A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2K^b-restricted cytotoxic and interferon (IFN)γ-producing CD8⁺ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a⁺ CD8⁺ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ⁺ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease.     

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process,which is partially reversed by benznidazole treatment

BackgroundSigns of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4⁺ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4⁺ T cells.Methodology/Principal findingsThe functional capacity of CD4⁺ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4⁺ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4⁺ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4⁺ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24–48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9–12 months after treatment, an increase in the CD4⁺ T cell subset coproducing three molecules, which were mainly granzyme B⁺, perforin⁺ and IFN-γ⁺ (1.4% versus 4.5%).Conclusions/SignificanceA CD4⁺ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9–12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4⁺ T cells.     

Developing lisocabtagene maraleucel chimeric antigen receptor T-cell manufacturing for improved process,product quality and consistency across CD19+ hematologic indications

Background aimsAutologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated substantial clinical benefit across several hematologic malignancies. However, patient-to-patient variability and heterogeneity of starting cellular material across patient populations and disease indications pose challenges to manufacturing consistency. Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, defined-composition, 4-1BB CAR T-cell product administered at equal target doses of CD8⁺ and CD4⁺ CAR⁺ T cells. Here the authors describe the optimization of the liso-cel manufacturing platform for product quality and consistency.MethodsLeukapheresis starting materials were collected from patients with large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia treated with liso-cel in clinical trials (NCT02631044 and NCT03331198). The liso-cel manufacturing process involves selection of CD8⁺ and CD4⁺ T cells from leukapheresis material followed by independent CD8⁺ and CD4⁺ T-cell activation, transduction, expansion, formulation and cryopreservation. Multivariate design of experimental approaches was utilized to optimize process conditions at both specific unit operations and across the process. Flow cytometry methods were used to assess cellular composition, memory phenotypes and cell proliferation. Antigen-specific functions, including cytokine secretion, cytolytic activity and proliferation, were assessed using endpoint assays after independent stimulation of CD8⁺ and CD4⁺ CAR⁺ T-cell product components.ResultsReductions in process duration time, optimization of drug product container and formulation and activation signal optimization led to significantly increased CAR⁺ T-cell product viability. The heterogeneity of patient-derived starting material, including low absolute lymphocyte counts in some samples, was reduced through early T-cell purification, leading to median T-cell frequencies >95% in selected materials across disease indications and limited non-T-cell impurities. These changes further increased lineage purity in CD8⁺ and CD4⁺ CAR⁺ T-cell drug products. CD8⁺ and CD4⁺ CAR⁺ T-cell component lot functional profiles demonstrated multifunctional mechanisms of action, including differential cytokine release, differential cytolytic kinetics and high frequencies of proliferating cells. Correlative analyses demonstrated strong underlying associations between starting material attributes and final CAR⁺ T-cell product phenotype.ConclusionsDespite substantial heterogeneity of starting leukapheresis material quality/composition between individual patients and across disease indications/histologies, the liso-cel manufacturing platform is robust and capable of generating a consistent drug product from diverse starting materials with a single manufacturing platform.     

Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation

BackgroundLeishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4⁺ and CD8⁺ T cell Line 1Lineepitopes. Identification of CD8⁺ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.ConclusionOur results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8⁺ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.     

CD73 Is Dispensable for the Regulation of Inflationary CD8+ T-Cells after Murine Cytomegalovirus Infection and Adenovirus Immunisation

The ecto-5''-nucleotidase (CD73) is expressed by T-cell subsets, myeloid derived suppressive cells and endothelial cells. It works in conjunction with CD39 to regulate the formation and degradation of adenosine in vivo. Adenosine has previously been shown to suppress the proliferation and cytokine secretion of T-cells and recent evidence suggests that inhibition of CD73 has the potential to enhance T-cell directed therapies. Here we utilised a CD73 knockout mouse model to assess the suppressive ability of CD73 on CD8⁺ T-cell classical memory and memory “inflation”, induced by murine cytomegalovirus (MCMV) infection and adenovirus immunisation. We show that CD73 is dispensable for normal CD8⁺ T-cell differentiation and function in both models. Thus CD73 as a suppressor of CD8⁺ T-cells is unlikely to play a deterministic role in the generation and functional characteristics of antiviral memory in these settings.     

T-cell receptor Vβ repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8⁺ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8⁺ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8⁺ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8⁺T-lymphocyte subsets showed high frequencies of LDE CD8⁺T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8⁺ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8⁺ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8⁺ T-cell clones that are selected during CL immune responses, suggesting that CD8⁺ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8⁺T-cells are associated with larger lesions.     

Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8⁺ T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8⁺ T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8⁺ T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8⁺ T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8⁺ T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8⁺ T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8⁺ T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8⁺ T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8⁺ T-cell responses by prophylactic AIDS vaccines.     

一例少见青少年T幼淋巴细胞白血病的病例报道及文献回顾

目的:报道一例少见幼年幼淋巴细胞白血病(T-PLL)患者的病例资料及诊疗过程,并通过文献复习总结了T-PLL的临床特点和诊疗措施。方法:对病例资料进行对比分析,同时通过文献回顾研究T-PLL的特点及发生、发展及诊疗情况。结果:本病例为少见青少年型幼淋巴细胞白血病,细胞以成熟小淋巴细胞为主,特征免疫表型为CD7~+CD5~+CD4~+CD8~+CD3~+,无染色体异常,有TCR基因重排。结论:T-PLL病例具有多态性及复杂性,在临床诊断中需要密切联系临床特点及实验室诊断,综合判断疾病情况,做出最优治疗方案。     

Holoendemic Malaria Exposure Is Associated with Altered Epstein-Barr Virus-Specific CD8+ T-Cell Differentiation

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8⁺ T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8⁺ T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8⁺ T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8⁺ T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8⁺ T cells or the global CD8⁺ memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8⁺ T-cell compartment that illuminates the etiology of eBL.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Co-ordinated isolation of CD8+ and CD4+ T cells recognizing a broad repertoire of cytomegalovirus pp65 and IE1 epitopes for highly specific adoptive immunotherapy

Background aimsAdoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8⁺ and CD4⁺ T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type.MethodsActivation of CMV-specific CD8⁺ and CD4⁺ T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed.ResultsCMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8⁺ and CD4⁺ CMV-specific T cells, while full-length CMV protein only efficiently activated CD4⁺ CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8⁺ and CD4⁺ T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation.ConclusionsThis study provides a feasible strategy for the rapid generation of clinical-grade CD8⁺ and CD4⁺ T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy.     

Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression

The important role of the CD8⁺ T-cells on HIV control is well established. However, correlates of immune protection remain elusive. Although the importance of CD8⁺ T-cell specificity and functionality in virus control has been underscored, further unraveling the link between CD8⁺ T-cell differentiation and viral control is needed. Here, an immunophenotypic analysis (in terms of memory markers and Programmed cell death 1 (PD-1) expression) of the CD8⁺ T-cell subset found in primary HIV infection (PHI) was performed. The aim was to seek for associations with functional properties of the CD8⁺ T-cell subsets, viral control and subsequent disease progression. Also, results were compared with samples from Chronics and Elite Controllers. It was found that normal maturation of total and HIV-specific CD8⁺ T-cells into memory subsets is skewed in PHI, but not at the dramatic level observed in Chronics. Within the HIV-specific compartment, this alteration was evidenced by an accumulation of effector memory CD8⁺ T (T_EM) cells over fully differentiated terminal effector CD8⁺ T (T_TE) cells. Furthermore, higher proportions of total and HIV-specific CD8⁺ T_EM cells and higher HIV-specific T_EM/(T_EM+T_TE) ratio correlated with markers of faster progression. Analysis of PD-1 expression on total and HIV-specific CD8⁺ T-cells from PHI subjects revealed not only an association with disease progression but also with skewed memory CD8⁺ T-cell differentiation. Most notably, significant direct correlations were obtained between the functional capacity of CD8⁺ T-cells to inhibit viral replication in vitro with higher proportions of fully-differentiated HIV-specific CD8⁺ T_TE cells, both at baseline and at 12 months post-infection. Thus, a relationship between preservation of CD8⁺ T-cell differentiation pathway and cell functionality was established. This report presents evidence concerning the link among CD8⁺ T-cell function, phenotype and virus control, hence supporting the instauration of early interventions to prevent irreversible immune damage.     

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Subclinical doses of Paclitaxel (PTX) given 1 day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu⁺ tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8⁺ T cells with enhanced lytic activity against neu⁺ tumors. PTX treatment also enhances maturation marker expression on CD11c⁺ DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8⁺ T cells with greater lytic capability than DC’s from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-γ-secreting CD8⁺ T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.     

Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

Quantification of T-cell dynamics during latent cytomegalovirus infection in humans

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8⁺ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8⁺ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8⁺ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8⁺ T-cell populations. The lifespans of circulating CMV-specific CD8⁺ T-cells did not differ significantly from those of bulk memory CD8⁺ T-cells, and the lifespans of bulk memory CD8⁺ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4⁺ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8⁺ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8⁺ T-cells are not due to longer lifespans of these cells.     

CD4(+) CD28(+) lymphocytes on day 5 after high-dose melphalan for multiple myeloma predict a low risk of infections during severe neutropenia and are associated with the number of reinfused T lymphocytes of the autologous stem cell graft

Background aimsNeutropenia following high-dose chemotherapy is associated with a substantial risk of infectious complications. The aim of this study was to identify variables in residual leukocyte subsets during neutropenia that are predictive for neutropenic fever.MethodsResidual leukocytes in the peripheral blood on day 5 after autologous blood stem cell transplantation were analyzed by three-color flow cytometry in 55 consecutive patients with multiple myeloma. Furthermore, the number of T cells transfused with the autografts was determined.ResultsNeutrophil counts at day 5 and neutrophil engraftment were similar in patients with and without neutropenic fever. Low absolute lymphocyte, CD4⁺ CD28⁺ and CD45RO⁺ CD28⁺ counts at day 5 were associated with neutropenic fever. T-cell counts at day 5 correlated with the CD3⁺ cell number in the graft.ConclusionsOur data show that the absolute lymphocyte, CD4⁺ CD28⁺ and CD45RO⁺ CD28⁺ counts play a role in host defense during severe neutropenia. The T-cell number in the graft may help to identify patients at high risk of neutropenic infections.     

Interferon-α and -γ Differentially Reduce Rapid Immature T-Cell Death by Contact with HIV-1 Carrier Cell Clones In Vitro

The non-antigen specific rapid cytotoxic (CT) death of immature TdT⁺CD4⁺CD8⁺ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as “effectors,” with IFN-α but not IFN-γ, or of the susceptible immature TdT⁺CD4⁺CD8⁺ T cells, referred to as “targets,” with IFN-γ but not IFN-α, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-α treated effector (gp120⁺CD8⁺ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-γ treated target (CD4⁺CD8⁺ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-γ treated effectors or IFN-α treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.     

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3⁺ T-cells than photo-damaged skin, however, the abundance of CD3⁻CD56⁺ Natural Killer (NK) cells, CD11c⁺HLA-DR⁺ conventional Dendritic Cells (cDC), BDCA-2⁺HLA-DR⁺ plasmacytoid DC (pDC), FoxP3⁺ Regulatory T-cells (T-reg), Vα24⁺Vβ11⁺ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4⁺ T-cells were associated with SCC, yet CD8⁺ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8⁺ T-cells than IEC. We propose that differences in CD8⁺ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4⁺ T-cells to CD8⁺ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.     

Dynamics of Peripheral Blood Lymphocyte Subpopulations in the Acute and Subacute Phase of Legionnaires’ Disease

Study Objective

Absolute lymphocytopenia is recognised as an important hallmark of the immune response to severe infection and observed in patients with Legionnaires’ disease. To explore the immune response, we studied the dynamics of peripheral blood lymphocyte subpopulations in the acute and subacute phase of LD.

Methods and Results

EDTA-anticoagulated blood was obtained from eight patients on the day the diagnosis was made through detection of L. pneumophila serogroup 1 antigen in urine. A second blood sample was obtained in the subacute phase. Multiparametric flow cytometry was used to calculate lymphocyte counts and values for B-cells, T-cells, NK cells, CD4⁺ and CD8⁺ T-cells. Expression of activation markers was analysed. The values obtained in the subacute phase were compared with an age and gender matched control group. Absolute lymphocyte count (×10⁹/l, median and range) significantly increased from 0.8 (0.4–1.6) in the acute phase to 1.4 (0.8–3.4) in the subacute phase. B-cell count showed no significant change, while T-cell count (×10⁶/l, median and range) significantly increased in the subacute phase (495 (182–1024) versus 979 (507–2708), p = 0.012) as a result of significant increases in both CD4⁺ and CD8⁺ T-cell counts (374 (146–629) versus 763 (400–1507), p = 0.012 and 119 (29–328) versus 224 (107–862), p = 0.012). In the subacute phase of LD, significant increases were observed in absolute counts of activated CD4⁺ T-cells, naïve CD4⁺ T-cells and memory CD4⁺ T-cells. In the CD8⁺ T-cell compartment, activated CD8⁺ T-cells, naïve CD8⁺ T-cell and memory CD8⁺ T-cells were significantly increased (p<0.05).

Conclusion

The acute phase of LD is characterized by absolute lymphocytopenia, which recovers in the subacute phase with an increase in absolute T-cells and re-emergence of activated CD4⁺ and CD8⁺ T cells. These observations are in line with the suggested role for T-cell activation in the immune response to LD.     

Prospective Immune Dynamics during the First 24 Weeks of Efavirenz Based-Antiretroviral Therapy in HIV-1-Infected Subjects,According to CD4+ T-Cell Counts at Presentation: The IMMUNEF Clinical Trial

BackgroundLongitudinal characterization of immune recovery in the first-phase of antiretroviral therapy (ART) is poorly described. We compared immune kinetics in individuals who were diagnosed early or late with HIV-1 infection, (thus commencing ART with different CD4⁺ T-cell counts), in order to investigate possible mechanisms involved in subsequent poor immune recovery.MethodsImmunophenotyping, immune activation, proliferation, apoptosis, regulatory T-cells and intracellular cytokine production were compared at baseline and during 24-week follow-up in two groups of HIV-1-infected patients initiating the same ART (tenofovir/emtricitabine/efavirenz) and divided according to baseline CD4⁺ T-cell counts (late: ≤200/μL; early: >200/μL). Wilcoxon-rank sum test and analysis for repeated measures were used to evaluate differences between groups over time.ResultsTwenty-four out of 30 enrolled subjects were evaluable for the analysis, 13 late and 11 early presenters. Significantly lower CD4⁺ naïve and memory T-cells, and higher plasma viral load, as well as augmented percentages of activated (CD4⁺/CD25⁺ cells), apoptotic (CD4⁺/AnnexinV⁺/7AAD⁻, CD4⁺/caspase 8⁺ and CD4⁺/caspase 9⁺), and proliferating (CD8⁺/Ki67⁺ cells) lymphocytes were present at baseline in late presenters; ART resulted in a reduction of apoptotic and proliferating lymphocytes within the follow-up period.ConclusionsA skewing towards memory/activated/apoptotic phenotype is seen in HIV-1-infected subjects starting ART at low CD4⁺ T-cell counts; ART results in early (24 weeks) trend towards normalization of these parameters. Antiretroviral therapy may play a role in rapidly limiting aberrant immune exhaustion even in late presenters, while requiring more time for re-population of highly depleted naïve T-cells.

Trial Registration

EU Clinical Trial Register EUDRACT number 2008-006188-35 https://www.clinicaltrialsregister.eu/ctr-search/trial/2008-006188-35/IT