Memory Cell Subsets Research Articles

Initiation of Antiretroviral Therapy Restores CD4+ T Memory Stem Cell Homeostasis in Simian Immunodeficiency Virus-Infected Macaques.

Treatment of human immunodeficiency virus (HIV) infection with antiretroviral therapy (ART) has significantly improved prognosis. Unfortunately, interruption of ART almost invariably results in viral rebound, attributed to a pool of long-lived, latently infected cells. Based on their longevity and proliferative potential, CD4(+) T memory stem cells (TSCM) have been proposed as an important site of HIV persistence. In a previous study, we found that in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM), CD4(+) TSCM are preserved in number but show (i) a decrease in the frequency of CCR5(+) cells, (ii) an expansion of the fraction of proliferating Ki-67(+) cells, and (iii) high levels of SIV DNA. To understand the impact of ART on both CD4(+) TSCM homeostasis and virus persistence, we conducted a longitudinal analysis of these cells in the blood and lymph nodes of 25 SIV-infected RM. We found that ART induced a significant restoration of CD4(+) CCR5(+) TSCM both in blood and in lymph nodes and a reduction in the fraction of proliferating CD4(+) Ki-67(+) TSCM in blood (but not lymph nodes). Importantly, we found that the level of SIV DNA in CD4(+) transitional memory (TTM) and effector memory (TEM) T cells declined ∼100-fold after ART in both blood and lymph nodes, while the level of SIV DNA in CD4(+) TSCM and central memory T cells (TCM-) did not significantly change. These data suggest that ART is effective at partially restoring CD4(+) TSCM homeostasis, and the observed stable level of virus in TSCM supports the hypothesis that these cells are a critical contributor to SIV persistence. Understanding the roles of various CD4(+) T cell memory subsets in immune homeostasis and HIV/SIV persistence during antiretroviral therapy (ART) is critical to effectively treat and cure HIV infection. T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into other memory T cell subsets, such as central and transitional memory T cells (TCM and TTM, respectively). CD4(+) TSCM are disrupted but not depleted during pathogenic SIV infection. We find that ART is partially effective at restoring CD4(+) TSCM homeostasis and that SIV DNA harbored within this subset contracts more slowly than virus harbored in shorter-lived subsets, such as TTM and effector memory (TEM). Because of their ability to persist long-term in an individual, understanding the dynamics of virally infected CD4(+) TSCM during suppressive ART is important for future therapeutic interventions aimed at modulating immune activation and purging the HIV reservoir.

Constitutive LcK activity drives sensitivity differences between CD8+ memory T cell subsets

Abstract CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential T cell receptor (TCR) signaling between memory subsets contributes to sensitivity differences is unclear. We show that constitutive activity of lymphocyte-specific protein tyrosine kinase (Lck) is greater in effector memory T cells (TEM) compared with central memory T cells (TCM), leading to enhanced activation signaling, including Zeta-chain-associated protein kinase 70 (Zap-70) phosphorylation and intracellular calcium influx, and resulting in increased cytotoxic effector function in TEM. We provide evidence that the differences in Lck activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk). Together, this work demonstrates a role for constitutive Lck activity in controlling antigen sensitivity, and suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM.

A pilot study showing associations between frequency of CD4+ memory cell subsets at diagnosis and duration of partial remission in type 1 diabetes

In some patients with type 1 diabetes the dose of insulin required to achieve euglycemia is substantially reduced soon after diagnosis. This partial remission is associated with β-cell function and good glucose control. The purpose of this study was to assess whether frequencies of CD4+ T cell subsets in children newly diagnosed with type 1 diabetes are associated with length of partial remission. We found that the frequency of CD4+ memory cells, activated Treg cells and CD25+ cells that express a high density of the IL-7 receptor, CD127 (CD127hi) are strongly associated with length of partial remission. Prediction of length of remission via Cox regression is significantly enhanced when CD25+ CD127hi cell frequency is combined with either Insulin Dependent Adjusted A1c (IDAA1c), or glycosylated hemoglobin (HbA1c), or C-peptide levels at diagnosis. CD25+ CD127hi cells do not express Foxp3, LAG-3 and CD49b, indicating that they are neither Treg nor Tr1 cells.

Ipilimumab reshapes T cell memory subsets in melanoma patients with clinical response

ABSTRACTPurpose: Therapy targeting CTLA-4 immune checkpoint provides increased survival in patients with advanced melanoma. However, immunotherapy is frequently associated with delayed and heterogeneous clinical responses and it is important to identify prognostic immunological correlates of clinical endpoints. Experimental design: 77 patients with stage III/IV melanoma were treated with ipilimumab alone every 3 weeks, during 9 weeks. Blood samples were collected at the baseline and before each dose for in depth immune monitoring. Results: The median follow-up was 28 mo with a median survival of 7 mo. Survival and clinical benefit were significantly improved when absolute lymphocyte count at the baseline was above 1 × 109/L. Notably, ipilimumab had a global effect on memory T cells, with an early increase of central and effector subsets in patients with disease control. By contrast, percentages of stem cell memory T cells (TSCM) gradually decreased despite stable absolute counts and sustained proliferation, suggesting a process of differentiation. Higher proportions of eomes+ and Ki-67+ T cells were observed, with enhanced skin homing potential and induction of cytotoxic markers. Conclusion: These results suggest that CTLA-4 blockade is able to reshape the memory subset with the potential involvement of Eomes and memory subsets including TSCM.

CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression.

In order to determine if the expression of the activation marker CD38 can correlate with HIV disease progression independently of cycling, we performed a cluster-based multivariate correlation analysis of total circulating CD4+ T cell counts and viral loads with frequencies of CD38 and Ki67 expression on CD4+ lymphocytes from patients with untreated HIV infection, stratified in maturation subpopulations, and subpopulation subsets defined by the expression of CXCR5, CXCR3, and CCR4. The frequencies of the activated phenotypes %CD38+ Ki67− and %CD38+ Ki67+ of the CXCR5− CXCR3− CCR4+ (“pre-Th2”) central memory (TCM) cell subset clustered together, comprising a significant negative correlate of total circulating CD4+ T cell counts and a positive correlate of viral load in multivariate analysis. Frequency of cycling-uncoupled CD38 expression in “pre-Th2” TCM cells was a negative correlate of total circulating CD4+ T cell counts in univariate analysis, which was not the case of their %CD38+ Ki67+. CXCR5+ CXCR3− CCR4− TCM cells were underrepresented in patients, and their absolute counts correlated negatively with their %CD38+ Ki67− but not with their % CD38+ Ki67+. Our results may imply that CD38 expression either reflects or participates in pathogenic mechanisms of HIV disease independently of cell cycling.

T Stem Cell Memory Dynamics during Acute Graft-Versus-Host Disease

T stem cell memory (Tscm) represents a subset of T cells whose phenotype sits between those conventionally thought of as naive and those designated central memory. Functionally, they exhibit the capacity for self-renewal and can generate other memory cell subsets. As such, Tscm cells replenish and support the memory cell pool during infections, and contribute to immune reconstitution in patients following hematopoietic cell transplantation (HCT). Their fate and biological role in Graft-Versus-Host Disease (GVHD) is not yet understood. To address this question, we determined the dynamics and tissue-specific homeostasis of Tscm following allogeneic HCT in the translational non-human primate model of GVHD.In this study, we tracked Tscm cells by flow cytometry at baseline, and longitudinally after MHC haploidentical HCT. Recipients were divided into 2 large groups based on clinical outcome: "Primary GVHD": Animals receiving subtherapeutic GVHD prophylaxis (either no prophylaxis, or monotherapy with either Rapamycin or CTLA4-Ig; neither of which significantly prevented GVHD); "Breakthrough GVHD": Animals receiving combined Rapamycin/CTLA4-Ig or Tacrolimus/Metotrexate (both of which prevented primary GVHD but were eventually associated with breakthrough disease). We have previously shown that reconstituting T cells in animals with primary versus breakthrough GVHD display highly significant differences in their immunophenotype and transcriptome profiles.We have now discovered that primary GVHD is associated with a robust expansion of Tscm cells that peaks at day 5 post HCT (Fig 1A and B) and is associated with a decrease in the number of naive T lymphocytes. The Tscm compartment subsequently exhibited a contraction that accompanied an increase in the number of differentiated memory/effector T cell subsets (not shown). In contrast, animals that initially controlled GVHD maintained a delayed and prolonged expansion of Tscm cells (Fig 1A and B) that was associated with the significant preservation of naive T lymphocyte counts. Importantly, upon development of breakthrough GVHD, recipients demonstrated a transition of Tscm cells toward effector cells (Fig 1C). At the time of necropsy, animals with both primary and breakthrough GVHD displayed similar Tscm tissue distribution profiles, with these cells residing in the peripheral blood > lymph nodes > spleen > bone marrow > colon ~ liver ~ lungs.These data suggest that during GVHD, naive T cells transit to memory/effector lymphocytes via Tscm, and that one of the key functions of GVHD immunoprophylaxis may be to limit the rate at which naive T cells enter into the Tscm and subsequent effector T cell pool. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differentiated Human CD8+ Memory T Cells Can Revert to a Naive-like Phenotype with Memory Stem Cell Functions.

The mechanisms leading to the establishment of the memory T-cell pool in humans remain to be fully elucidated. Murine models of memory T-cell generation propose an irreversible differentiation process from naïve to memory and effector cells. T-memory stem cells (TSCM) have been recently described in humans as constituting a key precursor subset of memory cells along this pathway.To study the generation of memory T-cells, primary naive T-cells (TN) derived from human cord blood (CB) were exposed to different activating stimuli, and then incubated with various cytokine combinations, and monitored throughout for phenotypic and functional changes. Between 3 to 9 days after antigen activation, the proportion of CD45RA+/CCR7+ CD8+ TN cells dropped from 86.64±5.84 (mean±1SD, n=50) to less than 20% (10.36±6.23). Specific cytokines mixtures were then added to the cultures and within 13 to 28 days after initial activation CD8+ cells re-expressed CD45RA, reverting back to a TN-like phenotype, characterized by the co-expression of CCR7, CD62L, CD27 and CD45RA, and loss of CD45RO. These constituted 71%±11.68 (1SD n=50) of the CD8+ T-cell population. The kinetics of 3 representative cultures are shown in Figure 1.Recently differentiated central memory (TCM) and effector memory (TEM) CD8+ T-cells could be both induced to revert to a naïve-like phenotype (TNrev). Functional analysis showed that compared to TN, CD8+ TNrev displayed higher proliferative capacity upon antigenic stimulation, differentiated more quickly into progeny memory cells and acquired cytolytic effector function. CD8+ TNrev also had the ability to undergo several cycles of activation and differentiation whilst retaining the ability to revert back to TNrev.CD8+ TNrev had an extended phenotype almost identical to TSCM, including the expression of CD95, and differed from TN in the expression of CXCR3 and Integrin b7. However, the expression of the latter markers and of CD95, became virtually undetectable following prolonged steady state culture and/or after modifying the cytokine milieu.Here we describe for the first time the capacity of CD8+ TCM and TEM cells that have recently differentiated from primary TN to revert back to a naïve-like phenotype. CD8+ TNrev are functionally and phenotypically indistinguishable from TSCM, but with further changes to the cytokine milieu, they reacquire, with time, a phenotype resembling TN. TN Reversion may represent a way for the immune system to maintain a pool of antigen-experienced CD8+ T-cells with naïve characteristics, with implications for current models of memory T-cell generation. We demonstrate how understanding the T-cell differentiation process made possible the large scale in vitro generation of TSCM-like cells that can be used for adoptive immunotherapy strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P81: Recovery of distinct T cell subsets under severe lymphopenic conditions in hemoblastosis patients

Numerous studies have shown that high-dose chemotherapy and autologous hematopoietic stem cell transplantation (AHSCT) led to a profound and long-lasting state of immunodeficiency characterized by persisting low levels of T cells in hemoblastosis patients. Well-timed T-cell reconstitution is crucial for early restoration of anti-infectious and anti- tumor immune response. Lymphocyte recovery is mediated through the two main mechanisms – a homeostatic proliferation of T cells and generation of new naive T cells via thymopoiesis. It is known, that homeostatic proliferation is important for the restoration of T cell count in immune competent host during the 1st year following AHSCT. Thymus begins to fill up T cell repertoire approximately from the 6th month following AHSCT. We have investigated dynamics of CD4+FOXP3+ Treg recovery following AHSCT and possible relationship between Tregs and clinical outcomes since the suppressive activity of Tregs under lymphopenic conditions may influence on peripheral expansion of T cells. Thymic activity following AHSCT has been evaluated by measuring amounts of CD4+ CD45RA+CD31+ naive T cells, i.e. “recent thymic emigrants” (RTEs).109 patients with non-Hodgkin’s lymphomas, Hodgkin’s lymphoma and multiple myeloma underwent AHSCT in 2009–2014. The content of circulating CD4+FOXP3+ Tregs and CD4+CD45RA+CD31+ T cells was evaluated using flow cytometry before AHSCT, at the day of engraftment, and following 6 and 12 months. Pre-transplant count of CD4+FOXP3+ Tregs was significantly higher compared to healthy controls (5.4 ± 2.9 vs 3.8 ± 1.9%; pU = 0.011; here and below data presented as Mean ± SD). Percentage of Tregs restored rapidly and reached initially high level at the time of engraftment, and then subsequently decreased within a year until it lowered to healthy donors‘ values. CD4+FOXP3+ Tregs at the time of engraftment were increased in patients with relapse or progression of disease within 6 and 12 months following AHSCT compared to non-relapsed patients (11.0 ± 6.1 vs 6.2 ± 3.0%; pU = 0.016, and 10.1 ± 5.2 vs 6.1 ± 3.8%; pU = 0.008). Pre-transplant count of CD4+CD45RA+CD31+ T cells was significantly lower compared to healthy controls (17.1 ± 11.4 vs 30.3 ± 11.2%, pU = 0.0005) and did not reach donors‘ values following 12 month (23.1 ± 13.5%, pU = 0.032). Relapsed patients had the same quantity of RTEs as the patients with remission within the 1st year following AHSCT. There was no any significant association between RTEs and Tregs counts. Surprisingly, we have found high levels of circulating CD4+CD45RA- T cells co-expressing CD31 molecule in patients before AHSCT, since this molecule is infrequent on memory subsets in healthy controls (20.7 ± 12.0 vs 8.2 ± 2.1%,pU Our data of Tregs reconstitution may confirm the earlier assumption that the presence of Tregs during the period of immune recovery preserves optimal T cell receptors diversity. However, the excess of these cells leads to the inhibition of proliferative activity and immune response and is associated with early relapse. Conversely, relatively slow recovery of RTEs determines theirlack of influence on survival within the 1st post-transplant year. The biological role and the way of appearance of CD31 molecule on T cell memory subset (CD4+CD45RA- and/or CD4+CD45RO+) still remain unclear. Further studies are required to enlighten the role of CD31+ memory T cells on lymphoproliferative disorders pathogenesis.

IMCT-16SERIAL EVALUATION OF LYMPHOCYTE SUBSETS IN PATIENTS WITH NEWLY DIAGNOSED HIGH GRADE GLIOMAS TREATED WITH STANDARD RADIATION AND TEMOZOLOMIDE

BACKGROUND: The immune system plays a significant role in cancer prevention and outcome. In high grade gliomas (HGG), severe lymphopenia is associated with shortened survival due to tumor progression. Here we report the first comprehensive study of serial changes in lymphocyte subsets in HGG following standard radiation (RT) and temozolomide (TMZ). METHODS: Adults (KPS > 60, HIV negative) with newly diagnosed HGG scheduled to receive standard RT and TMZ followed by adjuvant TMZ were eligible. Patients were enrolled at the Johns Hopkins Hospital and at Washington University. Blood was collected before beginning therapy and at weeks 6, 10, 18, and 26, and 3 months after completing adjuvant TMZ. Analysis of lymphocyte subsets was performed at NIH/NCI on a Beckman Coulter Gallios flow cytometer; T cell naive, memory and effector subsets were assessed within CD8, Treg and nonTreg CD4 T cells. Data was analyzed with FlowJo. RESULTS: All 20 patients have enrolled. Their median age is 53 years; baseline dexamethasone dose is 0.5 mg/day, and follow-up time is 9.5 months. 15% had lymphocyte counts < 1000 cells/mm3 before starting therapy. The most significant T-cell reductions were in naive CD4+ (60% of baseline at 26 weeks, p = 0.008) and CD4+ (49% of baseline at 26 weeks, p = 0.007) cells. CD8, Tregs, CD3, CD8 effector, and NK cells were less affected. B cells were also markedly reduced. The CD4 + /CD8+ ratio fell after RT/TMZ and remained low. CONCLUSIONS: This is the first comprehensive study to report serial trends in lymphocyte subsets following RT + TMZ. CD4+ and naive CD4+ T cells appear most affected and this lymphopenia is persistent after 9 months of follow up. This data provides insight into potential therapeutic approaches (i.e. IL-7) and the optimal timing for immunologic interventions in this patient population.

Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

BackgroundIn mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV).MethodsTo assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds.ResultsAt the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (103.46 EID50 /0.1 ml) and drastically decreased at day 5 pi (100.9 EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 105.35 EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain.ConclusionsIncreased expression of chicken IL-2 during virulent NDV replication in naïve chickens decreased viral titers in blood, spleens, oral and cloacal secretions on day 4–5 post infection. This is consistent with the previously described role of IL-2 in enhancing the clearance of viruses in mammals, such as human respiratory syncytial virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0353-x) contains supplementary material, which is available to authorized users.

Early pediatric atopic dermatitis shows only a cutaneous lymphocyte antigen (CLA)+ TH2/TH1 cell imbalance, whereas adults acquire CLA+ TH22/TC22 cell subsets

Identifying differences and similarities between cutaneous lymphocyte antigen (CLA)(+) polarized T-cell subsets in children versus adults with atopic dermatitis (AD) is critical for directing new treatments toward children. We sought to compare activation markers and frequencies of skin-homing (CLA(+)) versus systemic (CLA(-)) "polar" CD4 and CD8 T-cell subsets in patients with early pediatric AD, adults with AD, and control subjects. Flow cytometry was used to measure CD69/inducible costimulator/HLA-DR frequency in memory cell subsets, as well as IFN-γ, IL-13, IL-9, IL-17, and IL-22 cytokines, defining TH1/cytotoxic T (TC) 1, TH2/TC2, TH9/TC9, TH17/TC17, and TH22/TC22 populations in CD4 and CD8 cells, respectively. We compared peripheral blood from 19 children less than 5 years old and 42 adults with well-characterized moderate-to-severe AD, as well as age-matched control subjects (17 children and 25 adults). Selective inducible costimulator activation (P < .001) was seen in children. CLA(+) TH2 T cells were markedly expanded in both children and adults with AD compared with those in control subjects, but decreases in CLA(+) TH1 T-cell numbers were greater in children with AD (17% vs 7.4%, P = .007). Unlike in adults, no imbalances were detected in CLA(-) T cells from pediatric patients with AD nor were there altered frequencies of TH22 T cells within the CLA(+) or CLA(-) compartments. Adults with AD had increased frequencies of IL-22-producing CD4 and CD8 T cells within the skin-homing population, compared with controls (9.5% vs 4.5% and 8.6% vs 2.4%, respectively; P < .001), as well as increased HLA-DR activation (P < .01). These data suggest that TH2 activation within skin-homing T cells might drive AD in children and that reduced counterregulation by TH1 T cells might contribute to excess TH2 activation. TH22 "spreading" of AD is not seen in young children and might be influenced by immune development, disease chronicity, or recurrent skin infections.

Melatonin controls experimental autoimmune encephalomyelitis by altering the T effector/regulatory balance.

Experimental autoimmune encephalomyelitis (EAE), the experimental model for multiple sclerosis (MS), is triggered by myelin-specific Th1 and Th17 cells. The immunomodulatory activities of melatonin have been shown to be beneficial under several conditions in which the immune system is exacerbated. Here, we sought to elucidate the basis of the melatonin protective effect on EAE by characterizing the T effector/regulatory responses, particularly those of the memory cell subsets. Melatonin was tested for its effect on Th1, Th17 and T regulatory (Treg) cells in the lymph nodes and CNS of immunodominant peptide of myelin oligodendrocyte glycoprotein (pMOG)-immunized and EAE mice, respectively. The capacity of melatonin to ameliorate EAE as well as modifying both T cell response and effector/regulatory balance was surveyed. T cell memory subsets and CD44, a key activation marker involved in the EAE pathogenesis, were also examined. Melatonin protected from EAE by decreasing peripheral and central Th1/Th17 responses and enhancing both the Treg frequency and IL-10 synthesis in the CNS. Melatonin reduced the T effector memory population and its pro-inflammatory response and regulated CD44 expression, which was decreased in T effector cells and increased in Tregs. The alterations in the T cell subpopulations were associated with a reduced mononuclear infiltration (CD4 and CD11b cells) of the melatonin-treated mice CNS. For the first time, we report that melatonin protects against EAE by controlling peripheral and central T effector/regulatory responses, effects that might be partially mediated by CD44. This immunomodulatory effect on EAE suggests that melatonin may represent an effective treatment option for MS.

Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

Immunomodulatory effects of OX40 agonists in a defined antigen challenge in cynomolgus macaques.

3086 Background: Immunomodulatory antibodies targeting specific T cell costimulatory pathways have shown great potential as cancer therapeutics. OX40 agonists can enhance T cell proliferation and survival and cause tumor regressions in mouse models. While initial evaluations of pharmacodynamic activity of these compounds in non-human primate models have relied on non-specific T cell proliferation readouts (Ki67), additional sensitive biomarkers of pharmacodynamic activity would add value in evaluating optimal dosing and timing of candidate therapeutics. Methods: In this study, we compared the in vivo activity in cynomolgus macaques of intravenously infused OX40 agonist MEDI6469 (9B12, a murine IgG1 antibody) and MEDI6383, a human OX40 ligand fusion protein consisting of a dimer of OX40L trimers held together by Fcγ domains that also agonizes the OX40 receptor. Antigen-specific responses were evaluated to respiratory syncytial virus soluble F (sF) protein, which was administered intramuscularly on the same day as OX40 agonist treatment. Ki67 proliferation was additionally measured in whole blood. Results: Enhancement of F-specific T cell responses, serum anti-F IgG responses and functional antibodies was observed in animals that received the OX40 agonists. In addition, OX40 agonism resulted in a temporal enhancement of cellular proliferation (Ki67 staining) in CD4 and CD8 T cell memory subsets, as well as in NK cells and B cells. The strongest antigen-specific and Ki67 responses were observed in response to MEDI6383, with the exception of responses observed for NK cell proliferation which were similar between MEDI6469 and MEDI6383. Conclusions: Our study indicates that antigen-specific cellular and humoral responses may be useful biomarkers of OX40 agonist activity. Additionally, our study confirms that Ki67 readouts are informative in cynomolgus macaques, which was also demonstrated in separate studies using rhesus macaques.

Ipilimumab reshapes T cell memory subsets in melanoma patients with clinical response (TUM2P.1006)

Abstract CTLA-4 is the main negative regulator of T cell mediated antitumor immune responses. Treatment with Ipilimumab, a humanized anti-CTLA-4 mAb, was shown to provide increased survival in patients with melanoma. However, only a subset of patients benefit and it is therefore important to identify prognostic immunological correlates of clinical endpoints. 77 patients with stage III or IV melanoma were treated with ipilimumab every 3 weeks, during 9 weeks. Blood samples were collected at week 0, 3, 6, 9, 12. The clinical response was assessed at week 16. Lymphocyte phenotyping was performed on fresh blood samples. The median follow-up was 28 months with a median survival of 7 months, which was significantly improved when the absolute lymphocyte count (ALC) at the baseline was above 1000/uL. In addition ALC was predictive of disease control (DC) at week 16. Ipilimumab had a global effect on central, transitional and effector memory T cell subsets, with a marked increase of these populations between week 0 and 3 in patients with DC. In addition, higher proportions of Eomes+, Ki67+ and Granzyme B+ T cells were observed. These changes were associated with a significant decrease in the proportion of stem cell memory T cells (TSCM). These results suggest that Ipilimumab is able to reshape the memory subset at early time points and provide new insights into the mechanisms of memory T cell subset expansion with the potential involvement of TSCM.

Figuring Fact from Fiction: Unbiased Polling of Memory T Cells

Immunization generates several memory T cell subsets that differ in their migratory properties, anatomic distribution, and, hence, accessibility to investigation. In this issue, Steinert et al. demonstrate that what was believed to be a minor memory cell subset in peripheral tissues has been dramatically underestimated. Thus, current models of protective immunity require revision.

Virus-specific immune memory at peripheral sites of herpes simplex virus type 2 (HSV-2) infection in guinea pigs.

Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.

Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. DisclosuresNo relevant conflicts of interest to declare.

Absence of the memory response to encephalitogen following intergender adoptively transferred experimental autoimmune encephalomyelitis.

Animals that have recovered from adoptively transferred EAE develop clinical disease signs 2-3days earlier than controls when challenged with encephalitogen. This may be due to the reactivation of donor-derived memory cells or stimulation of recipient-derived memory cells primed during the adoptive disease episode. In order to determine the origin of the memory cell subset, we used a donor-recipient model where donor cells are rejected in recipients following a course of adoptively transferred disease. Our results suggest the early onset of disease seen in recipients recovered from adoptively transferred disease and challenged with encephalitogen is due to the sustained presence of donor-derived memory cells.

Abstract 2883: Improved generation of central memory CD8+ T cells with CD40L expressing recombinant vaccinia virus

Abstract Immunological memory is the ability of the immune system, upon re-encounter with the same antigen, to respond with greater speed and intensity and constitutes the basis for vaccination. Regarding CD8+ T-cells, the memory compartment has been divided in different subsets of memory cells: Central Memory CD8+ T cells (CD45RO+CD62L+; Tcm) and Effector Memory CD8+ T cells (CD45RO+CD62L- ; Tem). In cancer immunotherapy, studies performed on animal models showed that Tcm tumor-reactive CD8+ T cells, as compared to effector memory T cells, confer superior antitumor immunity leading to better outcome.. In the last decades has been clarified that CD4+T cells help, during CD8+ T cells memory differentiation, is mainly mediated by the CD40L expression on their surface upon activation and consecutive triggering of CD40 expressed by antigen presenting cells and activated CD8+ T cells. Recombinant vaccinia virus (rVV) is one of the most immunogenic viral vectors enabling specific targeting and modulation of anti-tumor immune responses. We previously demonstrated that infection of antigen presenting cells (APCs) , with a non-replicating rVV encoding human CD40L (rVV-CD40L) was leading to an efficient licensing of APC, as demonstrated by increased expression of surface ligand/receptors (MHC I/II, CD80 and CD86),Th1 and homeostatic cytokines (IL-12, TNFa, IL-7 and IL-15). Using sorted naive CD8+ Tcells (CD45RA+CD62L+) primed with infected allogeneic CD14+ we observed that activated cells had an increased central memory phenotype (CD45RO+ CD62L+) with rVV40L infection as compared to culture primed with wild type virus infected CD14+ cells or treated with soluble 40 Ligand recombinant protein (s40L). The majority of Tcm express even the a-chain of IL-7 receptor (IL-7Ra) which has been reported to be express on “Bona Fide” memory cells and is associated with an increased cells survival. In an autologous system targeting a tumor antigen response, we demonstrated how CD14+rVV40L, as compared controls or soluble 40L stimulation, strongly promote the expansion of MelanA/Mart-1 specific CD8+ characterized with a Tcm phenotype and expression of IL-7Ra. Functional characterization of these tumor specific CD8+ demonstrated higher levels of IFN-g and IL-2. Studies performed in murine models have shown that, upon activation, CD8+ T cells can also express the CD40 receptor and therefore become directly sensitive to CD40L activation. However,in our hands, induction and characterization of CD40 receptor, on human CD8+ T cells remained negative. Overall, these data are confirming the potent anti-tumor capacities of recombinant vaccinia viruses expressing CD40L notably via CD40 driven APC licensing which promotes an effective generation of antigen specific CD8+ T cells with a memory phenotype. These in-vitro observations validate the strong clinical potential of our recombinant vaccinia virus constructs co-expressing CD40L for cancer immunotherapies. Citation Format: Emanuele Trella, Evangelos Panoupolos, Swantje Heidtmann, Nermin Raafat, Giulio Cesare Spagnoli, Paul Zajac. Improved generation of central memory CD8+ T cells with CD40L expressing recombinant vaccinia virus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2883. doi:10.1158/1538-7445.AM2014-2883