Non-activated CD4 Research Articles

FRI614 Rescuing Aging-associated CD4+ T Cell Dysfunction Via Mitochondrial Transplantation

Abstract Disclosure: C.A. Headley: None. Mitochondrial dysfunction alters cellular metabolism, increases oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, we investigated whether the transfer of functional mitochondria into CD4+ T cells that were isolated from old mice and elderly humans, could abrogate aging-associated mitochondrial dysfunction and improve the functionality of aged CD4+ T cells. Our results show that the delivery of exogenous mitochondria to aged T cells led to cellular and mitochondrial reprogramming. This was evidenced by significant mitochondrial proteome alterations that coincided with improved aerobic metabolism and decreased mitoROS in non-activated CD4+ T cells from old mice that received healthy mitochondria. Compared to non-manipulated CD4+ T cells, CD4+ T cells that received functional mitochondria (i.e. mito-transferred CD4+ T cells from old mice) showed improvements in activation-induced TCR-signaling kinetics that correlated with increased frequencies of CD4+ T cells that displayed markers of activation (CD25), increased IL-2 production, as well as enhanced proliferation ex vivo. Immunodeficient mouse models (RAG-KO and TCR-KO) showed that the adoptive transfer of mito-transferred naive CD4+ T cells, protected recipient mice from influenza A and M. tuberculosis infections and promoted effector memory T cell differentiation. Further, mito-transfer improved CD4+ T cell activation in human T cells by decreasing activation-induced senescence. These findings support mitochondria as targets of therapeutic intervention in aging. Presentation: Friday, June 16, 2023

LBSUN86 Effects Of Hyperandrogenemia And Western-style Diet On Rhesus Macaque Tissue-resident Memory T-cells And Their Potential Role In Controlling Local Inflammation And Insulin Sensitivity

Abstract We have previously demonstrated that the combination of chronic hyperandrogenemia (elevated circulating testosterone; T) and an obesogenic Western-style diet (WSD) exerts synergistic effects on the metabolic and reproductive axis, leading to the development of insulin resistance, visceral obesity, and ovarian dysfunction in female rhesus macaques. The underlying cellular connection between T+WSD treatment and abnormal metabolic and reproductive function is not well understood. Tissue resident-memory T-cells (Trm) have emerged as potent regulators of local inflammation. Thus, we hypothesized that T+WSD treatment drives proinflammatory phenotypic changes in Trm cells. To address this hypothesis, we studied a cohort of adult female rhesus macaque exposed to a low-fat diet and no exogenous T (controls) versus those treated with T+WSD for one year. T+WSD animals became insulin resistant and had higher visceral adiposity than control animals. During the mid-luteal phase, all animals were necropsied and immune cells residing in visceral omental white adipose tissue (OM-WAT), the corpus luteum (CL), the contralateral ovary not containing the CL (OV), endometrium (ENDO), lymph nodes (LN), bone marrow (BM), and peripheral blood mononuclear cells (PBMC) were characterized by flow cytometry. Non-activated CD4+ and CD8+ naïve/central memory cells expressing CD28 were detected in all tissue examined. However, the tissue retention receptor CD69 was expressed only on CD4+ and CD8+ cells residing in OM-WAT, CL, OV, and ENDO. These Trm subsets expressed high levels of the C-C Motif Chemokine Receptor 5 (CCR5), whose role in inflammation has been previously reported. Combined T+WSD treatment resulted in a significant increase in CD8+ Trm cell populations in OM-WAT. Notably, the frequencies of Trm in OM-WAT were positively correlated with HOMA-IR, the indicator of insulin resistance. Collectively, our data indicate that combined hyperandrogenemia and WSD can lead to selective recruitment or local differentiation of Trm cells in visceral fat. Trm activation in fat may drive the development of a proinflammatory environment that negatively affects adipose metabolic function, leading to altered systemic metabolic parameters (i. e., hyperinsulinemia) that influence ovarian and uterine processes. Future studies are warranted to define how Trm cell-adipocyte interactions affect adipose metabolic processes. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Prolonged administration of maraviroc reactivates latent HIV in vivo but it does not prevent antiretroviral-free viral rebound

Human immunodeficiency virus (HIV) remains incurable due to latent viral reservoirs established in non-activated CD4 T cells that cannot be eliminated via antiretroviral therapy. Current efforts to cure HIV are focused on identifying drugs that will induce viral gene expression in latently infected cells, commonly known as latency reversing agents (LRAs). Some drugs have been shown to reactivate latent HIV but do not cause a reduction in reservoir size. Therefore, finding new LRAs or new combinations or increasing the round of stimulations is needed to cure HIV. However, the effects of these drugs on viral rebound after prolonged treatment have not been evaluated. In a previous clinical trial, antiretroviral therapy intensification with maraviroc for 48 weeks caused an increase in residual viremia and episomal two LTR-DNA circles suggesting that maraviroc could reactivate latent HIV. We amended the initial clinical trial to explore additional virologic parameters in stored samples and to evaluate the time to viral rebound during analytical treatment interruption in three patients. Maraviroc induced an increase in cell-associated HIV RNA during the administration of the drug. However, there was a rapid rebound of viremia after antiretroviral therapy discontinuation. HIV-specific T cell response was slightly enhanced. These results show that maraviroc can reactivate latent HIV in vivo but further studies are required to efficiently reduce the reservoir size.

Maraviroc reactivates HIV with potency similar to that of other latency reversing drugs without inducing toxicity in CD8 T cells

Human immunodeficiency virus (HIV) remains incurable due to latent reservoirs established in non-activated CD4 T cells. Current efforts to achieve a functional cure rely on immunomodulatory strategies focused on enhancing the functions of cytotoxic cells. Implementation of these actions requires a coordinated activation of the viral transcription in latently infected cells so that the reservoir became visible and accessible to cytotoxic cells. As no latency reversing agent (LRA) has been shown to be completely effective, new combinations are of increasing importance. Recent data have shown that maraviroc is a new LRA. In this work, we have explored how the combination of maraviroc with other LRAs influences on HIV reactivation using in vitro latency models as well as on the cell viability of CD8 T cells from ART-treated patients. Maraviroc reactivated HIV with a potency similar to other LRAs. Triple combinations resulted toxic and were rejected. No dual combination was synergistic. The combination with panobinostat or disulfiram maintained the effect of both drugs without inducing cell proliferation or toxicity. Maraviroc does not alter the viability of CD8 T cells isolated from patients under antiretroviral treatment. This finding enhances the properties of maraviroc as a LRA.

Abstract 2371: Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity

Abstract Tumor cells can induce immunotolerance, which is reversed by checkpoint blockade immunotherapy in some patients, although response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses with their conversion to a suppressed state impairing anti-tumor immune responses. CD4+ T cells function by activating and controlling various signal transduction pathways. Over the past decade we have developed tests that quantitatively measure functional activities of signal transduction pathways (e.g. Hedgehog, Wnt, TGFβ Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK). They are based on Bayesian computational model inference of pathway activities from measurements (expression microarray, qPCR) of mRNA levels of target genes of the transcription factor associated with the respective signalling pathways1,2. These tests were extensively biologically validated, including on immune cells, and can be used to characterize their functional activity status. In the present study, this approach was used to investigate cellular mechanisms underlying breast cancer-induced immunosuppression of CD4+ T cells. Method: Generation of Affymetrix gene expression data has been previously described (J Clin Invest 2013;123(7):2873-92) and data is publically available (GEO dataset GSE36766). Briefly, breast cancer tissue sections from fresh surgical specimens were mechanically dissociated in X-VIVO 20. Following activation with anti-CD3/CD28, CD4+ T cells from healthy donor blood were incubated with primary tumor supernatants (SN) and compared to controls. Signaling pathway activities were measured using Affymetrix expression data from the individual CD4+ T cell treatment groups. Results: CD4+ T cell activation resulted in induction of PI3K, NFkB, JAK-STAT1/2, JAK-STAT3, Notch, and parallel decrease in TGFβ pathway activities. Incubation with primary tumor SN did not affect pathway activity in non-activated CD4+ T cells, but reduced activity of PI3K, NFκB, JAK-STAT1/2, JAK-STAT3, Notch, while increasing TGFβ pathway activity in activated CD4+ T cells. Conclusion: A soluble factor(s) from breast tumor tissues increases TGFβ and reduces effector immune pathway activity in activated CD4+ T cells and thereby can induce an immunotolerant state. Investigation into the nature of this soluble factor(s) is in progress. These data demonstrate that signaling pathway assays can be used to quantitatively measure the functional state of immune responses in CD4+ lymphocytes. The ultimate goal is to apply this approach for predicting and monitoring immunotherapy responses and identifying novel drug targets that can reverse tumor-induced immunosuppression. Ref: 1. Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 2. Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972. Citation Format: Anja Van De Stolpe, Wim Verhaegh, Arie van Doorn, Grégory Noël, Chunyan Gu-Trantien, Karen Willard-Gallo. Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2371.

HIV Controllers Exhibit Effective CD8+ T Cell Recognition of HIV-1-Infected Non-activated CD4+ T Cells

SummaryEven with sustained antiretroviral therapy, resting CD4+ T cells remain a persistent reservoir of HIV infection, representing a critical barrier to curing HIV. Here, we demonstrate that CD8+ T cells recognize infected, non-activated CD4+ T cells in the absence of de novo protein production, as measured by immune synapse formation, degranulation, cytokine production, and killing of infected cells. Immune recognition is induced by HLA-I presentation of peptides derived from incoming viral particles, and recognition occurred either following cell-free virus infection or following cell-to-cell spread. CD8+ T cells from HIV controllers mediate more effective immune recognition than CD8+ T cells from progressors. These results indicate that non-activated HIV-infected CD4+ T cells can be targeted by CD8+ T cells directly after HIV entry, before reverse transcription, and thus before the establishment of latency, and suggest a mechanism whereby the immune response may reduce the size of the HIV reservoir.

Glycan-Glycan Interaction Determines Shigella Tropism toward Human T Lymphocytes.

ABSTRACTDirect interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis.

Translocator Protein as an Imaging Marker of Macrophage and Stromal Activation in Rheumatoid Arthritis Pannus.

PET radioligands targeted to translocator protein (TSPO) offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus-monocytes, macrophages, fibroblastlike synoviocytes (FLS cells), and CD4-positive (CD4+) T lymphocytes (T cells)-to more accurately interpret TSPO PET signal from RA synovium. Methods: Three RA patients and 3 healthy volunteers underwent PET of both knees using the TSPO radioligand 11C-PBR28. Through 3H-PBR28 autoradiography and immunostaining of synovial tissue in 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO messenger RNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS cells, and CD4+ T cells. Results:11C-PBR28 PET signal was significantly higher in RA joints than in healthy joints (average SUV, 0.82 ± 0.12 vs. 0.03 ± 0.004; P < 0.01). Further, 3H-PBR28-specific binding in synovial tissue was approximately 10-fold higher in RA patients than in healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS cells, and CD4+ T cells. The in vitro study demonstrated the highest TSPO messenger RNA expression and 3H-PBR28-specific binding in activated FLS cells, nonactivated M0 macrophages, and activated M2 reparative macrophages, with the least TSPO expression being in activated and nonactivated CD4+ T cells. Conclusion: To our knowledge, this study was the first evaluation of cellular TSPO expression in synovium, with the highest TSPO expression and PBR28 binding being found on activated synovial FLS cells and M2 macrophages. TSPO-targeted PET may therefore have a unique sensitivity in detecting FLS cells and macrophage-predominant inflammation in RA, with potential utility for assessing treatment response in trials using novel FLS-cell-targeted therapies.

HIV Controllers Exhibit Effective Cd8+T Cell Recognition of Hiv-1 Infected Non-Activated Cd4+T Cells

Even with sustained antiretroviral therapy, resting CD4+ T cells remain a persistent reservoir of HIV infection, representing a critical barrier to HIV cure. Here we demonstrated that CD8+ T cells recognize infected, non-activated CD4+ T cells in the absence of de novo protein production, as measured by immune synapse formation, degranulation, cytokine production and killing of infected cells. Immune recognition was induced by HLA-I presentation of peptides derived from incoming viral particles, and recognition occurred either following cell-free virus infection or following cell-to-cell spread. Importantly, CD8+ T cells from HIV Controllers mediated more effective immune recognition than CD8+ T cells from Progressors. These results indicate that non-activated HIV infected CD4+ T cells can be targeted by CD8+ T cells directly after HIV entry, before reverse transcription and thus before establishment of latency, and suggest a mechanism whereby the immune response may impact the size of the HIV reservoir.

Human CD4(+) T-Cells: A Role for Low-Affinity Fc Receptors.

Both lymphoid and myeloid cells express Fc receptors (FcRs). Low-affinity FcRs engage circulating immune complexes, which results in the cellular activation and pro-inflammatory cytokine production. FcRs participate in the internalization, transport, and/or recycling of antibodies and antigens. Cytosolic FcRs also route these proteins to proteasomes and antigen-presentation pathways. Non-activated CD4+ T-cells do not express FcRs. Once activated, naive CD4+ T-cells express FcγRIIIa, which, upon IC ligation, provide a costimulatory signal for the differentiation of these cells into effector cell population. FcγRIIIa present on CD4+ T-cell membrane could internalize nucleic acid-containing ICs and elicit a cross-talk with toll-like receptors. FcγRIIIa common γ-chain forms a heterodimer with the ζ-chain of T-cell receptor complex, suggesting a synergistic role for these receptors. This review first summarizes our current understanding of FcRs on CD4+ T-cells. Thereafter, I will attempt to correlate the findings from the recent literature on FcRs and propose a role for these receptors in modulating adaptive immune responses via TLR signaling, nucleic acid sensing, and epigenetic changes in CD4+ T-cells.

CD8+ T cells from elite controllers recognize and kill HIV abortively infected CD4+ T cells (VIR6P.1168)

Abstract Understanding the underlying immunological mechanisms of spontaneous HIV control by HIV+ elite controllers (EC) remains an important step in the development of efficient HIV vaccines and functional cures. To understand control mechanisms triggered by CD8+ T cell we established a system in non-activated peripheral blood mononuclear cells (PBMC) using infectious HIV (NL4.3 IRES GFP) carrying the fusion protein Vpr-Beta lactamase (to assess viral entry) and a staining strategy to study the CD8+ T cell response by flow cytometry. First we showed that non-activated CD4+ T cells are permissive for HIV entry but this leads mostly to abortive infection (b-lac+ GFP-). CD8+ T cells from EC were able to recognize these HIV+ cells, became activated (IFNg+, MIP1b+) and underwent degranulation (CD107a+). This activation was initiated by the presentation of HIV peptides from incoming viruses and HLA-I blockade almost abolished this response. We confirmed by mass spectrometry HIV peptides processed in the cytoplasm of CD4+ cells shortly after HIV entry. These results suggest that the ability of CD8+ T cells to form immunological synapses allows them to be activated by low levels of HIV antigens from incoming particles at the surface of early-infected CD4+ T cells. Killing of abortively HIV-infected cells early after HIV entry might avoid inflammation due to pyroptosis and therefore contribute to HIV control.

An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

Integration is a central event in the replication of retroviruses, yet ≥ 90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4(+) T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4(+) T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4(+) T cells. We found that infection of cytokine-treated resting CD4(+) T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.

The infectious synapse formed between mature dendritic cells and CD4+T cells is independent of the presence of the HIV-1 envelope glycoprotein

BackgroundSince cell-mediated infection of human immunodeficiency virus type 1 (HIV-1) is more efficient than cell-free infection, cell-to-cell propagation plays a crucial role in the pathogenesis of HIV-1 infection. Transmission of HIV-1 is enabled by two types of cellular contacts, namely, virological synapses between productively infected cells and uninfected target cells and infectious synapses between uninfected dendritic cells (DC) harboring HIV-1 and uninfected target cells. While virological synapses are driven by expression of the viral envelope glycoprotein on the cell surface, little is known about the role of envelope glycoprotein during contact between DC and T cells. We explored the contribution of HIV-1 envelope glycoprotein, adhesion molecules, and antigen recognition in the formation of conjugates comprising mature DC (mDC) and CD4+ T cells in order to further evaluate their role in mDC-mediated HIV-1 transmission at the immunological synapse.ResultsUnlike virological synapse, HIV-1 did not modulate the formation of cell conjugates comprising mDC harboring HIV-1 and non-activated primary CD4+ T cells. Disruption of interactions between ICAM-1 and LFA-1, however, resulted in a 60% decrease in mDC-CD4+ T-cell conjugate formation and, consequently, in a significant reduction of mDC-mediated HIV-1 transmission to non-activated primary CD4+ T cells (p < 0.05). Antigen recognition or sustained MHC-TcR interaction did not enhance conjugate formation, but significantly boosted productive mDC-mediated transmission of HIV-1 (p < 0.05) by increasing T-cell activation and proliferation.ConclusionsFormation of the infectious synapse is independent of the presence of the HIV-1 envelope glycoprotein, although it does require an interaction between ICAM-1 and LFA-1. This interaction is the main driving force behind the formation of mDC-CD4+ T-cell conjugates and enables transmission of HIV-1 to CD4+ T cells. Moreover, antigen recognition boosts HIV-1 replication without affecting the frequency of cellular conjugates. Our results suggest a determinant role for immune activation driven by mDC-CD4+ T-cell contacts in viral dissemination and that this activation likely contributes to the pathogenesis of HIV-1 infection.

Blood CD4+CD45RO+CXCR5+ T cells are decreased but partially functional in signal transducer and activator of transcription 3 deficiency

The generation of high-affinity antibodies requires the presence of a population of CD4+ T cells (follicular TH [TFH] cells) in the lymph node follicles. These cells differ from TH1, TH2, and TH17 effector cells in that they strongly express activation markers and the chemokine receptor CXCR5 and secrete large amounts of IL-21 and CXCL13. Small numbers of nonactivated CD4+CD45RO+CXCR5+ T cells are also found in the blood. We sought to obtain in vitro a population close to the TFH cells and to study the presence of this cell population among patients with autosomal dominant hyper-IgE syndrome carrying heterozygous signal transducer and activator of transcription 3 (STAT3) mutations that impair the IL-21 signaling required for B-cell differentiation. CD4+CD45RO+CXCR5+ T cells were isolated from blood and activated by CD3/T-cell receptor. We found that CD4+CD45RO+CXCR5+ activated T cells corresponding to circulating bona fide memory TFH cells and that STAT3-deficient patients have abnormally low numbers of "TFH-like" blood T cells. However, STAT3-deficient TFH cells have much the same phenotypic and functional characteristics as TFH cells from healthy control subjects. The ability of STAT3-deficient TFH cells to produce IL-21 on CD28/T-cell receptor activation and to proliferate did not differ from that observed for control TFH cells in vitro. Although the STAT3-deficient TFH cells were also able to help control B cells to produce IgG and IgA, induction of IgG production by naive B cells was impaired. Heterozygous mutations in STAT3 lead to reduced numbers of circulating TFH-like cells, a finding that might account (at least in part) for the observed defect in antibody production.

The intensity of immune activation is linked to the level of CCR5 expression in human immunodeficiency virus type 1‐infected persons

Immune activation is a main driver of AIDS- and non-AIDS-linked morbidities in the course of HIV-1 infection. As CCR5, the main HIV-1 co-receptor, is not only a chemokine receptor but also a co-activation molecule expressed at the surface of T cells, it could be directly involved in this immune activation. To test this hypothesis, we measured by flow cytometry the mean number of CCR5 molecules at the surface of non-activated CD4(+) T cells (CCR5 density), which determines the intensity of CCR5 signalling, and the percentage of CD8(+) T cells over-expressing CD38 (CD38 expression), a major marker of immune activation, in the blood of 67 HIV-1-infected, non-treated individuals. CCR5 density was correlated with CD38 expression independently of viral load (P=0.016). CCR5 density remained unchanged after highly active anti-retroviral therapy (HAART) introduction or cessation, whereas CD38 expression decreased and increased, respectively. Moreover, pre-therapeutic CCR5 density was highly predictive (r=0.736, P<10(-4) ) of residual CD38 over-expression after 9 months of HAART. Hence, CCR5 might play an immunological role in HIV-1 infection as a driver of immune activation. This could explain why CCR5 antagonists may have an inhibitory effect on immune activation.

A specific type of membrane microdomains is involved in the maintenance and translocation of kinase active Lck to lipid rafts

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4+ T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4+ T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4+ T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

Calculated withdrawal of low-dose immunosuppression based on a detailed immunological monitoring after kidney transplantation between monocygotic twins

Background Pretransplant screening in living donor kidney transplantation includes human leukocyte antigen matching, and panel reactive antibody analysis, whereas T cell mediated anti-donor reactivity is not assessed routinely. We investigated T cell reactivity after living related kidney transplantation between two monocygotic twins and in consequence correlated the withdrawal of individual immunosuppressive medication with immunological findings. Methods Immunosuppression consisted of mycophenolate mofetil, glucocorticoid single shot, and induction therapy with antithymocyte immunoglobulin. Results FACS analysis of recipient peripheral blood cells revealed a normal haemogram after transplantation, showing non-activated CD4 and CD8 cells. Mixed lymphocyte reaction did not reveal donor-specific T cell activity. IFN-γ and IL-10 ELISA of supernatants of recipient cells cocultivated with donor cells support the lack of Th1 and Th2 cell differentiation. Conclusion Based on immunological findings on days 5 and 20 MMF-therapy was reduced and stopped. Immunological monitoring on day 90 confirmed the absence of immune reactions directed against donor tissue.

Oligoclonal activation of CD4+ T lymphocytes in posterior uveitis.

Several lines of evidence support an important role for activated T lymphocytes in the perpetuation of autoimmune intraocular inflammatory disease (posterior uveitis). In this study peripheral blood lymphocytes (PBL) were examined by three-colour flow cytometry to assess the distribution of IL-2 receptors (IL-2R) among CD4+ and CD8+ T cell subsets in patients with active posterior uveitis and control subjects. Patients with uveitis (n = 70) showed a significant increase in PBL expressing the alpha-chain (Tac) of the IL-2R compared with controls (n = 28) (34.2% versus 29.6%) (P < 0.05). This increased Tac expression was present on both the CD4+ subset (25.7% versus 20.9%) (P < 0.05) and the CD8+ subset (2.5% versus 1.8%) (P < 0.05) of lymphocytes. We also examined whether the activated CD4+ PBL from uveitis patients (n = 30) showed a dominant pattern of T cell receptor (TCR) gene rearrangement, suggestive of an oligoclonal response to a small number of antigenic peptides. A significant increase in the usage of the V alpha 2.3 TCR family by activated but not by non-activated CD4+ PBL was detected in patients (3.9% versus 3.4%) (P < 0.05) compared with controls. There was evidence of oligoclonal activation of CD4+ PBL in 11/30 patients (36.7%) but in none of the controls (n = 10). However, different V alpha or V beta TCR families were selectively activated among and even within individual patients. The heterogeneity in TCR expression among patients with active intraocular inflammatory disease is discussed.

Mitochondrial DNA Decline in T Cells of HIV‐1 Seroconverters May Be Dependent on Immune Activation

Earlier reports have indicated that human immunodeficiency virus type 1 (HIV-1) infection itself might cause mitochondrial DNA (mtDNA) decline in peripheral blood mononuclear cells (PBMCs). However, the mtDNA dynamics within this heterogeneous cell population during HIV-1 infection are not fully understood. mtDNA content was assessed longitudinally in PBMCs and in isolated CD4(+) and CD8(+) T cells from 16 documented HIV-1 seroconverters who were naive to antiretroviral therapy. The correlation between the mtDNA content of CD4(+) and CD8(+) T cells and their immunologically activated proportion was studied. Additionally, mtDNA content was measured within isolated activated and nonactivated CD4(+) and CD8(+) T cells obtained from 5 antiretroviral-naive men with chronic HIV-1 infection. In the seroconverter group, mtDNA content in CD8(+) T cells decreased 5 years after seroconversion (P=.007). mtDNA content in either CD4(+) or CD8(+) T cells did not correlate with the proportion of activated cells within either population. However, for the chronically infected men, mtDNA content in activated CD8(+) T cells was lower than that in nonactivated cells (P=.043). A similar trend was observed in the CD4(+) T cell fraction. These findings indicate that HIV-1 infection affects mtDNA content, particularly in the most immunologically activated cells. Furthermore, the importance of measuring mtDNA in specific cell fractions rather than in the heterogeneous PBMC population is emphasized.

Regulation of Hematopoietic Colony Forming Cells by CD4+CD25+ T Cells: A Role for T Regulatory Cells in Hematopoiesis?.

CD4+CD25+ T cells (Treg) comprise a small population within the normal peripheral CD4 T cell compartment. Their primary physiological role appears to be the regulation of autoimmune responses, however, in recent years it has been established that they can modulate anti-tumor as well as transplantation responses. Treg cells have been found to exert their affects on multiple types of immunologically relevant cells including CD4, CD8 and NK populations. Although model dependent, cytokines including TGFβ and IL-10 have been identified as mediators of this population's regulatory activity and ex-vivo, the inhibition effected is generally contact dependent. Based upon the expanding application of Treg cells in stem cell transplants for the control of GVHD, rejection (HVG) and GVL responses, we hypothesized that following T cell receptor engagement and activation in recipients, CD4+CD25+ cells may modulate hematopoietic responses via production of effector cytokines. To address this question, various populations of CD4+CD25+ T cells were initially co-cultured with unfractionated syngeneic bone marrow cells (BMC) for 24–48 hours in medium supplemented with growth factors to maintain progenitor cell (i.e. CFU) function. Following co-culture, cells were collected and replated in triplicate in methylcellulose containing medium together with hematopoietic growth factors and five-seven days later, colonies were counted. CD4+CD25+ T cells were purified from BALB/c or B6–CD8−/− mice which were then activated for 3–8 days with anti-CD3/CD28 beads (a gift of Dr. B. Blazar, U. Minn.) These cells inhibited syngeneic CFU-IL3 colony ($25 cells) formation at ratios as low as 2:1 and 0.5:1 CD4+CD25+: BMC. Notably, Tregs from B6-CD8−/− mice exhibited comparable inhibition of allogeneic (BALB/c) CFU-IL3. Non-activated CD4+CD25+ T cells co-cultured with BMC did not exhibit this inhibitory activity nor did CD4+CD25− cells which contaminated (<10%) CD4+CD25+ populations. Activated Treg cells were also found to inhibit the production of CFU-HPP, a multi-potential marrow progenitor cell population. Contact dependency was found to be required for this effect as separation of activated CD4+CD25+ T cells from BMC “targets” in trans-well cultures abrogated inhibition. Prior depletion of CD25+ cells in vivo resulted in increases in CFU-GM 7–9 days after syngeneic BMT in mice suggesting that Tregs can inhibit hematopoietic reconstitution in vivo. To examine a potential contribution of TGFβ in this model, neutralizing anti-TGFβ mab was added during CD4+CD25+ T cell + BMC co-culture. The inhibition of CFU activity was abrogated in the presence of this antibody. To begin investigating the role of MHC class II molecules in this Treg cell activity, c-kit+ enriched (>85%) BMC from B6-MHC class II KO and B6-wt mice were co-cultured with B6 Treg cells from CD8−/− mice. In contrast to B6-wt c-kit enriched populations, CFU inhibition was not detected against the MHC class II deficient c-kit enriched BMC population. Antibody experiments are in progress to determine if cognate interaction is required between c-kit enriched cells and CD4+CD25+ T cells. In summary, this is the first report demonstrating that CD4+CD25+ T cells can alter hematopoietic progenitor cell activity. We hypothesize that membrane bound TGFβ may participate in effecting such regulation via direct Treg cell interactions with progenitor cell populations.