T-cell Compartment Research Articles

Expansion of the CD4+ effector T-cell repertoire characterizes peanut-allergic patients with heightened clinical sensitivity

Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. We studied the T-cell receptor β-chain (TCRβ) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRβ sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. TCRβ analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.

Effect of Seasonal Malaria Chemoprevention on Immune Markers of Exhaustion and Regulation.

Seasonal malaria chemoprevention (SMC) is a novel strategy to reduce malaria infections in children. Infection with Plasmodium falciparum results in immune dysfunction characterized by elevated expression of markers associated with exhaustion, such as PD1 and LAG3, and regulatory CD4+FOXP3+ T cells. In the current study, the impact of seasonal malaria chemoprevention on malaria-induced immune dysfunction, as measured by markers associated with exhaustion and regulatory T cells, was explored by flow cytometry. Children that received seasonal malaria chemoprevention had fewer malaria episodes and showed significantly lower fold changes in CD4+PD1+ and CD4+PD1+LAG3+ compared to those that did not receive SMC. Seasonal malaria chemoprevention had no observable effect on fold changes in CD8 T cells expressing PD1 or CD160. However, children receiving SMC showed greater increases in CD4+FOXP3+ T regulatory cells compared to children not receiving SMC. These results provide important insights into the dynamics of malaria-induced changes in the CD4 T-cell compartment of the immune system and suggest that the reduction of infections due to seasonal malaria chemoprevention may also prevent immune dysfunction. NCT02504918.

Recovery of central memory and naive peripheral T cells in Follicular Lymphoma patients receiving rituximab-chemotherapy based regimen

Preclinical models and clinical studies have shown that anti-CD20-based treatment has multifaceted consequences on T-cell immunity. We have performed a prospective study of peripheral T-cell compartment in FL patients, all exhibiting high tumor burden and receiving rituximab-chemotherapy-based regimen (R-CHOP). Before treatment, FL patients harbor low amounts of peripheral naive T cells, but high levels of CD4+ TEM, CD4+ Treg and CD8+ TEMRA subsets and significant amounts of CD38+ HLA-DR+ activated T cells. A portion of these activated/differentiated T cells also expressed PD-1 and/or TIGIT immune checkpoints. Hierarchical clustering of phenotyping data revealed that 5/8 patients with only a partial response to R-CHOP induction therapy or with disease progression segregate into a group exhibiting a highly activated/differentiated T cell profile and a markedly low proportion of naive T cells before treatment. Rituximab-based therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype and of CD8+ T cells to a naive phenotype. In parallel, a decrease in the number of peripheral T cells expressing both PD-1 and TIGIT was detected. These observations suggest that the standard rituximab-based therapy partially reverts the profound alterations observed in T-cell subsets in FL patients, and that blood T-cell phenotyping could provide a better understanding of the mechanisms of rituximab-based treatment.

CD8+CD28- T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection.

During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28- T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28- T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28- T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28- T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28- T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28- T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28- T-cell killing. Both CD28+ and CD28- T cells in CHB expressedCXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28- T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28- T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28- T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28- T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

Tuberculosis Antigen-Specific T-Cell Responses During the First 6 Months of Antiretroviral Treatment.

The reconstitution of Mycobacterium tuberculosis antigen-specific CD4 T cells in a cohort of HIV-infected persons starting antiretroviral treatment (ART) in a high tuberculosis endemic area is described. Restoration of the antigen-specific CD4 T-cell subsets mirrored the overall CD4 T-cell compartment. Activation (assessed by HLA-DR expression) decreased during ART but remained elevated compared to HIV-uninfected persons. Despite known M. tuberculosis sensitization determined by interferon-γ release assay, 12/23 participants had no M. tuberculosis-specific CD4 T cells detectable by flow cytometry, combined with overall elevated T-cell activation and memory differentiation, suggesting heightened turnover. Our data suggest early ART initiation to maintain polyfunctional immune memory responses.

Concentric and Eccentric Endurance Exercise Reverse Hallmarks of T-Cell Senescence in Pre-diabetic Subjects.

The peripheral T-cell pool undergoes a striking age associated remodeling which is accelerated by progressive insulin resistance. Exercise training is known to delay several aspects of T-cell senescence. The purpose of the current study was to investigate the effect of 3 weeks regular concentric or eccentric endurance exercise training on the composition of the T-cell compartment in pre-diabetic subjects. Sixteen male older adults with impaired glucose tolerance were recruited and performed either concentric exercise (CE) or eccentric exercise (EE) walking 3 times a week for 3 weeks. Fasting venous blood sampling was performed before training and after the training intervention. Various T-cell subpopulations were analyzed by flow cytometry. We did not find significant time × group effects (interaction) but found several significant time effects for cell type ratios and cell subsets proportions. There was an increase of the CD4+/CD8+ (0.55 ± 0.85%; p = 0.033) and CD4+/CD3+ ratio (5.63 ± 8.44%; p = 0.018) and a decrease of the CD8+/CD3+ ratio (-0.95 ± 1.64%; p = 0.049) after training. We found proportional increases of CD4+/CCR7+/CD45RO+ central memory cells (5.02 ± 7.68%; p = 0.030), naïve CD8+/CCR7+/CD45RO- (3.00 ± 6.68%; p = 0.047) and CD8+/CCR7+/CD45RO+ central memory cells (3.01 ± 3.70%; p = 0.009), while proportions of CD4+/CCR7-/CD45RO- TEMRA cells (-2.17 ± 4.66%; p = 0.012), CD8+/CCR7-/CD45RO- TEMRA cells (-5.11 ± 7.02%; p = 0.018) and CD16+ cells (-4.67 ± 6.45%; p = 0.016) decreased after training. 3 weeks of either CE or EE were effective in reversing hallmarks of T-cell senescence in pre-diabetic subjects. It is suggested that exercise stimulates production and mobilization of naïve T-cells, while differentiated TEMRA cells might disappear by apoptosis.

Naïve regulatory T cells in infancy: Associations with perinatal factors and development of food allergy.

In previous studies, deficits in regulatory T-cell (Treg) number and function at birth have been linked with subsequent allergic disease. However, longitudinal studies that account for relevant perinatal factors are required. The aim of this study was to investigate the relationship between perinatal factors, naïve Treg (nTreg) over the first postnatal year and development of food allergy. In a birth cohort (n=1074), the proportion of nTreg in the CD4+ T-cell compartment was measured by flow cytometry at birth (n=463), 6 (n=600) and 12 (n=675)months. IgE-mediated food allergy was determined by food challenge at 1year. Associations between perinatal factors (gestation, labour, sex, birth size), nTreg at each time point and food allergy at 1year were examined by linear regression. A higher proportion of nTreg at birth, larger birth size and male sex was each associated with higher nTreg in infancy. Exposure to labour, as compared to delivery by prelabour Caesarean section, was associated with a transient decrease nTreg. Infants that developed food allergy had decreased nTreg at birth, and the labour-associated decrease in nTreg at birth was more evident among infants with subsequent food allergy. Mode of birth was not associated with risk of food allergy, and there was no evidence that nTreg at either 6 or 12months were related to food allergy. The proportion of nTreg at birth is a major determinant of the proportion present throughout infancy, highlighting the importance of prenatal immune development. Exposure to the inflammatory stimulus of labour appears to reveal differences in immune function among infants at risk of food allergy.

A Tumor-Peptide-Based Nanoparticle Vaccine Elicits Efficient Tumor Growth Control in Antitumor Immunotherapy.

Recognition of immunoactive oligonucleotides by the immune system, such as Toll-like receptor ligand CpG, leads to increased antibody and T-cell responses. Systemic application often results in unwanted generalized nonantigen-specific activation of the immune system. Nanoparticles are ideal carriers for small and large molecules. Recently, we have demonstrated that calcium phosphate (CaP) nanoparticles functionalized with CpG, and viral antigens are able to induce specific T-cell immunity that protects mice against viral infection and efficiently reactivates the exhausted CD8+ T-cell compartment during chronic retroviral infection. Therefore, CaP nanoparticles are promising vaccine vehicles for therapeutic applications. In this study, we investigated the therapeutic potential use of these nanoparticles in a murine xenograft colorectal cancer model. Therapeutic vaccination with CaP nanoparticles functionalized with CpG and tumor model antigens increased the frequencies of cytotoxic CD8+ T cells in the tumor in a type I interferon-dependent manner. This was accompanied with significantly repressed tumor growth in contrast to the systemic administration of soluble CpG and antigens. Combination therapy of CaP nanoparticles and immune checkpoint blocker against PD-L1 further enhanced the cytotoxic CD8+ T-cell response and eradicated the tumors. Strikingly, vaccination with CaP nanoparticles functionalized with CpG and a primary tumor cell lysate was also sufficient to control the tumor growth. In conclusion, our results represent a translational approach for the use of CaP nanoparticles as a potent cancer vaccine vehicle.

Using single cell genomics to change the treatment of lung cancer.

e20563 Background: Lung cancer (LC) is common with a dismal prognosis. Although treatment with immunotherapy (IO) has improved survival outcomes, these therapies remain expensive. Even using biomarker selection, response rates still fall short of 50%. The majority of immune cell profiling done previously uses samples taken from early stage patients focusing on a single immune cell subtype. Here we use single cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) to characterise the innate and adaptive immune cell activation state and assess the response to exposure to IO in vitro from patients with advanced LC. Methods: Patients with locally advanced or metastatic LC having an Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) have biopsies collected for analysis. Cells are grown in culture +/- nivolumab for 48 hours. scRNA-seq and CITE-seq is conducted using established protocols. Transcriptomic and proteomic data on the innate and adaptive immune cell subsets assess markers of immune activation and/or suppression and the changes after nivolumab are quantified. Results are correlated with clinical outcomes. Results: In a locally advanced/metastatic population, EBUS-TBNA biopsies yield highly cellular samples with a heterogeneous population of immune cells able to be cultured +/- IO using novel, inclusive techniques. Transcriptomic clustering reveals distinct sub-populations within the T-cell, B-cell and innate immune cell compartments. Within these clusters, CITE-seq shows protein expression on individual cells can determine states of exhaustion, cytolytic ability, migratory potential and innate immune activation. Conclusions: Single cell genomics is feasible and informative in patients with advanced LC. This work will form the basis of a functional, real-time assay to assess individualised responses to IO therapy that has the potential to predict IO response.

Intratumoral CD3+ and CD8+ T-cell densities in patients with deficient DNA mismatch repair (dMMR) metastatic colorectal cancer (mCRC) receiving programmed death-1 (PD-1) blockade.

3532 Background: Colorectal cancer with dMMR display heterogeneity in the extent of intratumoral T-cell infiltration which may explain their variable responsiveness to PD-1 blockade. We examined the association of intratumoral CD3+ and CD8+ T-cell densities (TCD) with objective response rate (ORR) and response duration in patients with dMMR mCRCs receiving pembrolizumab (PEM). Methods: Record review was performed on 12 patients with dMMR mCRC treated with PEM (200 mg intravenously every 3 weeks) after failure of prior chemotherapy [median no. of regimens was 1 (range 1-4)] between 01/2015 and 12/2017. CD3+ and CD8+ TCDs were analyzed in the primary tumor core (CT) and at the invasive margin (IM) by immunohistochemistry and automated image analysis to determine density score (0 to 100) for each T-cell subtype and compartment (Ventana Medical Systems, Inc.). Patients were categorized as 1) responders [CR (complete response) + PR (partial response)] vs. non responders [SD (stable disease) + PD (progressive disease)] per RECIST version 1.1, and 2) by duration of response (< or > 12 months). Results: Median follow-up post PEM was 19.5 (9-41) months. Responders included 2 CR and 5 PR; non-responders included 4 SD and 1 PD. The ORR and median time to response were 58.3% (7/12) and 12 weeks (range 9-40), respectively. CD3+ and CD8+ TCD scores were higher in responders vs. in non-responders as well as in patients who had disease control for > 12 months; differences were greatest for CD8+ CT (Table). Conclusions: Among patients treated with PEM, data suggest higher intratumoral CD3+ and CD8+ TCDs in responders versus non-responders and in those with a longer duration of disease control. If confirmed, TCDs may potentially predict responsiveness to PD-1 blockade in dMMR mCRCs. [Table: see text]

KEYNOTE-826: A phase 3, randomized, double-blind, placebo-controlled study of pembrolizumab plus chemotherapy for first-line treatment of persistent, recurrent, or metastatic cervical cancer.

TPS5595 Background: Cervical cancer arises in the setting of persistent infection with high-risk human papillomavirus subtypes. Many patients with early-stage and locally advanced carcinoma can be salvaged with radical surgery and chemoradiation, respectively. However, women with recurrent/metastatic disease represent a poor prognostic group with high unmet clinical needs. Incorporation of anti-angiogenesis therapy has emerged as a therapeutic option, but the survival benefit of 3.7 months over chemotherapy (CT) alone is modest (Tewari et al. NEJM 2014). Because viral tumor antigen-specific T cells reside predominantly in programmed cell death 1–expressing T-cell compartments, checkpoint inhibition may unleash a diverse antitumor T-cell response. Based on the 14.3% objective response in KEYNOTE-158, the US FDA granted accelerated approval to pembrolizumab (pembro) in June 2018 for second-line therapy and beyond. Methods: KEYNOTE-826 is a phase 3, randomized, double-blind, placebo-controlled, multinational trial of CT with pembro or with placebo for first-line treatment of recurrent, persistent, or metastatic cervical cancer. Patients not previously treated with CT for recurrence who are not amenable to curative treatment will be randomized 1:1 to CT + pembro 200 mg or placebo every 3 weeks. The CT regimen (paclitaxel 175 mg/m2 + cisplatin 50 mg/m2 or carboplatin AUC 5, with or without bevacizumab 15 mg/kg) will be selected by the investigator before randomization. Stratification factors include metastasis status at diagnosis, bevacizumab use (yes/no), and tumor PD-L1 status (combined positive score <1, 1 to <10, or ≥10). Treatment will continue until disease progression, unacceptable toxicity, or voluntary patient withdrawal for up to 35 cycles (~2 years). Primary endpoints are progression-free survival (PFS) per RECIST v1.1 assessed by blinded independent central review and overall survival. Secondary endpoints are objective response, duration of response, 12-month PFS, patient-reported quality of life, and safety. Enrollment is ongoing. Clinical trial information: NCT03635567.

Therapeutic effects of artesunate on lupus-prone MRL/lpr mice are dependent on T follicular helper cell differentiation and activation of JAK2–STAT3 signaling pathway

BackgroundAnti-malarial drug artesunate (ART), a semi-synthetic derivative of artemisnin, has immunosuppressive effects on several autoimmune diseases, including Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), and Colitis. However, molecular mechanisms of ART, especially on follicular helper T cells (Tfh), central players in SLE pathology, are far from clear. PurposeThe object for this work is to investigate the therapeutic effect of ART on lupus-prone MRL/lpr mice and its regulatory function on Tfh cells. Study design and methodsMRL/lpr mice were used to explore therapeutic effects of ART on lupus-prone MRL/lpr mice and its regulatory functions on Tfh cells. Then, experiments of renal function were accomplished using the biochemical kits. Effects of ART on histopathology of kidneys, inflammatory factors and autoantibodies were examined using H&E staining, ELISA and real-time PCR. Flow cytometry and western blot analysis were used to examine effects of ART on Tfh differentiation and Jak2–Stat3 signaling pathway. ResultsUpon oral administration, ART significantly prolonged the survival of MRL/lpr mice, ameliorated the lupus nephritis symptoms, decreased the levels of anti-dsDNA antibodies deposited in the kidney, and the levels of pathogenic cytokines (IL-6, IFN-γ and IL-21). After ART treatment, T-cell compartment in the spleen of MRL/lpr mice was restored in terms of reduction in the number of Tfh cells and in the maintenance of the ratio of Tfr to follicular regulatory T cells (Tfh). In addition, ART has significantly inhibited the phosphorylation levels of Jak2 and Stat3 in the MRL/lpr mice. ConclusionART showed therapeutic effects on lupus-prone MRL/lpr mice by inhibiting the differentiation of Tfh cells as well as altering the activation status of Jak2–Stat3 signaling cascade.

Human Tc17 cells harbor potent immune suppressive potential, whereas Tc1 cells lack suppressive ability

Abstract Regulatory T-cells keep untoward immune responses under control. In several autoimmune diseases, there is a substantial regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that acute relapses of Multiple Sclerosis are characterized by a deficit of CD8+ T-cell mediated immune-suppression. Similar to CD4+ T-cells, lineages for CD8+ cytotoxic T-cells (Tc) are predominantly determined by the cytokine milieu during differentiation. We hypothesized that distinct Tc lineages may harbor differential ability to suppress effector CD4+ T-cell responses. To test this hypothesis, we obtained purified human naïve CD8+ T-cells and activated them in different in-vitro conditions to differentiate them toward Tc0 (control), Tc1, Tc2 and Tc17 lineages. We then tested their ability to suppress various types of CD4+ T-cells in flow cytometric suppression assays. We observed that Tc0 controls and Tc2 cells had comparable suppression. However, Tc1 cells showed significant loss of suppressive ability. Interestingly, Tc17 cells exhibited significantly greater suppression of effector cells of various lineages (including bulk ex-vivo CD4+ T-cells as well as in-vitro differentiated Th1 and Th17 cells). Tc17 cells also showed the greatest proportion of CD8+ T-cells with a terminally differentiated phenotype (CD27−/CD45RO−), which we have previously shown to harbor the highest suppressive ability in a neuroantigen-specific setting. These studies illustrate that Tc17 cells, known to play a vital role in infection, tumor and autoimmunity, may have an important immune-regulatory function against effector CD4+ T-cells that could be harnessed in the design of CD8+ T-cell based immune-modulatory therapy.

Choline acetyltransferase-expressing T cells are required to control chronic viral infection

Abstract Although widely studied as a neurotransmitter, recent advances have revealed an important role for T-cell derived acetylcholine (ACh) in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of chat within the T-cell compartment ablated vasodilation in response to infection, impaired the migration of anti-viral T cells into infected tissues, and ultimately compromised the control of chronic LCMV-Cl13. Our results reveal a genetic proof-of-function for ChAT in T cells during viral infection and identify a pathway of T-cell migration that sustains anti-viral immunity.

The Way Forward: Potentiating Protective Immunity to Novel and Pandemic Influenza Through Engagement of Memory CD4 T Cells.

Potentially pandemic strains of influenza pose an undeniable threat to human populations. Therefore, it is essential to develop better strategies to enhance vaccine design and predict parameters that identify susceptible humans. CD4 T cells are a central component of protective immunity to influenza, delivering direct effector function and potentiating responses of other lymphoid cells. Humans have highly diverse influenza-specific CD4 T-cell populations that vary in stimulation history, specificity, and functionality. These complexities constitute a formidable obstacle to predicting immune responses to pandemic strains of influenza and derivation of optimal vaccine strategies. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors of immunity in the CD4 T-cell compartment will aid in both predicting susceptible hosts and in development of vaccination strategies that will poise most human subjects to respond to pandemic influenza strains with protective immune responses.

Accelerated thymopoiesis and improved T-cell responses in HLA-A2/-DR2 transgenic BRGS-based human immune system mice.

Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2-/- Il2rg-/- SirpaNOD (BRGS) HIS mouse model expressing human HLA-A2 and -DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34+ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T-cell development in the mouse thymus. Development of CD4+ and CD8+ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T-cell compartments in peripheral lymphoid organs. Both B- and T-cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen-specific T-cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B- and T-cell homeostasis and function in the BRGS-based HIS mouse model.

Targeted Delivery of IL2 to the Tumor Stroma Potentiates the Action of Immune Checkpoint Inhibitors by Preferential Activation of NK and CD8+ T Cells.

Recombinant human IL2 is being considered as a combination partner for immune checkpoint inhibitors in cancer therapy, but the product only has a narrow therapeutic window. Therefore, we used F8-IL2, an antibody-IL2 fusion protein capable of selective localization to the tumor site, in combination with antibodies against murine CTLA-4, PD-1, and PD-L1. In immunocompetent mice bearing CT26 tumors, the combination of F8-IL2 with CTLA-4 blockade was efficacious, leading to increased progression-free survival and protective immunity against subsequent tumor rechallenges. The combination with anti-PD-1 induced substantial tumor growth retardation, but tumor clearance was rare, whereas the combination with anti-PD-L1 exhibited the lowest activity. A detailed high-parametric single-cell analysis of the tumor leukocyte composition revealed that F8-IL2 had a strong impact on NK-cell activity without collateral immune activation in the systemic immune compartment, whereas CTLA-4 blockade led to significant changes in the T-cell compartment. Leukocyte depletion studies revealed that CD8+ T and NK cells were the main drivers of the therapeutic activity. We extended the experimental observations to a second model, treating MC38 tumor-bearing mice with F8-IL2 and/or CTLA-4 blockade. Only the combination treatment displayed potent anticancer activity, characterized by an increase in cytolytic CD8+ T and NK cells in tumors and draining lymph nodes. A decrease in the regulatory T cell frequency, within the tumors, was also observed. The results provide a rationale for the combined use of engineered IL2 therapeutics with immune checkpoint inhibitors for cancer therapy.

Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression.

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell-mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.

Concise Review: Boosting T-Cell Reconstitution Following Allogeneic Transplantation-Current Concepts and Future Perspectives.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for a large number of malignant and nonmalignant (inherited) diseases of the hematopoietic system. Nevertheless, non‐HLA identical transplantations are complicated by a severe T‐cell immunodeficiency associated with a high rate of infection, relapse and graft‐versus‐host disease. Initial recovery of T‐cell immunity following HSCT relies on peripheral expansion of memory T cells mostly driven by cytokines. The reconstitution of a diverse, self‐tolerant, and naive T‐cell repertoire, however, may take up to 2 years and crucially relies on the interaction of T‐cell progenitors with the host thymic epithelium, which may be altered by GvHD, age or transplant‐related toxicities. In this review, we summarize current concepts to stimulate reconstitution of a peripheral and polyclonal T‐cell compartment following allogeneic transplantation such as graft manipulation (i.e., T‐cell depletion), transfusion of ex vivo manipulated donor T cells or the exogenous administration of cytokines and growth factors to stimulate host‐thymopoiesis with emphasis on approaches which have led to clinical trials. Particular attention will be given to the development of cellular therapies such as the ex vivo generation of T‐cell precursors to fasten generation of a polyclonal and functional host‐derived T‐cell repertoire. Having been tested so far only in preclinical mouse models, clinical studies are now on the way to validate the efficacy of such T‐cell progenitors in enhancing immune reconstitution following HSCT in various clinical settings. stem cells translational medicine 2019;00:1–8

Deep Profiling of the CD8+ T-cell Compartment Identifies Activated Cell Subsets and Multifunctional Responses Associated With Control of Cytomegalovirus Viremia.

Human cytomegalovirus (HCMV) is a common opportunistic pathogen in transplant recipients. Patterns of viremia and reactivation are influenced by the host immune response, including CD8 T cells. However, the cellular deficits or phenotypic differences that account for differential outcomes during HCMV viremia are incompletely understood. Peripheral blood mononuclear cells were collected from 20 transplant recipients (10 viremia controllers and 10 noncontrollers) at onset of HCMV viremia and 4 weeks postonset. We used mass cytometry to perform in-depth characterization of cell surface and intracellular CD8 T cell markers and to compare frequencies of these cells between groups. Deep profiling identified 2 central memory T cell subsets at onset and 5 terminally differentiated memory T (TEMRA) cell subsets at 4 weeks that were associated with control of HCMV viremia, in addition to 6 TEMRA subsets at onset and 4 weeks associated with relapsing or remitting HCMV viremia. In general, CD8 T-cell clusters associated with poorly controlled HCMV viremia lacked markers of activation or terminal differentiation including CD38, CD69, CD25, CD57, and HLA-DR. We also measured the production of 8 HCMV-specific effector molecules (TNFα, IFNγ, interleukin 2, granzyme B, perforin, macrophage inflammatory protein 1β, interleukin 10, and CD107a) in CD8 T cells. Viremia controllers had greater diversity of HCMV-specific multifunctional responses at both time points, including significantly higher frequencies of HCMV-specific TNFαIFNγ CD8 T cells at onset. These multifunctional cells had a phenotype consistent with activated TEM/TEMRA cells. Uncontrolled CMV viremia is associated with specific clusters of memory T-cell subsets and lower frequencies of HCMV-specific multifunctional CD8 T cells.