Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells’ recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells’ predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses

Apoptotic programs are determined during lineage commitment of CD4(+) T effectors: Selective regulation of T effector-memory apoptosis by inducible nitric oxide synthase

Lineage-committed T effectors generated in response to Ag during the inflammatory phase are destined to die during termination of the immune response. We present evidence to suggest that molecular signatures of lineage commitment are reflected in apoptotic cascades activated in CD4(+) T effectors. Exemplifying this, ablation of inducible NO synthase (iNOS) protected effector-memory T (TEM) cells, but not T-Naive or central-memory T cells, activated in vitro, from apoptosis triggered by cytokine deprivation. Furthermore, attrition of T effectors generated in the secondary, but not the primary, response to Ag was substantially reduced in mice, which received iNOS inhibitors. Distinct patterns of iNOS expression were revealed in wild-type TEM effectors undergoing apoptosis, and ablation of iNOS protein in primary and TEM wild-type effectors confirmed observations made in iNOS(-/-) cells. Describing molecular correlates of this dependence, mitochondrial damage, activation of the protein Bax, and release from mitochondria of the apoptosis-inducing factor were selectively abrogated in iNOS(-/-) TEM effectors. Suggesting that iNOS dependence was linked to the functional identity of T cell subsets, both iNOS induction and apoptosis were compromised in IFN-gamma(-/-) TEM effectors, which mirrored the response patterns of iNOS(-/-) TEM. Collectively, these observations suggest that programs regulating deletion and differentiation are closely integrated and likely encoded during lineage commitment of T effectors. The Journal of Immunology, 2013, 190: 97-105

Efficient expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells\u27 recognition of immunodominant but variable regions of the virus, which facilitates viral escape mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells an cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing -expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells\u27 predominant CD8 effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses

Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

<p>Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab</p

T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

<div><p>CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4⁺ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in <i>in vitro</i> experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15–35% of CD4⁺ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab’)2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.</p></div

Mouse anti CD6 domain 1 antibody (m CD6D1 mAb) ameliorates EAE in mice.

<p>EAE disease was induced in C57BL/6 normal female mice using MOG_35-55 emulsified in CFA and pertussis toxin. Randomized mice were injected with 60 μg in 100 μl volume of either control m Iso Ab (shown in circles) or m CD6D1 mAb (shown as squares) between day 15 and day 27 (arrows indicate days of dosing). 7 animals were used in each group. (A) Clinical scores are represented as Mean ± SEM. Fig is representative of six independent studies (*p≤0.05). The statistical significance was determined using 2 way ANOVA followed by Sidak’s multiple comparison analysis. (B-C) Splenocytes of these mice were stimulated with plate bound anti-mouse CD3 mAb. (B) After 96 h, proliferation of these splenocytes was estimated using Alamar Blue reagent <b>**</b>p≤0.01. Statistical significance was determined using non-parametric unpaired t-test followed by Mann-Whitney test (C) Supernatants were collected after 72 h and 6 cytokines were analyzed using Cytokine bead array (CBA) kit. All cytokines except IL-12 showed significant difference using unpaired t-test (*p≤0.05). (D) m Iso Ab and m CD6D1 mAb treated EAE-induced mice were sacrificed on day 32 post immunization and transverse lumbar spinal cord sections were prepared (middle and lower panel). Naive mouse without EAE induction was used as additional control (top panel). Left panel showed light microscopy of sections stained with hematoxylin and eosin (H&E), while the right panel showed luxol fast blue staining for demyelination of neurons. Arrows in the middle panel show infiltrating lymphocytes among neutrophils migrating into the spinal cord in mice with disease and treated with m Iso Ab. Representative Fig shown.</p

Differential expression of genes in presence of Itolizumab at different phases relative to control (gene expression custom 15K).

<p>Differential expression of genes in presence of Itolizumab at different phases relative to control (gene expression custom 15K).</p

Differential expression profiles over time for Itolizumab.

<p>Scatter plots for all the 15K genes in the array for control group versus Itolizumab in early, middle (Average expression for days 6 and 8) or late phases (Average expression for days 10 and 14) are shown. Regression line represents R-Squared value between Itolizumab and Iso Ab groups. The colored region is > log₂ 1 over unstimulated in the control group. Green region depicts the genes down regulated by at least 20% in the Itolizumab arm over the Iso Ab group.</p