Jacob Couturier

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV

Objective:The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. Design:We examined memory CD4+ T cells and HIV DNA in adipose tissue–stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4+ T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4+ T cells. Methods:AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4+ T-cell activation and HIV production were measured by flow cytometry and ELISA. Results:AT-SVF CD3+ T cells were activated (>60% CD69+) memory CD4+ and CD8+ T cells in uninfected and HIV-infected persons, but the AT-SVF CD4+/CD8+ ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4+ T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-&agr;1&bgr;1. Adipocytes also enhanced T-cell viability. Conclusion:Adipose tissues of ART-treated patients harbour activated memory CD4+ T cells and HIV DNA. Adipocytes promote CD4+ T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis

ABSTRACT Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB.

Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells

BackgroundGranzyme B (GrzB) is a serine proteinase expressed by memory T cells and NK cells. Methods to measure GrzB protein usually involve intracellular (flow cytometry) and extracellular (ELISA and ELISpot) assays. CD8 T cells are the main source of GrzB during immunological reactions, but activated CD4 T cells deploy GrzB as well. Because GrzB is an important mediator of cell death, tissue pathology and disease, clarification of differences of GrzB expression and secretion between CD4 and CD8 T cells is important for understanding effector functions of these cells.ResultsMemory CD4 and memory CD8 T cells were purified from human peripheral blood of healthy donors, and production of GrzB was directly compared between memory CD4 and memory CD8 T cells from the same donors using parallel measurements of flow cytometry (intracellular GrzB), ELISpot (single cell secretion of GrzB), and ELISA (bulk extracellular GrzB). Memory CD8 T cells constitutively stored significantly more GrzB protein (~25%) compared to memory CD4 T cells as determined by flow cytometry (~3%), and this difference remained stable after 24 hrs of activation. However, measurement of extracellular GrzB by ELISA revealed that activated memory CD4 T cells secrete similar amounts of GrzB (~1,000 pg/ml by 1x105 cells/200 μl medium) compared to memory CD8 T cells (~600 pg/ml). Measurement of individual GrzB-secreting cells by ELISpot also indicated that similar numbers of activated memory CD4 (~170/1×105) and memory CD8 (~200/1×105) T cells secreted GrzB. Expression of CD107a further indicated that Grzb is secreted similarly by activated CD4 and CD8 T cells, consistent with the ELISA and ELISpot results. However, memory CD8 T cells expressed and secreted more perforin compared to memory CD4 T cells, suggesting that perforin may be less associated with GrzB function for memory CD4 T cells.ConclusionsAlthough measurement of intracellular GrzB by flow cytometry suggests that a larger proportion of CD8 T cells have higher capacity for GrzB production compared to CD4 T cells, ELISpot and ELISA show that similar numbers of activated CD4 and CD8 T cells secrete similar amounts of GrzB. Secretion of GrzB by activated CD8 T cells may be more tightly controlled compared to CD4 T cells.

Selective Expansion of Memory CD4+ T cells By Mitogenic Human CD28 Generates Inflammatory Cytokines and Regulatory T cells

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co‐ligation or cross‐linking; ANC28 selectively expanded CD4+CD25+FOXP3– (Teff) and CD4+CD25+FOXP3+ (Treg) cells. ANC28 stimulated the CD45RO+ CD4+ (memory) population, whereas CD45RA+CD4+ (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25– T cells through a contact‐dependent mechanism. Purity influenced the response of CD4+CD25+ cells because bead‐purified CD4+CD25+ cells (85–90% pure) responded strongly to ANC28, whereas 98% pure FACS‐sorted CD4+CD25bright (Treg) did not respond. Purified CD4+CD25int cells responded similarly to the bead‐purified CD4+CD25+ cells. Thus, pre‐activated CD4+ T cells (CD25int) respond to ANC28 rather than Treg (CD25bright). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.

Granzyme B‐ and Fas ligand‐mediated cytotoxic function induced by mitogenic CD28 stimulation of human memory CD4+ T cells

Some human memory CD4+ T cells have cytotoxic functions best understood in the context of viral infections; however, their possible role in pathologic processes is understudied. The novel discovery that mitogenic CD28 antibodies induced proliferation and expansion of Tregs offered therapeutic promise for autoimmune disorders. However, the failed TGN1412 trial forced reassessment of this concept. As memory CD4+ T cells are known to produce toxic molecules, including granzyme B (GrzB) and FasL, we wondered whether mitogenic CD28 was able to induce these cytotoxic molecules. A commercially available mitogenic human CD28 mAb (clone ANC28.1) was used to determine whether mitogenic CD28 induces cytotoxic function from human memory CD4+ T cells. We found that stimulation of memory CD4+ T cells by ANC28.1, as well as by conventional costimulation (CD3/CD28 mAb), robustly induced enzymatically active GrzB, along with increased surface expression of FasL. These functional phenotypes were induced in association with increased expression of T cell activation markers CD69 and CD25, and elimination of target cells by ANC28.1‐activated memory CD4+ T cells involved both GrzB and FasL. Additionally, ANC28.1‐activated memory CD4+ T cells caused disruption of epithelial cell monolayer integrity, which was partially mediated by GrzB. These findings reveal functions of memory CD4+ T cells previously unknown to be induced by mitogenic CD28, and suggest that these pathogenic mechanisms may have been responsible for some of the widespread tissue destruction that occurred in the TGN1412 trial recipients.

HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: Implications for bystander cell and tissue pathologies

Granzyme B (GrzB) is expressed by activated T cells and mediates cellular apoptosis. GrzB also acts as an extracellular protease involved in tissue degradation. We hypothesized that GrzB production from activated memory CD4 T cells may be associated with HIV pathogenesis. We found that stimulated memory CD4 T cells (via costimulation, cytokines, and TLR ligands) concomitantly produced GrzB and HIV. Both GrzB and HIV expression were mainly restricted to CCR5-expressing memory CD4+CD45RO+ T cells, including Th1 and Th17 subsets. Activated memory CD4 T cells also mediated tissue damage, such as disruption of intestinal epithelial monolayers. In non-human primates, CD4 T cells of rhesus macaques (pathogenic SIV hosts) expressed higher GrzB compared to African green monkeys (non-pathogenic SIV hosts). These results suggest that GrzB from CCR5+ memory CD4 T cells may have a role in cellular and tissue pathologies during HIV infection.

Allele Specific Expression of MICA Variants in Human Fibroblasts Suggests a Pathogenic Mechanism

The major histocompatibility complex class I chain-related gene A (MICA) is involved in immune responses of both nature killer (NK) cells and subsets of T cells with its receptor NKG2D. MICA is highly polymorphic in sequence which leads to MICA protein variants with distinct features. Specific polymorphisms of MICA have been associated with inflammatory diseases, including ankylosing spondylitis (AS), ulcerative colitis (UC) and Behçet’s disease. Studies herein characterize expression features of three MICA variants including MICA*008, a common variant in general population, and *MICA*007 and *019, which are associated with susceptibility to inflammatory diseases. MICA*019 was highly expressed on the surface of fibroblasts whereas expression of MICA*007 was the lowest in the culture supernatant. MICA*008 had low cell surface expression but was the only MICA allele in which exosomal material was detected. Surface or membrane-bound MICA activates NKG2D-mediated cytotoxicity, whereas soluble and exosomal MICAs down-regulate NKG2D. Therefore, comparisons of these three MICA variants in fibroblasts provides insight into understanding how MICA associated immune responses could be regulated to influence levels of inflammation.

HIV Persistence in Adipose Tissue Reservoirs

Purpose of ReviewThe purpose of this review is to examine the evidence describing adipose tissue as a reservoir for HIV-1 and how this often expansive anatomic compartment contributes to HIV persistence.Recent FindingsMemory CD4 T cells and macrophages, the major host cells for HIV, accumulate in adipose tissue during HIV/SIV infection of humans and rhesus macaques. Whereas HIV and SIV proviral DNA is detectable in CD4 T cells of multiple fat depots in virtually all infected humans and monkeys examined, viral RNA is less frequently detected, and infected macrophages may be less prevalent in adipose tissue. However, based on viral outgrowth assays, adipose-resident CD4 T cells are latently infected with virus that is replication-competent and infectious. Additionally, adipocytes interact with CD4 T cells and macrophages to promote immune cell activation and inflammation which may be supportive for HIV persistence. Antiviral effector cells, such as CD8 T cells and NK/NKT cells, are abundant in adipose tissue during HIV/SIV infection and typically exceed CD4 T cells, whereas B cells are largely absent from adipose tissue of humans and monkeys. Additionally, CD8 T cells in adipose tissue of HIV patients are activated and have a late differentiated phenotype, with unique TCR clonotypes of less diversity relative to blood CD8 T cells. With respect to the distribution of antiretroviral drugs in adipose tissue, data is limited, but there may be class-specific penetration of fat depots.SummaryThe trafficking of infected immune cells within adipose tissues is a common event during HIV/SIV infection of humans and monkeys, but the virus may be mostly transcriptionally dormant. Viral replication may occur less in adipose tissue compared to other major reservoirs, such as lymphoid tissue, but replication competence and infectiousness of adipose latent virus are comparable to other tissues. Due to the ubiquitous nature of adipose tissue, inflammatory interactions among adipocytes and CD4 T cells and macrophages, and selective distribution of antiretroviral drugs, the sequestration of infected immune cells within fat depots likely represents a major challenge for cure efforts.

The effect of extracorporeal photopheresis alone or in combination therapy on circulating CD4+Foxp3+CD25- T cells in patients with leukemic cutaneous T-cell lymphoma

Extracorporeal photopheresis (ECP) alone or in combination therapy is effective for treatment of leukemic cutaneous T‐cell lymphoma (L‐CTCL), but its mechanism(s) of action remain unclear. This study was designed to investigate the effect of ECP on regulatory T cells and CD8+ T cells in L‐CTCL patients.

HIV-1 replication in CD4+ T cells exploits the down-regulation of antiviral NEAT1 long non-coding RNAs following T cell activation

The related NEAT1_1 and NEAT1_2 long noncoding RNAs (lnc RNAs) have been recently implicated in innate immunity against viral infection. We used CRISPR-Cas9 to generate Jurkat CD4+ T cell lines with a knockout (KO) of the NEAT1 gene. Viabilities of NEAT1 KO Jurkat lines were indistinguishable from parental Jurkat cells, as was the induction of CD69 after T cell activation. The KO lines were however more sensitive to the induction of apoptosis than parental Jurkat cells. HIV-1 replication was higher in the KO lines than parental Jurkat cells, demonstrating an anti-HIV function of NEAT1 lncRNAs. We observed a strong down-regulation of NEAT1 lncRNAs following activation of resting peripheral blood mononuclear cells and purified CD4+ T cells. These findings indicate that HIV-1 infection exploits the normal down-regulation of anti-viral NEAT1 lncRNAs in activated CD4+ T cells to enhance viral replication.