A Novel Adhesion Molecule in the Murine Thymic Microenvironment: Functional and Biochemical Analysis

The rat monoclonal antibody (mAb) 4F1, raised against mouse thymic stromal cells, recognizes cortical epithelium in tissue sections of mouse thymus; however, in flow cytometry, activated leucocytes (T cells, B cells, and macrophages) and transformed thymocytes are also positive for the 4F1-antigen (4F1-Ag). Western blotting, under both reducing and nonreducing conditions, demonstrates that the molecule to which 4F1 binds is expressed in four forms, 29, 32, 40, and 43 kD, all of which carry N-linked carbohydrate; and that the structure is identical on epithelium and lymphocytes. The 4F1-Ag on cortical epithelium is partially sensitive to PI-PLC treatment, whereas on transformed epithelial and lymphoid cell lines, it was resistant to this enzyme. The molecule, therefore, may exist in both transmembrane and phosphoinositol-linked forms. In functional blocking experiments, mAb 4F1 gave inhibition of both T-cell proliferation in MLR and of cytotoxic T-cell killing of alloantigenic targets; it also blocked adhesion of transformed thymocytes to thymic epithelial cells in vitro. These molecular and functional characteristics suggest that the 4F1-Ag is a novel adhesion molecule that may be involved both in intrathymic T lymphocyte differentiation and in peripheral T-cell function

Effects of recombinant human growth hormone on HIV-1-specific T-cell responses, thymic output and proviral DNA in patients on HAART: 48-week follow-up

Background: Efficacious immune-based therapy in treated chronic HIV-1 infection requires the induction of virus-specific CD4+ T cells and subsequent maturation and maintenance of specific memory CD8+ T cells. Concomitant daily administration of recombinant human growth hormone (rhGH) with highly active antiretroviral therapy (HAART) was used in chronically infected patients with lipodystrophy in an attempt to reconstitute these virus-specific T-cell responses. Methods: Individuals with chronic HIV-1 infection on HAART were enrolled on a randomized, double-blinded, placebo-controlled study to receive rhGH therapy. We assessed HIV-1-specific proliferative CD4+ and interferon-gamma (IFN-γ)-producing CD8+ T-cell responses, quantified thymic output and proviral HIV-1 DNA at the following time points: baseline; after 12 weeks of rhGH therapy; at 24 weeks, after randomization into three groups [placebo weeks 12-24 (Group A), alternate-day dosing weeks 12-24 (Group B), and twice-per-week dosing weeks 12-24 (Group C)]; and at 48 weeks after all patients had received HAART alone for the final 24 weeks. Results: We found significant increases in both proliferative CD4+ and IFN-γ-producing CD8+ HIV-1-specific T-cell responses after daily administration of rhGH. This increase was focused on HIV-1 Gag-specific T-cell responses. Following subsequent randomisation into different dosing regimens, HIV-1-specific proliferative CD4+ T-cell responses declined in patients receiving less frequent dosing of rhGH, while virus-specific IFN-γ-producing CD8+ T-cell responses were maintained for longer periods of time. There was no significant change in thymic output and the cell-associated HIV-1 DNA remained stable in most patients. An increased anti-HIV-1 Nef-specific CD4+ T-cell proliferative response was correlated to a decrease in proviral load, and an increased HIV-1 Gag-specific IFN-γ-producing CD8+ T-cell response correlated with an increase in proviral load. Conclusion: The implication of these data is that daily dosing of rhGH with HAART, in addition to improving HIV-1-associated lipodystrophy, may reverse some of the T-lymphocyte dysfunction seen in most treated HIV-1-positive patients, in a dose-dependent manner. Such immune-based therapeutic strategies used in treated, chronic HIV-1 infection may enable the induction of virus-specific CD4+ T cells essential for the subsequent kick-start and expansion of virus-specific CD8+ T cells. © 2008 Herasimtschuk et al; licensee BioMed Central Ltd

Are Long-Term Non-Progressors Very Slow Progressors? Insights from the Chelsea and Westminster HIV Cohort, 1988–2010

Define and identify long-term non-progressors (LTNP) and HIV controllers (HIC), and estimate time until disease progression. LTNP are HIV-1+ patients who maintain stable CD4+ T-cell counts, with no history of opportunistic infection or antiretroviral therapy (ART). HIC are a subset of LTNP who additionally have undetectable viraemia. These individuals may provide insights for prophylactic and therapeutic development. Records of HIV-1+ individuals attending Chelsea and Westminster Hospital (1988–2010), were analysed. LTNP were defined as: HIV-1+ for >7 years; ART-naïve; no history of opportunistic infection and normal, stable CD4+ T-cell counts. MIXED procedure in SAS using random intercept model identified long-term stable CD4+ T-cell counts. Survival analysis estimated time since diagnosis until disease progression. Subjects exhibiting long-term stable CD4+ T-cell counts with history below the normal range (<450 cells/µl blood) were compared to LTNP whose CD4+ T-cell count always remained normal. Within these two groups subjects with HIV-1 RNA load below limit of detection (BLD) were identified. Of 14,227 patients, 1,204 were diagnosed HIV-1+ over 7 years ago and were ART-naïve. Estimated time until disease progression for the 20% (239) whose CD4+ T-cell counts remained within the normal range, was 6.2 years (IQR: 2.0 to 9.6); significantly longer than 4.0 years (IQR: 1.0 to 7.3) for patients with historical CD4+ T-cell count below normal (Logrank chi-squared = 21.26; p<0.001). Within a subpopulation of 312 asymptomatic patients, 50 exhibited long-term stable CD4+ T-cell counts. Of these, 13 were LTNP, one of whom met HIC criteria. Of the remaining 37 patients with long-term stable low CD4+ T-cell counts, 3 controlled HIV-1 RNA load BLD. Individuals with stable, normal CD4+ T-cell counts progressed less rapidly than those with low CD4+ T-cell counts. Few LTNP and HIC identified in this and other studies, endorse the need for universal definitions to facilitate comparison

A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy

BACKGROUND: Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection. DESIGN: Chronically infected patients with low CD4 counts T-cell counts who commenced antiretroviral therapy (ART) were subsequently treated with combined interleukin-2 and therapeutic vaccination. METHODS: Thirty six anti-retroviral naive patients were recruited and initiated on combination ART for 17 weeks before randomization to: A) ongoing ART alone; B) ART with IL-2 twice daily for 5 days every four weeks starting at week 17 for 3 cycles; C) ART with IL-2 as in group B and Remune HIV-1 vaccine administered once every 3 months, starting at week 17; and D) ART with Remune vaccine as in group C. Patients were studied for 65 weeks following commencement of ART, with an additional prior 6 week lead-in observation period. CD4 and CD8 T-cell counts, evaluations of HIV-1 RNA levels and proliferative responses to recall and HIV-1 antigens were complemented with assessment of IL-4-secretion alongside quantification of anti-HIV-1 CD8 T-cell responses and neutralizing antibody titres. RESULTS: Neither IL-2 nor Remune™ vaccination induced sustained HIV-1-specific T-cell responses. However, we report an inverse relationship between HIV-1-specific proliferative responses and IL-4 production which continuously increased in patients receiving immunotherapy, but not patients receiving ART alone. CONCLUSION: Induction of HIV-1-specific cell-mediated responses is a major challenge in chronically HIV-1-infected patients even when combining immunisation with IL-2 therapy. An antigen-specific IL-4-associated suppressive response may play a role in attenuating HIV-specific responses

K21-Antigen: A Molecule Shared by the Microenvironments of the Human Thymus and Germinal Centers

The mouse IgG1 monoclonal antibody (mAb) K21 recognizes a 230-kD molecule (K21-Ag) on Hassall's corpuscles in the human thymus. This mAb also stains cultured thymic epithelial cells as well as other epithelial cell lines, revealing a predominant intracellular localization. Further analysis with mAb K21 on other lymphoid tissues showed that it also stains cells within the germinal centers of human tonsils, both lymphoid (B) cells and some with the appearance of follicular dendritic cells. Double immunostaining of tonsil sections shows that K21-Ag is not expressed by T cells, whereas staining with anti-CD22 and -CD23 mAb revealed some doublepositive cells. A subpopulation of the lymphoid cells express the K21-Ag much more strongly. This K21++/CD23++ subpopulation of cells is localized in the apical light zone of germinal centers, suggesting that K21-Ag may be an important marker for the selected centrocytes within germinal centers and may play a role in B-cell selection and/or development of B-cell memory. Flow cytometric analysis showed that K21-Ag is expressed on the surface of a very low percentage of thymocytes, tonsillar lymphocytes, and peripheral blood mononuclear cells. Analysis of purified/separated tonsillar T and B lymphocytes showed that T cells do not express the K21-Ag; in contrast, B cells express low levels of the K21-Ag, and this together with CD23 is upregulated after mitogenic stimulation. Our data therefore raise the possibility that the K2l- Ag may play a role in B-lymphocyte activation/selection

Transient Nature of Long-Term Nonprogression and Broad Virus-Specific Proliferative T-Cell Responses with Sustained Thymic Output in HIV-1 Controllers

HIV-1(+) individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4(+) T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-gamma ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1(+) individuals originally identified as "Long-term non-progressors" in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-gamma producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-gamma production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1(+) patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-gamma production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory

The role of CD8+ T cell clones in immune thrombocytopenia

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP

NK cell and T-cell immunity and TLR responsiveness during chronic HIV-1 infection

This project investigates the potential of Natural Killer (NK) cells to respond to pathogen associated molecular patterns (PAMP) as model adjuvants and establishes the potential of PAMP to aid the recovery of antigen specific T-cell responses in chronic HIV-1 infection. Innate immunity is mediated by through the recognition of conserved PAMP recognised by receptors such as the toll-like receptors (TLR), expressed by accessory cells including blood DC, which are responsible for initiating adaptive immune responses, but also activate NK cells. NK cells are no longer seen solely as killers, but instead produce cytokines, including IFN-γ, and participate in the generation of adaptive immune responses with DC. HIV-1 infection potentially chronically activates TLR pathways, via recognition of the viral genome and as a result of damage to mucosal surfaces leading to microbial translocation and systemic recognition of PAMP including bacterial lipopolysaccharides. NK cell activation, cytokine production, differentiation and proliferation in response to PAMP have therefore been examined. NK cell responsiveness to certain TLR agonists was refractory in HIV-1 infected individuals although responses were maintained within CD56+CD16- NK cells, potentially providing help for antigen specific T-cell responses. The effects of HIV-1 infection on NK cell maturation were also investigated. T-cell activation by TLR agonists was also refractory in HIV-1 infected individuals. The impact of TLR ligation on antigen specific T-cell responses was investigated, combining TLR agonists with peptide pools (influenza, Epstein-Barr virus and cytomegalovirus or HIV-1 gag antigens). Of the TLR agonists tested, only CpG DNA resulted in enhanced frequencies of IFN-γ producing T-cells in HIV-1 infected individuals, whereas LPS demonstrated consistently reduced IFN-γ production. In conclusion, maintenance of responsiveness of CD56+CD16- NK cells and enhancement of HIV-1 antigen specific T-cell responses by CpG DNA have implications for both repopulation of the NK cell compartment and reconstitution of acquired immunity during HIV-1 infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

NK cell and T-cell immunity and TLR responsiveness during chronic HIV-1 infection

This project investigates the potential of Natural Killer (NK) cells to respond to pathogen associated molecular patterns (PAMP) as model adjuvants and establishes the potential of PAMP to aid the recovery of antigen specific T-cell responses in chronic HIV-1 infection. Innate immunity is mediated by through the recognition of conserved PAMP recognised by receptors such as the toll-like receptors (TLR), expressed by accessory cells including blood DC, which are responsible for initiating adaptive immune responses, but also activate NK cells. NK cells are no longer seen solely as killers, but instead produce cytokines, including IFN-γ, and participate in the generation of adaptive immune responses with DC. HIV-1 infection potentially chronically activates TLR pathways, via recognition of the viral genome and as a result of damage to mucosal surfaces leading to microbial translocation and systemic recognition of PAMP including bacterial lipopolysaccharides. NK cell activation, cytokine production, differentiation and proliferation in response to PAMP have therefore been examined. NK cell responsiveness to certain TLR agonists was refractory in HIV-1 infected individuals although responses were maintained within CD56+CD16- NK cells, potentially providing help for antigen specific T-cell responses. The effects of HIV-1 infection on NK cell maturation were also investigated. T-cell activation by TLR agonists was also refractory in HIV-1 infected individuals. The impact of TLR ligation on antigen specific T-cell responses was investigated, combining TLR agonists with peptide pools (influenza, Epstein-Barr virus and cytomegalovirus or HIV-1 gag antigens). Of the TLR agonists tested, only CpG DNA resulted in enhanced frequencies of IFN-γ producing T-cells in HIV-1 infected individuals, whereas LPS demonstrated consistently reduced IFN-γ production. In conclusion, maintenance of responsiveness of CD56+CD16- NK cells and enhancement of HIV-1 antigen specific T-cell responses by CpG DNA have implications for both repopulation of the NK cell compartment and reconstitution of acquired immunity during HIV-1 infection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo