expansion of Cd45+ Cd11b+ cells. Moreover, the TME of aged mice was enriched in tumor-associated macrophages polarized towards a pro-inflammatory phenotype as well as more abundance of Ly6C+ Ly6G+ myeloid-derived suppressor cells (MDSC). Aged TMEs were also characterized by increased stiffness and extracellular matrix changes affecting collagen and proteoglycans. These TME changes, were associated with intrinsic signaling and transcriptional changes in lymphoma cells like increased activation of NF-kB pathway. Overall, these alterations were compatible with the TME of elderly DLBCL patients showing a global increase in the proportion of inflammatory and immunosuppressive (IN-LME) category (41% in 75-85 y.o. vs. 26% in 50-65 y.o.) and specifically in Tregs, MDSC and CD8 PD1HIGH cells in 75-85 y.o. This data indicates that low-grade chronic inflammation that has been associated with age-related diseases determines molecular and cellular characteristics of the DLBCL in the elderly population. Citation Format: Nahuel Zamponi, Maria Victoria Revuelta, Rossella Marullo, Nicolas Di Siervi, Nikita Kotlov, Wendy Béguelin, Leandro Cerchietti. Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR02.

CD4 T cell responses in persistent Borrelia burgdorferi infection.

Infection of mice with Borrelia burgdorferi (Bb), a tick-transmitted spirochete and the pathogen that causes Lyme disease in humans, triggers CD4 T cell activation in secondary lymphoid tissues, from which they disseminate into various infected tissues. Despite their activation and the appearance of CD4 T cell-dependent antibody responses, Bb establishes persistent infection in natural Bb reservoir hosts in the absence of overt disease, raising the question of the effectiveness of the anti-Bb T cell responses. Reviewing the existing literature, we propose that CD4 T cells might constitute a host cell target of Bb-mediated immune evasion, rendering these cells ineffective in orchestrating effective inflammatory responses and in supporting highly functional Bb-specific antibody induction. Supporting the induction of more effective CD4 T cell responses may help overcome Bb persistence.

CD4+ Cytotoxic T cells – Phenotype, Function and Transcriptional Networks Controlling Their Differentiation Pathways

The two major subsets of peripheral T cells are classically divided into the CD4+ T helper cells and the cytotoxic CD8+ T cell lineage. However, the appearance of some effector CD4+ T cell populations displaying cytotoxic activity, in particular during viral infections, has been observed, thus breaking the functional dichotomy of CD4+ and CD8+ T lymphocytes. The strong association of the appearance of CD4+ cytotoxic T lymphocytes (CD4 CTLs) with viral infections suggests an important role of this subset in antiviral immunity by controlling viral replication and infection. Moreover, CD4 CTLs have been linked with anti-tumor activity and might also cause immunopathology in autoimmune diseases. This raises interest into the molecular mechanisms regulating CD4 CTL differentiation, which are poorly understood in comparison to differentiation pathways of other Th subsets. In this review, we provide a brief overview about key features of CD4 CTLs, including their role in viral infections and cancer immunity, and about the link between CD4 CTLs and immune-mediated diseases. Subsequently, we will discuss the current knowledge about transcriptional and epigenetic networks controlling CD4 CTL differentiation and highlight recent data suggesting a role for histone deacetylases in the generation of CD4 CTLs.

Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that localize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971−990, and cardiac myosin heavy chain-α (Myhc) 334−352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21−40 dextramer+ T cells were inconsistently detected, the β1-adrenergic receptor 181−200/211−230 or branched chain α-ketoacid dehydrogenase kinase 111−130 dextramer+ cells were absent. Interestingly, SERCA2a 971−990, Myhc 334−352 and ANT 21−40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971−990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Long-term effects of acute cadmium exposure on testis immune privilege

Cadmium (Cd) is a widespread and non-biodegradable pollutant of great concern to human health. This element can affect cellular signal transduction and cell-to-cell interaction in the testis. Immune tolerance towards auto- and alloantigens is an important component of testis immunity. It is involved in spermatogenesis and hormone secretion. Plus, the immune tolerance may help to reveal the changes in testis immunity over a long period after Cd exposure. The current research was aimed at investigating the long-term effects of acute Cd exposure on testis immunity by means of elicitation of testicular immune cell composition shift induced by Cd. Cadmium chloride was intraperitoneally injected at 3 mg Cd/kg to mice. After that testis interstitial cells were stained with surface markers for leukocyte and lymphocyte subpopulations (CD45, CD11b, CD3, CD4, CD8, CD25) and analyzed cytofluorimetrically by week 4, 6, 8 and 12 after Cd administration (Cd group). To identify the delayed effects of cadmium on immune tolerance two groups of animals were subjected to intratesticular allotransplantation of neonatal testis (groups ITT and Cd+ ITT). One of the groups was administered with Cd four weeks before the transplantation (Cd+ITT group). I group served as a control that did not undergo any transplantation or Cd injection. For a better demonstration of the phenomenon of immunological tolerance of the testicles, an additional group (UKT group) was used which got grafts under the kidney capsule (non-immune privileged site).Investigation of the cell population showed that CD45+, CD11b+, CD4+, CD8+ cells were permanently present in testicular interstitial tissue in I group. Intratesticular testis transplantation increased the proportion of CD11b+ but did not have such a pronounced effect on CD8+ cells in ITT group. Moreover, the transplantation elevated CD4+ CD25+ cells known for their immunosuppressive property and promoted graft development by week 2 (histological data). Cd injection resulted in severe inflammation that quenched by week 4 (Cd and Cd+ ITT groups). This time point was chosen for transplantation in Cd+ ITT group. Such Cd pretreatment led to a high CD8+ cell proportion and to the delayed appearance of CD4+ CD25+ cells by week 2 (Cd+ ITTgroup). The finding is consistent with the impairment of graft development in Cd+ ITTgroup pretreated with Cd. Observation suggest that Cd pretreatment was associated with disproportion of interstitial immune cell populations which resulted in the impairment of immunoprotective function of the testis. The impairment of testis immunity showed itself only after several weeks of Cd administration, and only when the recipient testis immunity was provoked by alloantigens of donor testes.

Induction of Intestinal Th17 Cells by Flagellins From Segmented Filamentous Bacteria.

T-helper-17 (Th17) cells are a subset of CD4+ T cells that can produce the cytokine interleukin (IL)-17 and play vital roles in protecting the host from bacterial and fungal infections, especially at the mucosal surface. These are abundant in the small intestinal lamina propria (SILP) and their differentiation are associated with the colonization of the intestinal flora. Segmented filamentous bacteria (SFB) drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the SI LP of mice. Recent work has highlighted that SFB used microbial adhesion-triggered endocytosis (MATE) to transfer SFB antigenic proteins into small intestinal epithelial cells (SI ECs) and modulate host immune homeostasis. However, which components of SFB are involved in this immune response process remains unclear. Here, we examined the roles of SFB flagellins in Th17 cells induction using various techniques, including ELISA, ELISPOT, and RNA-seq in vitro and in vivo. The results show that the immune function of SFB flagellins is similar to SFB, i.e., induces the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the SI LP. Furthermore, treatment of mice with SFB flagellins lead to a significant increase in the expression of genes associated with the IL-17 signaling pathway, such as IL-6, IL-1β, TNF-α, IL-17A, IL-17F, and IL-22. In addition, SFB flagellins have an intimate relationship with intestinal epithelial cells, influencing the expression of epithelial cell-specific genes such as Nos2, Duox2, Duoxa2, SAA3, Tat, and Lcn2. Thus, we propose that SFB flagellins play a significant role in the involvement of SFB in the induction of intestinal Th17 cells.

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Improved connective integration of a degradable 3D-nano-apatite/agarose scaffold subcutaneously implanted in a rat model.

In this work, we evaluate the tissue response and tolerance to a designed 3D porous scaffold composed of nanocrystalline carbonate-hydroxyapatite and agarose as a preliminary step in bone repair and regeneration. These scaffolds were subcutaneously implanted into rats, which were sacrificed at different times. CD4+, CD8+ and ED1+ cells were evaluated as measurements of inflammatory reaction and tolerance. We observed some inflammatory response early after subcutaneous implantation. The 3D interconnected porosity increased scaffold integration via the formation of granulation tissue and the generation of a fibrous capsule around the scaffold. The capsule is initially formed by collagen which progressively invades the scaffold, creating a network that supports the settlement of connective tissue and generating a compact structure. The timing of the appearance of CD4+ and CD8+ cell populations is in agreement with the resolved inflammatory response. The appearance of macrophage activity evidences a slow and gradual degradation activity. Degradation started with the agarose component of the scaffold, but the nano-apatite was kept intact for up to 30 days. Therefore, this apatite/agarose scaffold showed a high capacity for integration by a connective network that stabilizes the scaffold and results in slow nano-apatite degradation. The fundamental properties of the scaffold would provide mechanical support and facilitate bone mobilization, which is of great importance in the masticatory system or large bones.

Salmonella infection induces autoantibody production in MyD88-deficient host: Identification of candidate bacterial proteins with homology to murine antigens

Abstract Persistent bacterial infections have been causally linked to the development of autoimmune diseases, but the precise mechanisms underlying this remain largely unknown. Of note, we demonstrated that systemic infection by attenuated Salmonella enterica serovar Typhimurium induces a breakdown in B cell tolerance in MyD88-deficient mice, as evidenced by the production of autoantibodies to nuclear and cytoplasmic antigens and immune complex deposition in the kidneys. The splenic niche of infected MyD88−/− mice revealed increased counts of activated myeloid cell subpopulations as well as CD4+ and CD8+ T cells. Moreover, this was associated with increased Th21 cell differentiation and the appearance of CD4+ T cells co-expressing IFN-g/IL-4 and IFN-g/IL-10. Importantly, infection with other bacteria (e.g. Acinetobacter baumanii, Streptococcus agalactiae or Escherichia coli) was unable to trigger the autoimmune phenomenon. We reasoned that the autoimmune response could be triggered by a process of molecular mimicry between mouse and Salmonella proteins. Bioinformatic analysis was used to identify potential Salmonella antigens with a high degree of homology to mouse proteins. This analysis revealed several candidate bacterial proteins with high homology to murine immune system proteins. The identified bacterial proteins were all uniquely expressed by Salmonella but not by the other three bacterial species that were incapable of inducing the autoimmune response in MyD88−/− mice. Therefore, our data suggest a mechanism for pathogen-specific induction of autoantibody production in the absence of MyD88 signaling pathway and provide important clues concerning the genotype-environmental factor correlations.

PD-L1 Monoclonal Antibody Treats Ischemic Stroke by Controlling Central Nervous System Inflammation.

Both pathogenic and regulatory immune processes are involved in the middle cerebral artery occlusion (MCAO) model of experimental stroke, including interactions involving the programmed death 1 (PD-1) receptor and its 2 ligands, PD-L1 and PD-L2. Although PD-1 reduced stroke severity, PD-L1 and PD-L2 appeared to play pathogenic roles, suggesting the use of anti-PD-L monoclonal antibody therapy for MCAO. Male C57BL/6 mice were treated with a single dose of anti-PD-L1 monoclonal antibody 4 hours after MCAO and evaluated for clinical, histological and immunologic changes after 96 hours of reperfusion. Blockade of the PD-L1 checkpoint using a single injection of 200 μg anti-PD-L1 monoclonal antibody given intravenously 4 hours after occlusion significantly reduced MCAO infarct volumes and improved neurological outcomes after 96 hours of reperfusion. Treatment partially reversed splenic atrophy and decreased central nervous system infiltrating immune cells concomitant with enhanced appearance of CD8(+) regulatory T cells in the lesioned central nervous system hemisphere. This study demonstrates for the first time the beneficial therapeutic effects of PD-L1 checkpoint blockade on MCAO, thus validating proposed mechanisms obtained in our previous studies using PD-1- and PD-L-deficient mice. These results provide strong support for the use of available humanized anti-PD-L1 antibodies for treatment of human stroke subjects.

Evaluation of CD4+ Cell Recovery in Vivo Using Single Photon Emission Tomography (SPECT) in Real Time Following CD34+ Cell Transplantation in Rhesus Macaques

It has not been possible to evaluate immune recovery in tissues without invasive biopsy. Investigators have had to rely on peripheral blood lymphocytes (PBLs) for analysis. PBLs, however, represent only a very small percentage of cells in lymphoid tissues (LTs). Using a combination of SPECTanda radiotracer, rhesus IgG1 anti-CD4R1 Tc-99m(Fab’)2,we sequentially imaged CD4+ cell recovery in rhesus macaques following varying doses of total body irradiation (TBI) and reinfusion of vector transduced, autologous CD34+ cells.Seven rhesus were used in this study. Two received a dose of 3Gy on two sequential days (3Gyx2days) of TBI (6Gy total), three received 4Gyx2days TBI, and two 5Gyx2days TBI. CD34+ cells were immunoselected from a leukapheresis product following G-CSF and SCF mobilization over 5 days. On the last day of TBI a mean (SD) of 5.9(1.7)x106/kg autologous CD34+ cells were reinfused after being transduced once (moi=50) with a SIV/HIV chimeric lentiviral vector expressing EGFP. SPECT/CT images were taken prior to transplant (Figure 1) and at approximately 6, 30, 90, 150, 260 and 380 days post-TBI. Development of immunogenicity to the radiotracer was monitored using size exclusion HPLC and an in vitro cell binding assay.Following 3Gyx2days TBI, both animals rejected their CD34+ cell graft despite achieving a PBL nadir of <150 Lcs/mcL. Both animals also developed an immune response to the radiotracer either within 4 weeks of the baseline scan or 182 days following TBI. SPECT/CT imaging revealed significant retention of CD4+ cells within immune tissues at d.6 post-TBI (Figure 2). At 4Gyx2days TBI, all 3 rhesus experienced dramatic depletion of PBLs. Flow cytometric analysis, however, showed a residual population of CD4+ cells, but a complete depletion of CD20+ cells. SPECT imaging revealed variability in LT CD4 depletion between and within hosts, with more dramatic decreases observed in axillary lymph nodes (73-92%) and milder decreases in the spleen (36-40%). The PB CD4+ count returned to baseline within 4-9 months, however, in vivo imaging revealed sub-optimal reconstitution of the CD4 pool in LNs one year from TBI (mean 54% recovery from baseline), consistent with LN immunohistochemistry (IHC). Similar levels of splenic radioresistance were observed at 5Gyx2 days TBI (Figure 2), and confirmed by IHC (Figure 3). Of particular interest was the discordance between rapid recovery of CD4+ EGFP- lymphocytes (Lcs) and the delayed appearance of CD4+EGFP+ Lcs (not observed until 5 months post-transplant), documenting endogenous CD4+ recovery from LTs. This contrasts with other Lc subsets that were more effectively depleted by irradiation. CD20+ EGFP+ Lcs recovered a mean(SD) of 69(48) days, consistent with the reappearance of CD20+ cells in general in the circulation. Thus, Lc subset and LT sensitivity to irradiation appear to differ.Unlike the 3Gyx2days TBI animals, no immunogenicity to the radiotracer was detected in the 4Gyx2days TBI rhesus monkeys until at least one year post transplant. After the 6 or 7th exposure, all three animals developed an immune response to the radiotracer. This suggests immune recovery remained incomplete in the animals receiving a dose of 4Gyx2days TBI or higher for at least one year following transplant. No immune response has been observed at 5Gyx2 at d.245.Our results present for the first time a non-invasive method in real time to evaluate CD4+ cell immune recovery in LTs following hematopoietic stem cell transplantation using SPECT/CT imaging. Despite myeloablation of circulating leukocytes following TBI, we did not observe total depletion of CD4+ cells in LTs such as the spleen. This is problematic if one is concerned about residual disease or endogenous contributions to recovery. The methodologies presented here have direct applications to the evaluation of immunosuppressive therapies and other immunosuppressive disorders, such as those associated with viral infections. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Histone Acetylation Mediated by Brd1 Is Crucial for Cd8 Gene Activation during Early Thymocyte Development

During T cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficient enhancer activity during initial Cd8 activation is known to cause a variegated CD8 expression, thereby generating a CD4+CD8-TCRb-/low population that should appear as CD4+CD8+ double-positive (DP) thymocytes. However, epigenetic and molecular mechanisms involved in initial Cd8 gene activation remain elusive. We previously reported that Brd1, also known as Brpf2, is responsible for the global acetylation of H3K14 as a subunit of the Hbo1 histone acetyltransferase (HAT) complex.In this study, we generated conditional Brd1 knockout mice (Tie2-Cre;Brd1fl/fl mice), in which Brd1 is inactivated in all hematopoietic cells. Although Tie2-Cre;Brd1fl/fl mice were born and grew normally, detailed analysis revealed an abnormal thymocyte differentiation. Deletion of Brd1 resulted in the appearance of CD4+CD8-TCRβ-/low thymocytes that was indistinguishable from DP thymocytes in their properties. Hierarchical clustering of gene expression profile showed that Brd1Δ/Δ CD4+CD8-TCRβ-/low thymocytes retain an expression profile nearly identical to one observed in DP thymocytes. These results indicate that Brd1Δ/Δ CD4+CD8-TCRβ-/low thymocytes correspond to immature thymocytes that would normally appear as CD4+CD8+ DP thymocytes, and was generated because of a variegated activation of Cd8 gene. Importantly, in the gut intraepithelial lymphocyte (IEL) compartment, the population of CD4+CD8aa+ aβT cells, whose generation requires Cd8 gene reactivation in CD4+CD8− helper T cells, were significantly decreased in Tie2-Cre;Brd1fl/fl mice compared to the Tie2-Cre control mice. In addition, the percentage of CD8aa expressing gdT cells was significantly reduced. These findings suggest that Brd1 is also required to initiate Cd8a activation in gdT cells as well as Cd8 reactivation in CD4+T cells.We further conducted retroviral transduction of Brd1 into Brd1Δ/Δ DN3 cells that were flowed by culture on TSt-4/DLL stromal cells. Upon exogenous Brd1 expression, CD8 expression was completely restored and thereby Brd1Δ/Δ DN3 cells differentiated into DP cells.ChIP analysis demonstrated that Brd1 and Hbo1 co-localize at the known enhancers in the Cd8 locus and that their bindings are responsible for acetylation at H3K14. Biochemical results confirmed a presence of Brd1 in Hbo1 HAT complexes. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which would serve as an epigenetic mark that promotes the recruitment of transcription machineries to the Cd8 enhancers. DisclosuresNo relevant conflicts of interest to declare.

Histone acetylation mediated by Brd1 is crucial for Cd8 gene activation during early thymocyte development.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRβ(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.

Runt-related transcription factor 3 is involved in the altered phenotype and function in ThPok-deficient invariant natural killer T cells

The interplay between the CD4-lineage transcription factor ThPok and the CD8-lineage transcription factor, runt-related transcription factor 3 (Runx3), in T-cell development has been extensively documented. However, little is known about the roles of these transcription factors in invariant natural killer T (iNKT) cell development. CD1d-restricted iNKT cells are committed to the CD4(+)CD8(-) and CD4(-)CD8(-) sublineages, which respond to antigen stimulation with rapid and potent release of T helper (Th) 1 and Th2 cytokines. However, previous reports have demonstrated a new population of CD8(+) NKT cells in ThPok-deficient mice. In the current study, we sought to determine whether Runx3 was involved in the re-expression of CD8 and function of iNKT cells in the absence of ThPok. We used mice lacking Runx3, ThPok or both and verified that Runx3 was partially responsible for the appearance of CD8(+) iNKT cells in ThPok knockout mice. Additionally, Runx3 participated in the immune response mediated by iNKT cells in a model of α-galactosylceramide-induced acute hepatitis. These results indicate that Runx3 is crucial for the phenotypic and functional changes observed in ThPok-deficient iNKT cells.

Porcine urinary bladder matrix-polypropylene mesh: a novel scaffold material reduces immunorejection in rat pelvic surgery

The present study set out to modify polypropylene vaginal surgical material using porcine urinary bladder matrix (UBM) in order to improve biocompatibility. The aim was to develop a compound scaffold that induced less vaginal erosion and to evaluate host immunoreactivity to this material in vivo. Forty-eight Sprague-Dawley rats were randomly divided into four equal groups. One group underwent a sham operation, and the other groups underwent vaginal implantation with different materials: UBM (U); UBM + polypropylene (UP); or polypropylene (P). The host tissue response was determined by macro-observation, and by histological and immunohistochemical methods at 7, 14, 21, or 28 days after surgery. The inflammation reaction was strongest throughout the entire observation time in Group P, but was weaker and had a tendency to decrease with time in Groups U and UP. The presence of the UBM material in the compound scaffold allowed the polypropylene to fuse with newly proliferating surrounding tissue and resulted in less rejection of the material by the host, as indicated by the reduced appearance of CD4-, and CD8-positive cells. Porcine UBM allowed mechanical isolation of polypropylene, and also reduced the immune reaction to polypropylene. This study suggests that the UBM + polypropylene compound scaffold may be a promising material for clinical use in pelvic reconstruction surgery.

Cutting Edge: Rag Deletion in Peripheral T Cells Blocks TCR Revision

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria

The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

CD8+ Cell Anti-HIV Activity Rapidly Increases Upon Discontinuation of Early Antiretroviral Therapy

CD8+ lymphocytes can suppress HIV replication without killing the infected cells. This CD8+ cell noncytotoxic anti-HIV response (CNAR) is associated with a beneficial clinical course. In this longitudinal study of 16 participants in the Options Project at UCSF, we measured the ability of CD8+ lymphocytes to suppress HIV replication in CD4+ cells during primary HIV infection, early antiretroviral therapy, and after treatment. CD8+ lymphocytes from subjects with untreated primary HIV-1 infection strongly suppressed HIV replication. Initiation of antiretroviral therapy during primary HIV-1 infection caused a marked decline in this CNAR. CD8+ cells from these subjects regained anti-HIV activity when early therapy was discontinued. The timing of the appearance of CD8+ cell anti-HIV activity directly correlated with the emergence of detectable virus levels. Maximal CNAR activity coincided with a decay in the kinetics of HIV replication. In addition, peak viral loads during treatment interruption were lower than pre-treatment virus levels (median reduction = 0.8 logs, p = 0.005) and CD4+ T cell counts were maintained for a 24-week period of follow-up. These results suggest that CNAR plays an important role in suppressing HIV replication in the setting of antiretroviral treatment interruption in HIV-infected individuals.

A critical function for TGF-β signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells

The molecular mechanisms directing the development of 'natural' CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) in the thymus are not thoroughly understood. We show here that conditional deletion of transforming growth factor-beta receptor I (TbetaRI) in T cells blocked the appearance of CD4+CD25+Foxp3+ thymocytes at postnatal days 3-5. Paradoxically, however, beginning 1 week after birth, the same TbetaRI-mutant mice showed accelerated expansion of thymic CD4+CD25+Foxp3+ populations. This rapid recovery of Foxp3+ thymocytes was attributable mainly to overproduction of and heightened responsiveness to interleukin 2, as genetic ablation of interleukin 2 in TbetaRI-mutant mice resulted in a complete absence of CD4+CD25+Foxp3+ cells from the thymus and periphery. Thus, transforming growth factor-beta signaling is critical to the thymic development of natural CD4+CD25+Foxp3+ T(reg) cells.