The role of FOXP3+ regulatory T cells in human autoimmune and inflammatory diseases.

CD4+ regulatory T cells (Treg ) expressing the forkhead box protein 3 (FOXP3) transcription factor (Tregs ) are instrumental for the prevention of autoimmune diseases. There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function. Numerous studies conducted in human autoimmune diseases have shown that Treg cells are impaired either in their suppressive function, in number, or both. However, the contribution of the FOXP3+ Treg subpopulations to the development of autoimmunity has not been delineated in detail. Rare genetic disorders that involve deficits in Treg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in Treg function. In patients with reduced Treg numbers (but no functional deficiency), the expansion of autologous Treg cells could be a suitable therapeutic approach: either infusion of in-vitro autologous expanded cells, infusion of interleukin (IL)-2/anti-IL-2 complex, or both. Treg biology-based therapies may not be suitable in patients with deficits of Treg function, unless their deficit can be corrected in vivo/in vitro. Finally, it is critical to consider the appropriate stage of autoimmune diseases at which administration of Treg cellular therapy can be most effective. We discuss conflicting data regarding whether Treg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself.

Gene transfer of two entry inhibitors protects CD4⁺ T cell from HIV-1 infection in humanized mice.

Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4⁺ T-cell infection in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically modified human T cells were grafted into immunodeficient NOD.SCID.γc⁻/⁻mice before infection and one in which genetically modified T cells were derived from CD34⁺ hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically modified CD4⁺ T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection. Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.

Epstein-Barr virus-driven B Cell Proliferation with CD4+ T Cell Expansion: A Lymphomatoid Granulomatosis-like Disease Related to Hyperinterleukin-10 Secretion of Remarkably Favourable Outcome with Rituximab.

Granulomatous lymphomatosis is an Epstein-Barr virus (EBV)-driven B cell proliferation associated with an exuberant CD4(+) T cell reaction with usually histopathological pictures of angiocentrism. So far, the characteristics of CD4(+) T cells in granulomatous lymphomatosis and the mechanism leading to their expansion remain poorly explored. We report a 56-year-old female with a past history of cold agglutinin disease, which was successfully treated with 4 weekly infusions of rituximab. She presented one year later with features of granulomatous lymphomatosis that resulted in severe lung and bone marrow infiltration. We provide evidence that CD4(+) T cell expansion was oligoclonal, involved anergic cells and did not result from an EBV-driven stimulation. Rather, it resulted possibly from a high production of interleukin-10 by immunoblastic EBV-positive B cells. The outcome was remarkably favourable with rituximab and steroids. Our results suggest that an EBV-driven B cell proliferation should be investigated in patients presenting with a CD4(+) T cells alveolitis or other systemic manifestations resulting from a CD4(+) T cell expansion. These features should prompt to introduce an immunosuppressive therapy including steroids and rituximab. Our results deserve further investigations to confirm our pathophysiological hypotheses in CD4(+) T cell expansions associated with EBV-driven B cell proliferations and to assess whether granulomatous lymphomatosis could result from comparable mechanisms.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy.

The T-cell antigen receptor (TCR) repertoire was studied longitudinally by analyzing the varying lengths of the beta chain CDR3 hypervariable region during the course of HIV-1 infection and following combination antiretroviral therapy. Drastic restrictions in CD8+ T-cell repertoire usage were found at all stages of natural progression and persisted during the first six months of treatment. In contrast, significant CD4+ T-cell repertoire perturbations were not found in early stages of infection but correlated with progression to AIDS. Out of ten patients presenting with pretreatment perturbations, normalization of the CD4+ repertoire was observed in eight good responders, but not in two cases of unsuccessful therapy. These results indicate that, besides CD4+ cell count rise, an efficient control of HIV replication may allow qualitative modifications of the CD4+ repertoire balance.

[Atypical progressive multifocal leukoencephalopathy (PML) in a patient with pulmonary sarcoidosis]. / Leucoencéphalopathie multifocale progressive atypique chez un patient atteint de sarcoïdose médiastinopulmonaire.

INTRODUCTION: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of central nervous system due to the JC virus. PML generally occurs in immunocompromised hosts and has a fatal outcome. OBSERVATION: We report a case of an atypical PML in a patient with pulmonary sarcoidosis: MRI showed multifocal and punctate contrast enhancements. The diagnostic was made by brain biopsy. CONCLUSION: The pathophysiology of this association is probably related to the immunodepression induced by sarcoidosis.

[Systemic lupus erythematosus and regulatory T cells]. / Cellules T régulatrices et lupus érythémateux systémique.

A global depletion of FoxP3+ CD25(bright) CD4+ regulatory T cell is observed during lupus flares. This phenomenon is not the consequence of the relocalization of Tregs in diseased organs but could be related to their specific sensitivity to Fas mediated apoptosis. Several therapeutic perspectives can be drawn taking into account these pathophysiological insights.

Oligoclonal expansion of HIV-specific cytotoxic CD8 T lymphocytes in the skin of HIV-1-infected patients with cutaneous pseudolymphoma.

A massive infiltration of the skin by activated CD8+ T lymphocytes involving both the dermis and the epidermis has been found in HIV-1-infected patients presenting with a chronic skin rash. We characterized the T cell receptor (TCR) BV-BJ junctional diversity of the skin-infiltrating lymphocytes (SILs) in four patients. The SILs expressed a limited set of TCRBV gene segments. Complementarity determining region 3 length analysis further emphasized their oligoclonality, suggesting that antigen stimulation might be responsible for the cutaneous T cell expansion. Furthermore, independent skin biopsies obtained from the same individual were shown to harbor distinct T cell repertoires, possibly reflecting the spatial heterogeneity of the antigenic stimuli. The CD8+ cytotoxic T lymphocyte (CTL) lines isolated from the skin rash in one patient exhibited a specific, class I MHC-restricted cytotoxic activity against HIV-1 Gag- and Pol-expressing target cells, whereas CTL lines derived from the skin lesions of a second patient were shown to be predominantly Env-specific. Taken together, these data demonstrate the infiltration of HIV-specific CTLs in the skin of HIV-infected patients, and suggest that in addition to their known role in controlling the retroviral infection, these CTLs may also be involved in the pathogenesis of cutaneous inflammatory disorders occurring during the course of HIV infection.

Impact of graft preservation solutions for liver transplantation on early cytokine release and postoperative organ dysfunctions. A pilot study.

INTRODUCTION: During liver transplantation, graft ischemia-reperfusion injury leads to a systemic inflammatory response producing postoperative organ dysfunctions. The aim of this observational and prospective study was to compare the impact of Solution de conservation des organes et tissus (SCOT) 15 and University of Wisconsin (UW) preservation solutions on early cytokine release, postreperfusion syndrome and postoperative organ dysfunctions. METHODS: Thirty-seven liver transplantations were included: 21 in UW Group and 16 in SCOT 15 group. Five cytokines were measured in systemic blood after anesthetic induction, 30minutes after unclamping portal vein and on postoperative day 1. RESULTS: Following unclamping portal vein, cytokines were released in systemic circulation. Systemic cytokine concentrations were higher in UW than in SCOT 15 group: Interleukin-10, Interleukine-6. In SCOT 15 group, significant reduction of postreperfusion syndrome incidence and acute kidney injury were observed. Alanine and aspartate aminotransferase peak concentrations were higher in SCOT 15 group than in UW group. However, from postoperative day 1 to day 10, aminotransferase returned to normal values and did not differ between groups. CONCLUSIONS: Compared to UW, SCOT 15 decreases systemic cytokine release resulting from graft ischemia-reperfusion injury and reduces incidence of postreperfusion syndrome and postoperative renal failure.

Regulatory T cell therapy: An option to induce operational tolerance in liver transplantation.

Regulatory T cells (Treg) may play an important role in operational (clinical) tolerance (OT), a stable graft function without immunosuppression in an otherwise immunocompetent host, that is spontaneously observed in some patients many years after transplantation. Several ongoing clinical trials are currently testing the effects of donor-specific or non-specific Treg infusion with the goal to induce this state of OT a few months after liver transplantation (LT). The preliminary results of two of these trials have been recently published, and raise a number of comments and issues: (1) These two papers demonstrate that a 100 to 1000-fold ex-vivo expansion of Treg is possible in humans after 2 weeks of culture. The optimal human Treg dose is however not clearly established, and might be higher than the dose that would be expected from translating murine data. (2) A lot of concerns are remaining regarding the Treg purity before expansion, the Treg stability during in vitro culture and the in vivo fate of infused cells. A strict monitoring of Treg should thus be done at each step. (3) Since Treg may play a detrimental role in some conditions, such as viral diseases and cancer, potential LT recipients with such diseases should probably be excluded from the initial trials of Treg infusion. (4) The follow-up of tolerant liver recipients should include repeated liver biopsies and detection of autoantibodies and humoral response, in addition to conventional liver graft assessment, in order to prevent the development of immune complications related to immunosuppression withdrawal. (5) The final issue raised by Treg therapy in LT is the choice of the immunosuppressive regimen used before tapering or withdrawal, appropriate to preserve OT establishment.

Expression of V beta gene segments by Sezary cells.

The T-cell receptor V beta repertoire expressed by Sezary cells was determined in a series of 16 patients whose samples have been shown to contain a majority of tumor cells. By using anti-V beta monoclonal antibodies, polymerase chain reaction analysis of expressed V beta, and, in selected cases, nucleotide sequencing, we have shown that the expressed V beta segments belong to five V beta families (V beta 5, V beta 6, V beta 8, V beta 13, and V beta 18), which contain a large fraction of the T-cell receptor V beta repertoire and do not share significant similarities in complementary determining region 4. V beta segments from these five families were also found to be strongly expressed by CD4 + CD7- peripheral blood cells obtained by fluorescence-activated cell sorting from two healthy donors. The diversity of the V beta repertoire expressed by Sezary cells appears to be similar to that expressed by circulating non-neoplastic T cells. These data do not support the hypothesis that a common superantigen is involved in the initiation of this form of cutaneous T-cell lymphoma.

Gene therapy approaches to HIV-infection: immunological strategies: use of T bodies and universal receptors to redirect cytolytic T-cells.

Combined regimens of classical antiviral treatments have not, until now, lead to the eradication of HIV-1. A specific anti-HIV immune response may have to be boosted or transferred to patients after suppression of viral replication, in order to eradicate residual infected cells from their sanctuaries. Cytotoxic T cells engineered to express recombinant chimeric receptors can be redirected against HIV-infected cells and could represent the basis of a new type of immunotherapy. Several HIV epitopes have been targeted successfully in vitro. Two types of binding domains (antibody fragments, CD4) fused with various signal transducing units (zeta chain of the CD3 complex, Fc epsilon RI gamma chain) have been tested for their ability to redirect effector cells to HIV infected lymphocytes. CD4-zeta-expressing myeloid and natural killer cells conferred SCID mice protection against challenge with tumor cells expressing HIV-env. Finally, the safety of the adoptive transfer of syngeneic CD4-zeta -modified T cells in HIV-infected individuals is currently under evaluation.

Phage display of peptide/major histocompatibility complex.

To date, there is no direct way to determine the antigenic specificity of T-cells. While B-cell epitopes can be selected from phage-displayed libraries of peptides, the corresponding molecular tool for identifying T-cell epitopes does not yet exist. The natural ligands of the T-cell antigen-receptor (TCR) are essentially antigenic peptides (P) associated with the products of the major histocompatibility complex (MHC). Here, we report phages displaying P-MHC complexes. Single-chain P-MHC class I molecules, produced in E. coli periplasm, stimulate T-cells in a peptide-specific fashion. The same P-MHC, fused at the tip of filamentous phage, directed their binding to a recombinant TCR restricted to the displayed MHC haplotype (H-2K(d)). Importantly, the binding of P-K(d)-fd to a K(d)-restricted TCR, and also to K(d)-restricted T-cell hybridomas, was modulated by the displayed peptide. Therefore, we suggest phage display of P-MHC as a direct molecular tool for probing T-cell specificity, and for selecting TCR ligands from genetic libraries encoding randomized or natural peptides.

Detection of apoptosis at the single-cell level by direct incorporation of fluorescein-dUTP in DNA strand breaks.

Apoptosis induced in different cell lines can be detected and quantified in one step. Direct labeling of apoptotic cells (DLAC) was performed by incorporation of fluorescein-dUTP (F-dUTP) in DNA strand breaks by terminal deoxynucleotidyl transferase (TdT). Nick-end-labeling using F-dUTP obviates the need for second-step revelation reagents without reducing the specificity of detection. Cells were analyzed by microscopy or flow cytometry for objective quantification of apoptotic cells in controlled dilution samples. Furthermore, we demonstrate that DLAC is compatible with surface labeling by monoclonal antibodies, allowing dual-color analysis. The data presented here illustrate the simplicity and potential of this method, which allows the preservation of fragile cells and the possibility of combining apoptosis detection with immunostaining.

Restoration of the immune system with anti-retroviral therapy.

Clinical benefits of highly active anti-retroviral treatments (HAART) are increasingly evidenced by resolving opportunistic infections and malignancies, as well as declining hospitalization and mortality rates [1]. This suggests that potent and sustained suppression of viral replication, at least to some extent, is associated with reconstitution of the immune system even in adult patients treated at advanced stages of the disease. Increased susceptibility to opportunistic infections and tumors mainly results from the loss of memory CD4+ T cell reactivity against recall antigens which is an early event in HIV disease progression. Primary responses of naive CD4+ T cells against new pathogens are suppressed even earlier in the course of HIV disease, and the progressive depletion in naive CD4+ T cells reflects profound alterations in T cell regeneration capacities. Previous studies revealed that monotherapy with ritonavir, a protease inhibitor, resulted in a slight improvement in memory CD4+ T cell responses to recall Ags only when detectable prior to onset of therapy, suggesting that the loss of CD4+ T cell reactivity might be irreversible at advanced stages of the disease [2]. In contrast our group demonstrated more recently that restoration in CD4+ T cell reactivity to specific antigens was feasible when HAART was administered in progressors [3]. Here we address some of the questions raised by immune restoration with HAART when administered at advanced stages of the disease.

Phenotype and function of peripheral blood and bone marrow T-cell colonies: identification of DC3-, 4-, 8-autoreactive T cells.

The existence of immature autoreactive T cells has been examined in peripheral blood (PB) and bone marrow (BM) derived T-lymphocyte colonies (TLC) that have previously been shown to be potentially generated from CD3-negative BM-T cells. An extensive phenotypical analysis of total and T-depleted TLC showed that both PB- and BM-derived TLC contained a mean of 5% immature T lymphocytes (ITL), which were negative for the CD3, CD4, and CD8 antigens but displayed the CD2 and CD7 antigens. The only detectable immune functions of these isolated ITL were an allo- and an autoreactivity without cytolytic activity. The self-reactivity of ITL was not detected in bulk non T-depleted TLC cells and seemed to be actively suppressed by autologous mature T cells. In addition, the auto-MLR of ITL was totally inhibited by anti-HLA class II but not by anti-class I monoclonal antibody (MoAb) and only partially by anti-CD4 moAb, whereas the anti-CD3 and anti-CD2 MoAbs gave no inhibition. Once activated, ITL could acquire in culture a mature T cell CD3 + CD4 + phenotype. The CD3-, 4-, 8-auto-reactive T cells present in T colonies could represent pre- or post-thymic cells that have not yet undergo or that have escaped the thymic selection.