Lalit Rane

Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial.

BACKGROUND Novel treatment options are urgently needed for multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, which are associated with immune dysfunction and poor treatment outcomes. Mesenchymal stromal cells (MSCs) are immunomodulatory and adjunct autologous treatment with bone marrow-derived MSCs might improve clinical outcome by transforming chronic inflammation into productive immune responses. Our aim was to assess the safety of infusion of autologous MSCs as an adjunct treatment in patients with tuberculosis. METHODS 30 patients with microbiologically confirmed MDR or XDR tuberculosis were treated with single-dose autologous bone marrow-derived MSCs (aimed for 1×10(6) cells per kg), within 4 weeks of the start of antituberculosis-drug treatment in a specialist centre in Minsk, Belarus. Inclusion patients were those with pulmonary tuberculosis confirmed by sputum smear microscopy, culture, or both; MDR or XDR tuberculosis confirmed by drug-susceptibility testing to first-line and second-line drugs; age older than 21 years to 65 years or younger; and absence of lesion compatible with a malignant process or ongoing tuberculosis in organs other than the lungs and pleura. In addition to the inclusion criteria, patients were excluded if they were pregnant, coinfected with HIV, or infected with hepatitis B, C, or both. The primary endpoint was safety measured by MSC-infusion related events; any tuberculosis-related event within the 6 month observation period that related to a worsening of the underlying infectious disease, measured by conversion of Mycobacterium tuberculosis culture or microscopic examination; or any adverse event defined clinically or by changes in blood haematology and biochemistry variables, measured monthly for 6 months after MSC infusion per protocol. This study is registered with the German Clinical Trials Registry, number DRKS00000763. FINDINGS The most common (grade 1 or 2) adverse events were high cholesterol levels (14 of 30 patients), nausea (11 of 30 patients), and lymphopenia or diarrhoea (ten of 30 patients). There were no serious adverse events reported. We recorded two grade 3 events that were transitory-ie, increased plasma potassium ion concentrations in one patient and a transitory grade 3 γ-glutamyltransferase elevation in another patient. INTERPRETATION MSCs as an adjunct therapy are safe and can now be explored further for the treatment of patients with MDR or XDR tuberculosis in combination with standard drug regimens. Adjunct treatment with MSCs needs to be evaluated in controlled phase 2 trials to assess effects on immune responses and clinical and microbiological outcomes.

Alternative splicing of interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Rα) in peripheral blood from patients with multiple sclerosis (MS)

IL-7 and IL-7Ralpha (IL-7R) form a non-redundant ligand receptor system which plays a crucial role in human T cell immunity. Both IL-7 and IL-7R are multi-exonal genes and exhibit alternative splicing. We measured the relative distribution of IL-7 and IL-7R spliced mRNA from patients with MS and healthy individuals and observed extensive alternative splicing of both genes with marked differences in proportional transcript expression levels. We report here for the first time that the IL-7 transcript, lacking exon 4, and not the full length IL-7 represents the dominant IL-7 RNA transcript in human PBMCs and a novel IL-7R splice variant lacking exons 5, 6 and 7.

Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-&ggr; production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V&bgr; was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8+ T cells. CD3+CD8+, CD3+CD4+, and CD3+CD4−CD8− [double-negative (DN) T cells] resided predominantly in central (CD45RA−CCR7+) and effector (CD45RA−CCR7−) memory subsets. CD8+ TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4+ TILs showed only up to 12% LAG-3+ staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8+) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-&agr; and IFN-&ggr; production. Twelve of 17 of CD8+ TILs showed preferential expansion of certain TCR V&bgr; families (eg, 99.2% V&bgr;13.2 in CD8+ TILs, 77% in the V&bgr;1, 65.9% in the V&bgr;22, and 63.3% in the V&bgr;14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8+ V&bgr;13.2) reacting strongly against autologous tumor defined by INF-&ggr; production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

Increased β-haemolytic group A streptococcal M6 serotype and streptodornase B-specific cellular immune responses in Swedish narcolepsy cases

Type 1 narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy associated with the HLA allele DQB1*06:02. Genetic predisposition along with external triggering factors may drive autoimmune responses, ultimately leading to the selective loss of hypocretin‐positive neurons.

Decreased IL-7 signaling in T cells from patients with PTLD after allogeneic HSCT.

Interleukin (IL)-7, a nonredundant cytokine for B and T cells, plays a central role in cell survival and immune memory formation. Peripheral blood mononuclear cells (PBMCs) from 7 patients after hematopoietic stem cell transplantation (HSCT) diagnosed with posttransplantation lymphoproliferative disease (PTLD) and from 10 Epstein-Barr virus (EBV) polymerase chain reaction-positive HSCT patients (controls) were evaluated for IL-7- and IL-2 induced Stat5 phosphorylation in CD4+ and CD8+ T cells. PBMCs from PTLD+ and control patients exhibited detectable EBV specific CD8+ T cells defined by tetramer analysis. CD4+ and CD8+ T cells from patients with PTLD showed statistically significant reduction in responsiveness to IL-7 compared with PBMCs obtained from controls defined by Stat5 phosphorylation. CD20+ B cells from patients with PTLD and from some EBV+ polymerase chain reaction control individuals exhibited IL-7R expression. Dysregulated immune surveillance, reflected by deficient Stat5 phosphorylation, may facilitate PTLD development despite the presence of EBV-reactive CD8+ T cells. Reduced IL-7 responsiveness will aid to monitor patients after HSCT for increased risk to develop EBV-associated PTLD.

Tumor-infiltrating lymphocytes (TILs) from patients with glioma

ABSTRACT Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+ CD4−CD8− T cells with a predominant central memory CD45RA−CCR7+ phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+ TIL, with low (1.25%) frequencies in CD4+ T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas.

Difference in immune response in vaccinated and unvaccinated Swedish individuals after the 2009 influenza pandemic

BackgroundPrevious exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009–2010.MethodsCellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test.ResultsPrevious seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms.ConclusionsStrong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.

Peptide microarray-based characterization of antibody responses to host proteins after bacille Calmette–Guérin vaccination

BACKGROUND Bacille Calmette-Guérin (BCG) is the worlds most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. METHODS The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. RESULTS IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor β (IL2Rβ), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFβR1) and, in agreement with the peptide microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination. CONCLUSIONS BCG vaccination modifies the hosts immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals.

Frontline Science: Placenta‐derived decidual stromal cells alter IL‐2R expression and signaling in alloantigen‐activated T cells

This study investigated how stromal cells affect the IL‐2 pathway in alloantigen‐activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL‐2 in cultures with alloantigen‐activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL‐2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL‐2Rβ) and CD132 (IL‐2Rγc) were reduced. Consequently, uptake of IL‐2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5+ T cells in response to IL‐15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD‐1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL‐2 in similar concentrations in the same setting increased the expression of CD95 and down‐regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL‐2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL‐2 production and IL‐2R expression and signaling, which may contribute to the stromal cell‐mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

TCR+CD4-CD8- T cells in antigen-specific MHC class I-restricted T-cell responses after allogeneic hematopoietic stem cell transplantation.

Human TCR&agr;&bgr;+ CD4−CD8− double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA+CCR7−) T-cell population and are polyclonal, determined by T-cell receptor V&bgr; CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4+ or CD8+ T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR+ and the CD8&agr; chain to study TCR+ DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCR&zgr; chain phosphorylation as compared with the TCR+CD8+ transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.