Immunomodulatory Therapeutic Strategies Research Articles

Immune signatures for HIV-1 and HIV-2 induced CD4+T cell dysregulation in an Indian cohort

BackgroundHIV-2 infection is characterised by a longer asymptomatic phase and slower AIDS progression than HIV-1 infection. Identifying unique immune signatures associated with HIV-2 pathogenesis may thus provide therapeutically useful insight into the management of HIV infection. This study examined the dynamics of the CD4+T cell compartment, critical in disease progression, focussing on chronic HIV-2 and HIV-1 infected individuals at various stages of disease progression.MethodsA total of 111 participants including untreated and treated HIV infected individuals and seronegative individuals were enrolled in this study. The relative proportion of CD4+T cell subsets, expressing CD25 (IL-2Rα) and CD127 (IL-7R), in HIV infected individuals and seronegative controls were assessed by multiparametric flow cytometry. Additionally, levels of immune activation and cytotoxic T lymphocytes in both the CD4+T and CD8+T cell compartments was evaluated.ResultsBoth treated and untreated, HIV-1 and HIV-2 infected individuals showed apparent dysregulation in CD4+ T cell subset frequency that was associated with disease progression. Furthermore, longitudinal sampling from a group of HIV-1 infected individuals on virologically effective ART showed no significant change in dysregulated CD4+T cell subset frequency. For both ART naïve and receiving groups associations with disease progression were strongest and significant with CD4+ T cell subset frequency compared to per cell expression of IL-2Rα and IL-7Rα. In untreated HIV-2 infected individuals, T cell activation was lower compared to ART naïve HIV-1 infected individuals and higher than seronegative individuals. Also, the level of Granzyme-B expressing circulating T cells was higher in both ART-naïve HIV-1 and HIV-2 infected individuals compared to seronegative controls.ConclusionDysregulation of IL-2 and IL-7 homeostasis persists in CD4+T cell subsets irrespective of presence or absence of viremia or antiretroviral therapy in HIV infection. Furthermore, we report for the first time on levels of circulating Granzyme-B expressing CD4+T and CD8+T cells in chronic HIV-2 infection. Lower immune activation in these individuals indicates that persistent immune activation driven CD4+T cell depletion, as observed in untreated HIV-1 infected individuals, may not be as severe and provides evidence for a disparate pathogenesis mechanism. Our work also supports novel immunomodulatory therapeutic strategies for both HIV-1 and HIV-2 infection.

Abstract B008: Treatment of IDO1 and TDO2 positive tumors with a kynurenine-degrading enzyme: A highly differentiated approach from IDO1 inhibition

Abstract Despite the sustained clinical benefit demonstrated by immune checkpoint inhibitors, a majority of patients derive minimal or no appreciable benefit, indicating the urgent need to incorporate novel immunomodulatory targets and therapeutic strategies. Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) catalyze the first and rate-limiting step in the immunosuppressive tryptophan/kynurenine pathway and are both upregulated in a number of tumor types. Although small-molecule IDO1 inhibitors are being clinically evaluated in several tumor types, so far they have not demonstrated significant clinical benefits either as a single agent or in combination with immune checkpoint inhibition. We are developing pegylated kynureninase (Kynase), a kynurenine degrading enzyme, to treat a broader population with IDO1 and/or TDO2 expressing tumors. We believe that a more robust antitumor immune response can be achieved by depleting kynurenine, produced by both IDO1 and TDO2, with Kynase, than by inhibiting only IDO1. The human Kynase (HsKYN) has been successfully engineered to exhibit vastly improved catalytic activity and stability toward kynurenine over the wild-type human enzyme. HsKYN achieved durable and near complete plasma kynurenine depletion in mice, rats and non-human primates. HsKYN demonstrated single agent efficacy in CT26, MC38 and B16-IDO syngeneic mouse models. Tumor gene expression analysis using NanoString revealed that HsKYN treatment upregulated T-cell and NK cell activation signature. More importantly, HsKYN significantly increased the tumor-infiltrating CD8 T-cells and their activation/polyfunctionality, and reduced the Treg population. As a direct comparison, the lead IDO1 inhibitor epacadostat did not impose any meaningful effects on the same immune cell populations. Furthermore, HsKYN showed beneficial combination efficacy with anti-PD-1 that was superior to combined Epacadostat / anti-PD-1. Evidence to date suggest that HsKYN is well tolerated in multiple species. Therefore, immunoprofiling, efficacy and safety results strongly support that Kynase is a more effective therapeutic approach than IDO1 inhibition. HsKYN is moving toward clinical development for treatment of cancers where IDO1 and/or TDO2 pathways play a significant immunosuppressive role. Citation Format: Silvia Coma, Jillian Cavanaugh, James Nolan, Jeremy Tchaicha, Karen McGovern, Everett Stone, John Blazeck, Candice Lamb, George Georgiou, Mark G Manfredi, Michelle Zhang. Treatment of IDO1 and TDO2 positive tumors with a kynurenine-degrading enzyme: A highly differentiated approach from IDO1 inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B008.

Parkinson's disease: Autoimmunity and neuroinflammation

Parkinson's disease is a neurodegenerative disease that causes the death of dopaminergic neurons in the substantia nigra. The resulting dopamine deficiency in the basal ganglia leads to a movement disorder that is characterized by classical parkinsonian motor symptoms. Parkinson's disease is recognized as the most common neurodegenerative disorder after Alzheimer's disease. PD ethiopathogenesis remains to be elucidated and has been connected to genetic, environmental and immunologic conditions. The past decade has provided evidence for a significant role of the immune system in PD pathogenesis, either through inflammation or an autoimmune response. Several autoantibodies directed at antigens associated with PD pathogenesis have been identified in PD patients. This immune activation may be the cause of, rather than a response to, the observed neuronal loss. Parkinsonian motor symptoms include bradykinesia, muscular rigidity and resting tremor. The non-motor features include olfactory dysfunction, cognitive impairment, psychiatric symptoms and autonomic dysfunction. Microscopically, the specific degeneration of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies, which are brain deposits containing a substantial amount of α-synuclein, have been recognized. The progression of Parkinson's disease is characterized by a worsening of motor features; however, as the disease progresses, there is an emergence of complications related to long-term symptomatic treatment. The available therapies for Parkinson's disease only treat the symptoms of the disease. A major goal of Parkinson's disease research is the development of disease-modifying drugs that slow or stop the neurodegenerative process. Drugs that enhance the intracerebral dopamine concentrations or stimulate dopamine receptors remain the mainstay treatment for motor symptoms. Immunomodulatory therapeutic strategies aiming to attenuate PD neurodegeneration have become an attractive option and warrant further investigation.

High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell-driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA-bound peptides has typically required billions of cells, still resulting in relatively few high-confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid-eluted HLA peptides after purification with the pan-reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high-confidence peptides could be identified, including melanoma-associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology-based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.

Recalcitrant Pseudotumoral Anogenital Herpes Simplex Virus Type 2 in HIV-Infected Patients: Evidence for Predominant B-Lymphoplasmocytic Infiltration and Immunomodulators as Effective Therapeutic Strategy

In patients with human immunodeficiency virus (HIV) infection, genital herpetic lesions may be extensive and tend to persist for longer periods; in addition, atypical hypertrophic, ulcerative, or pseudotumor forms have been reported, frequently showing resistance to acyclovir (ACV) treatment. Between 2003 and 2011, 10 HIV-1-infected patients presenting with chronic pseudotumoral anogenital herpes simplex type 2 (HSV-2) infections were studied. All patients developed chronic, hypertrophic HSV-2 anogenital lesions with multilesional presentation in 7 cases and involvement of 2 anatomical sites in 6 of them. At the time of diagnosis, the median CD3(+)CD4(+) absolute blood count was 480.5 cells/µL (range, 165-632 cells/µL), whereas the plasma HIV load was undetectable in all cases. Histopathologic analysis of lesion biopsies showed a moderately dense dermal polytypic plasma cell infiltrate. Detection of HSV-2 by culture and/or polymerase chain reaction was positive for all patients, with evidence for ACV-resistant strains in 6 of 8 cases. In addition, viral resistance to ACV was found only in HSV-2 isolated from ulcerative lesions, whereas purely pseudotumoral ones harbored sensitive strains. Durable control was observed with HSV DNA polymerase inhibitors in only 2 cases, and the immunomodulators imiquimod and thalidomide allowed 5 patients to reach sustained complete response. HSV-2-related pseudolymphoma in HIV-infected patients is characterized by a predominant polyclonal lymphoplasmacytic infiltration, and is frequently refractory to antiherpetic drugs. Immunomodulatory therapeutic strategies using thalidomide showed consistent efficacy, and should be considered early during the course of disease.

Rethinking the multiple roles of B cells in organ transplantation

To discuss the B-cell diverse functions in organ transplantation, highlighting the emerging debate on the role of regulatory B cells (Bregs). We underscore the need to re-examine and integrate data on B-cell functional activities, aiming to discriminate their regulatory (REG) and inflammatory (INFLAMMA) functions and to translate this knowledge for the development of novel immunomodulatory therapeutic strategies and to rethink the current ones. Data from both experimental models and clinical trials point that B cells of various phenotypes have immunoregulatory activity and play an important role in controlling graft inflammation. Data on the state of operational tolerance, in kidney transplantation, suggest the relevance of preserving a healthy B-cell compartment - in numbers and in the Breg capacity to activate the CD40/STAT3 signalling pathway - for achieving and maintaining homeostasis. Moreover, autoantibodies also comprise transplant immunobiology and it seems that not all alloantibodies are deleterious. The role of B cells, in organ transplantation, can no longer be taken as mere generators of plasma cells, which produce alloantibodies deleterious to the graft. B cells also seem to integrate a complex immunoregulatory network in organ transplantation, with Bregs of various phenotypes and possibly also antibodies. The functional discrimination of REG/INFLAMMA B-cell roles needs to be considered in the clinical setting.

Abstract 201: Changes In Inflammatory Response To Ischemic Stroke Across The Life Span

Background/Purpose- Inflammation plays a critical role in the response to stroke, and post-ischemic inflammatory responses strongly contribute to the extent of ischemic brain injury. Although much is known about the post stroke inflammatory cascade in brain; the contribution of age to post ischemic inflammation has been understudied. Aging is a non-modifiable risk factor for stroke and is a critical determinant to stroke outcome. Since 75-89% of strokes occur in the elderly, it is important to characterize the differential effects of aging on the inflammatory response to stroke before translation of immunomodulatory therapeutic strategies into the clinic. Therefore, the objective of this study was to study the impact of aging on post s troke inflammatory milieu of the brain . Methods- Young(6 months), aging(15 months) and aged(22 months) male and female mice were subjected to 60 minutes of MCAO (n=5-6/ group). The ischemic hemisphere was subjected to mechanical and enzymatic digestion and then brain mononuclear cells were harvested at the 30%-60% interphase of percoll gradient. Cells were stained with fluorophore conjugated antibodies for CD45,CD3,CD11b and Gr1 and counted on LSRII cytometer(BD Biosciences). Results- Blood derived leukocytes(CD45high) increased in the brain in stroke(7133+−4514) vs. sham(1081+−279.9). We found a significant main effect of age, F(2,38)=5,p<0.05 and stroke, F(2,38)=19.5,p<0.01 in the percentage of T cells(CD45highCD3+). There was also a significant age by stroke interaction in the percentage of T cells, F(2,38)=0.58,p<0.05. In the CD45intermediateCD11b+ microglia population, we saw a significant main effect of stroke, F(1,38)=18.6,p<0.01 (sham 19199.2+−3439 vs. stroke 38365.4+−1111 ). Similarly, there was a significant main effect of stroke on CD45intermediate/CD11b+/Gr1+ microglia numbers, F(1,38)=12.6,p<0.01 . Conclusions- T cells, microglia and Gr1+ microglia numbers increase in the brain after an ischemic stroke. However, only the CD45hiCD3+T cells significantly increased in the aging brain after an ischemic insult in the two sexes. Microglia and Gr1+microglia did increase after stroke in both sexes, but there was no significant effect of age. Our study suggests that aging creates a differential inflammatory milieu in the brain after an ischemic event and that elderly mount a profound T cell response to stroke. Since most of our stroke patients are old, bench to clinic translation of stroke therapies needs to account for this heterogeneity in inflammatory response across different ages.

Immune Reconstitution Of Regulatory T-Cells Following Autologous Hematopoietic Stem Cell Transplantation

The regulatory function of CD4+CD25highCD127dim T-cells (Tregs) has been associated with the risk of disease relapse, with GVHD in the allogeneic HSCT setting, and with the immunomodulatory capacity of autologous HSCT for autoimmune disorders. Despite their potentially important role in HSCT, their patterns of immune reconstitution postransplant remain unknown. In this study, we have analyzed the kynetics of immune reconstitution of Tregs in 42 adult (median age 56, 28–74), 27 male and 15 female, recipients of an autologous peripheral blood HSCT following conditioning with Melphalan-200 (multiple myeloma, n = 31) or BEAM (NHL, n = 11) from May 2006 to October 2007. Peripheral blood samples were prospectively collected prior to and at various time points after HSCT. Tregs were defined by their immunophenotype (CD3+CD4+CD25highCD127dim) and are expressed as a percentage of total CD4+ T-cells. FoxP3 and CTLA4 mRNA expression was performed in duplicate with 500ng mRNA in a LightCycler with TaqMan probes, and is presented in arbitrary units as a normalized ratio to the level of expression of β2-microglobulin. Immune reconstitution of Tregs occurs unexpectedly early after HSCT. The percentage of Tregs triples the pretransplant baseline level at only two weeks postransplant (6.35% vs 2.17%; p<0.001; table 1). Tregs remain high at day +90 when lymphocyte count recovery starts taking place. The level of early Tregs expansion at day +14 showed a strong correlation with the baseline Tregs level prior to HSCT (r = 0.74; p = 0.029). Markers of Tregs regulatory function such as FoxP3 (10.32 vs 3.0; p = 0.003) or CTLA4 (13.21 vs 6.35; p = 0.082) outline a profile of mRNA expression that mirrors the early expansion of Tregs, providing additional evidence of a true functional immune reconstitution of Tregs early after SCT.Tabled 1Table 1Time PointsCD3+ Lymph (×10e9/L)Tregs (% of CD4+)FoxP3 (β2-ratio)CTLA4 (β2-ratio)Pre-HSCT1.46 ± 0.672.17 ± 0.963.00 ± 4.786.35 ± 6.64Day +141.04 ± 1.286.35 ± 1.4510.32 ± 9.5613.21 ± 13.65Day +902.07 ± 1.155.87 ± 2.132.42 ± 1.856.22 ± 4.49ANOVA0.0610.0010.0030.082values in table are mean±S.D. Open table in a new tab This is the first study to analyze the kynetics of immune recovery of Tregs after autologous HSCT, and it shows that it takes place as early as two weeks into the procedure. Although FoxP3 and CD25-expression can be induced in non-regulatory T-cells by early allorreactive phenomena after allogeneic SCT, this should not be a confounding factor or compromise the analysis in the autologous setting. Understanding the early kynetics of Tregs postransplant reconstitution offers potential targets for immunomodulatory therapeutic strategies at the early phase of Tregs expansion postransplant or even at their baseline level which correlates with their subsequent expansion. values in table are mean±S.D.

Neuroimmunology of dancing eye syndrome in children

Clinical, histopathological, and laboratory evidence suggest that dancing eye syndrome/opsoclonus-mycoclonus syndrome is an immune-mediated and probably autoimmune disorder. There is a clear, but not invariable, association with neuroblastoma or ganglioneuroma in children, and with a miscellany of neoplasms in adults. Direct support, i.e. the identification of antibodies against cell-surface antigens in neurons, is lacking but the nature of other evidence is such that the case for use of immunomodulatory therapeutic strategies is compelling. Because at least 85% of children affected by the condition are left with permanent cognitive deficits, notwithstanding a favourable initial neurological response to, say, steroid or adrenocorticotrophic hormone, therapeutic stakes are high. There is an urgent need for controlled therapeutic trials which, because of the comparative rarity of the condition in children, will require international collaboration to expedite studies.

Neuroimmunology of dancing eye syndrome in children

Clinical, histopathological, and laboratory evidence suggest that dancing eye syndrome/opsoclonus-mycoclonus syndrome is an immune-mediated and probably autoimmune disorder. There is a clear, but not invariable, association with neuroblastoma or ganglioneuroma in children, and with a miscellany of neoplasms in adults. Direct support, i.e. the identification of antibodies against cell-surface antigens in neurons, is lacking but the nature of other evidence is such that the case for use of immunomodulatory therapeutic strategies is compelling. Because at least 85% of children affected by the condition are left with permanent cognitive deficits, notwithstanding a favourable initial neurological response to, say, steroid or adrenocorticotrophic hormone, therapeutic stakes are high. There is an urgent need for controlled therapeutic trials which, because of the comparative rarity of the condition in children, will require international collaboration to expedite studies.

Decreased HLA-A expression in prostate cancer is associated with normal allele dosage in the majority of cases

A comparison has been made of the phenotypic expression of MHC class I antigens with the corresponding HLA-A genotype in 15 cases of benign prostatic hyperplasia (BPH) and 34 cases of primary locally invasive prostatic carcinoma. Expression of class 1 protein, detected by immunocytochemistry, was partially or completely lost in approximately 90% of the tumours examined. Comparative genomic analysis of the beta2 microglobulin (beta2m) gene and 15 individual HLA-A haplotypes using a polymerase chain reaction (PCR)-based method demonstrated abnormal gene dosage in the minority of cases: homozygous deletion of the beta2m locus was detected in one case and HLA-A allele in two cases (HLA-A1 and HLA-A2, respectively), representing approximately 8% of the population studied. This first comparative study of gene dosage and expression of class 1 protein reported for prostate cancer reveals that deletion is not the cause of the partial or complete loss seen in the majority of cases. This raises the possibility, in the future, for novel selective immunomodulatory therapeutic strategies which stimulate a clinically significant re-expression of class 1 protein and associated cytotoxic T-cell response.

Decreased HLA-A expression in prostate cancer is associated with normal allele dosage in the majority of cases.

A comparison has been made of the phenotypic expression of MHC class I antigens with the corresponding HLA-A genotype in 15 cases of benign prostatic hyperplasia (BPH) and 34 cases of primary locally invasive prostatic carcinoma. Expression of class 1 protein, detected by immunocytochemistry, was partially or completely lost in approximately 90% of the tumours examined. Comparative genomic analysis of the beta2 microglobulin (beta2m) gene and 15 individual HLA-A haplotypes using a polymerase chain reaction (PCR)-based method demonstrated abnormal gene dosage in the minority of cases: homozygous deletion of the beta2m locus was detected in one case and HLA-A allele in two cases (HLA-A1 and HLA-A2, respectively), representing approximately 8% of the population studied. This first comparative study of gene dosage and expression of class 1 protein reported for prostate cancer reveals that deletion is not the cause of the partial or complete loss seen in the majority of cases. This raises the possibility, in the future, for novel selective immunomodulatory therapeutic strategies which stimulate a clinically significant re-expression of class 1 protein and associated cytotoxic T-cell response.