Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases.

Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops.

Detailed analysis of the cells that infiltrate lesional skin cannot be performed in skin biopsy specimens using immunohistochemistry or cell separation techniques because enzyme treatments applied during the isolation step can destroy small amounts of protein and minor cell populations in the biopsy specimen. Here, we describe a method for isolating T cells from drops of whole blood obtained from lesions during skin biopsy in patients with cutaneous T-cell lymphoma. Lesional blood is assumed to contain lesional resident cells, cells from capillary vessels, and blood overflowing from capillary vessels into the lesion area. The lesional blood showed substantial increases in distinct cell populations, chemokines, and the expression of various genes. The proportion of CD8+CD45RO+ T cells in the lesional blood negatively correlated with the modified severity-weighted assessment tool scores. CD4+CD45RO+ T cells in the lesional blood expressed genes associated with the development of cancer and progression of cutaneous T-cell lymphoma. In addition, CD8+CD45RO+ T cells in lesional blood had unique T-cell receptor repertoires in lesions of each stage. Assessment of lesional blood drops might provide new insight into the pathogenesis of mycosis fungoides and facilitate evaluation of the treatment efficacy for mycosis fungoides as well as other skin inflammatory diseases.

Cheap and Commonplace: Making the Case for BCG and γδ T Cells in COVID-19.

Antigen-specific vaccines developed for the COVID-19 pandemic demonstrate a remarkable achievement and are currently being used in high income countries with much success. However, new SARS-CoV-2 variants are threatening this success via mutations that lessen the efficacy of antigen-specific antibodies. One simple approach to assisting with this issue is focusing on strategies that build on the non-specific protection afforded by the innate immune response. The BCG vaccine has been shown to provide broad protection beyond tuberculosis disease, including against respiratory viruses, and ongoing studies are investigating its efficacy as a tool against SARS-CoV-2. Gamma delta (γδ) T cells, particularly the Vδ2 subtype, undergo rapid expansion after BCG vaccination due to MHC-independent mechanisms. Consequently, γδ T cells can produce diverse defenses against virally infected cells, including direct cytotoxicity, death receptor ligands, and pro-inflammatory cytokines. They can also assist in stimulating the adaptive immune system. BCG is affordable, commonplace and non-specific, and therefore could be a useful tool to initiate innate protection against new SARS-CoV-2 variants. However, considerations must also be made to BCG vaccine supply and the prioritization of countries where it is most needed to combat tuberculosis first and foremost.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Bispecific antibodies in acute lymphoblastic leukemia therapy.

INTRODUCTION: Blinatumomab, first in a class of bispecific T-cell engagers, revolutionized treatment paradigm of B-cell precursor relapsed/refractory or minimal residual disease positive acute lymphoblastic leukemia (ALL) in adults and children, inducing deep remissions in a proportion of patients. However, significant numbers of patients do not respond or eventually relapse. Strategies for improvement of treatment outcomes are required. AREAS COVERED: This review discusses the main structural and functional features of blinatumomab, and its place in the treatment of ALL. Furthermore, prospects to increase the efficacy of blinatumomab are addressed. The developments in the field of bispecific antibodies and their possible implications for treatment of ALL are reviewed. EXPERT OPINION: Better understanding the mechanisms of response and resistance to blinatumomab might help us to identify the group of patients benefiting most from treatment and to spare potentially toxic subsequent treatment strategies. Data emerging from ongoing clinical trials might change the treatment landscape of ALL and beyond. Early use of blinatumomab in frontline protocols with more advantageous treatment sequences and in combination with other targeted therapies might reduce the failure rates. Exponentially increasing number of novel treatment options and their possible combinations might complicate treatment decision-making without data from randomized trials.

Concepts in immuno-oncology: tackling B cell malignancies with CD19-directed bispecific T cell engager therapies.

The B cell surface antigen CD19 is a target for treating B cell malignancies, such as B cell precursor acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma. The BiTE® immuno-oncology platform includes blinatumomab, which is approved for relapsed/refractory B cell precursor acute lymphoblastic leukemia and B cell precursor acute lymphoblastic leukemia with minimal residual disease. Blinatumomab is also being evaluated in combination with other agents (tyrosine kinase inhibitors, checkpoint inhibitors, and chemotherapy) in various treatment settings, including frontline protocols. An extended half-life BiTE molecule is also under investigation. Patients receiving blinatumomab may experience cytokine release syndrome and neurotoxicity; however, these events may be less frequent and severe than in patients receiving other CD19-targeted immunotherapies, such as chimeric antigen receptor T cell therapy. We review BiTE technology for treating malignancies that express CD19, analyzing the benefits and limitations of this bispecific T cell engager platform from clinical experience with blinatumomab.

T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation-Updated Consensus and Review 2020.

Immune responses to protein and peptide drugs can alter or reduce their efficacy and may be associated with adverse effects. While anti-drug antibodies (ADA) are a standard clinical measure of protein therapeutic immunogenicity, T cell epitopes in the primary sequences of these drugs are the key drivers or modulators of ADA response, depending on the type of T cell response that is stimulated (e.g., T helper or Regulatory T cells, respectively). In a previous publication on T cell-dependent immunogenicity of biotherapeutics, we addressed mitigation efforts such as identifying and reducing the presence of T cell epitopes or T cell response to protein therapeutics prior to further development of the protein therapeutic for clinical use. Over the past 5 years, greater insight into the role of regulatory T cell epitopes and the conservation of T cell epitopes with self (beyond germline) has improved the preclinical assessment of immunogenic potential. In addition, impurities contained in therapeutic drug formulations such as host cell proteins have also attracted attention and become the focus of novel risk assessment methods. Target effects have come into focus, given the emergence of protein and peptide drugs that target immune receptors in immuno-oncology applications. Lastly, new modalities are entering the clinic, leading to the need to revise certain aspects of the preclinical immunogenicity assessment pathway. In addition to drugs that have multiple antibody-derived domains or non-antibody scaffolds, therapeutic drugs may now be introduced via viral vectors, cell-based constructs, or nucleic acid based therapeutics that may, in addition to delivering drug, also prime the immune system, driving immune response to the delivery vehicle as well as the encoded therapeutic, adding to the complexity of assessing immunogenicity risk. While it is challenging to keep pace with emerging methods for the preclinical assessment of protein therapeutics and new biologic therapeutic modalities, this collective compendium provides a guide to current best practices and new concepts in the field.

In vitro model for the assessment of human immune responses to subunit RSV vaccines.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.

Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis.

The local expression of WC1+ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1+ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1+ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1+ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.

Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen.

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.

AIDS prevention and control in the Yunnan region by T cell subset assessment.

BACKGROUND: Prior to being spread throughout broader China, multiple human immunodeficiency virus (HIV)-1 genotypes were originally discovered in the Yunnan Province. As the HIV-1 epidemic continues its spread in Yunnan, knowledge of the influence of gender, age, and ethnicity to instances of HIV reservoirs will benefit monitoring the spread of HIV. METHODS: The degree to which T cells are depleted during an HIV infection depends on the levels of immune activation. T-cell subsets were assessed in newly-diagnosed HIV/AIDS patients in Yunnan, and the influence of age, gender, and ethnicity were investigated. Patients that were newly diagnosed with the HIV-infection between the years 2015 and 2018 at the First Affiliated Hospital of Kunming Medical College were selected for this study (N = 408). The lymphocyte levels and T cell subsets were retrospectively measured in whole blood samples by FACS analysis. RESULTS: The median CD4 count was 224 ± 191 cells/µl. Significantly higher mean frequencies and absolute numbers were observed in CD3+, CD3+CD4+, CD3+CD8+, CD45+, and CD3+CD4+/CD45+ in females compared to males. Han patients showed a higher total number of CD3+T cells and the ratio of CD3+ /CD45+ cells compared to any other ethnic minority (P < 0.001). The numbers of CD3+ T-cells, CD3+CD8+ T cells, and CD45+ T cells were highest in the age group ≥ 60. Significant differences were observed in the counts of CD3+, CD3+CD8+, and CD45+ cells and the ratio of CD3+/CD45+ and CD3+CD4+/CD45+ cells between the ≤ 29 and 30-59 age groups. CONCLUSION: This study has revealed that low levels of CD4+ T cells can be observed in newly-diagnosed HIV/AIDS patients in the Yunnan province. It has also been demonstrated that gender, age, and ethnicity have a significant association with the ratio of T-cell subsets that may contribute to virus progression and disease prognosis in individuals belonging to certain subsets of the population. This study has highlighted the importance of HIV/AIDS screening in at-risk populations to ensure timely and adequate clinical management in Yunnan.

RepSeq Data Representativeness and Robustness Assessment by Shannon Entropy.

High-throughput sequencing (HTS) has the potential to decipher the diversity of T cell repertoires and their dynamics during immune responses. Applied to T cell subsets such as T effector and T regulatory cells, it should help identify novel biomarkers of diseases. However, given the extreme diversity of TCR repertoires, understanding how the sequencing conditions, including cell numbers, biological and technical sampling and sequencing depth, impact the experimental outcome is critical to proper use of these data. Here, we assessed the representativeness and robustness of TCR repertoire diversity assessment according to experimental conditions. By comparative analyses of experimental datasets and computer simulations, we found that (i) for small samples, the number of clonotypes recovered is often higher than the number of cells per sample, even after removing the singletons; (ii) high-sequencing depth for small samples alters the clonotype distributions, which can be corrected by filtering the datasets using Shannon entropy as a threshold; and (iii) a single sequencing run at high depth does not ensure a good coverage of the clonotype richness in highly polyclonal populations, which can be better covered using multiple sequencing. Altogether, our results warrant better understanding and awareness of the limitation of TCR diversity analyses by HTS and justify the development of novel computational tools for improved modeling of the highly complex nature of TCR repertoires.

Gender differences in the T-cell profiles of the airways in COPD patients associated with clinical phenotypes.

T lymphocytes are believed to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How T cells are recruited to the lungs and contribute to the inflammatory process is largely unknown. COPD is a heterogeneous disease, and discriminating disease phenotypes based on distinct molecular and cellular pathways may provide new approaches for individualized diagnosis and therapies. Bronchoalveolar lavage (BAL) and blood samples were obtained from 40 never-smokers, 40 smokers with normal lung function, and 38 COPD patients. T-cell chemokine receptor expression was analyzed with flow cytometry, and soluble BAL cytokines and chemokines were measured using a cytokine multiplex assay. Correlations with gender and clinical characteristics including lung imaging were investigated using multivariate modeling. Th1/Tc1- and Th2/Tc2-associated soluble analytes and T-cell chemokine receptors were analyzed as cumulative Th1/Tc1 and Th2/Tc2 immune responses. A higher expression of chemokine receptor CCR5 on CD8+ T cells in BAL and higher percentage of CXCR3+CD8+ T cells in blood was found in female smokers with COPD compared to those without COPD. CCR5 expression on CD4+ and CD8+ T cells was lower in BAL from male smokers with COPD compared to those without COPD. Among female smokers with COPD, Th1/Tc1 immune response was linked to BAL macrophage numbers and goblet cell density, and Th2/Tc2 response was associated with the measures of emphysema on high-resolution computed tomography. The highly gender-dependent T-cell profile in COPD indicates different links between cellular events and clinical manifestations in females compared to males. Our findings may reveal mechanisms of importance for the difference in clinical course in female COPD patients compared to males.

Assessment of the Plasmodium falciparum Preerythrocytic Antigen UIS3 as a Potential Candidate for a Malaria Vaccine.

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei-P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced.

Self-reactivity as the necessary cost of maintaining a diverse memory T-cell repertoire.

The adaptive immune system is expected to protect the host from infectious agents and malignancies, while avoiding robust activation against self-peptides. However, T cells are notoriously inept at protection whenever the pathogen or tumor is persistent in the body for longer periods of time. While this has been thought of as an adaptation to limit the immunopathology from continued effector T-cell responses, it is also likely an extension of the T cell's intrinsic mechanisms which evolved to tolerate self-peptides. Here we deliberate on how the need to tolerate self-peptides might stem from a paradoxical requirement-the utility of such molecules in maintaining a diverse repertoire of pathogen-specific memory T cells in the body. Understanding the mechanisms underlying this intriguing nexus, therefore, has the potential to reveal therapeutic strategies not only for improving immune responses to chronic infections and tumors but also the long-term efficacy of vaccines aimed at cellular immune responses.

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

The assessment of immune-regulatory effects of extracorporeal photopheresis in systemic sclerosis: a long-term follow-up study.

The therapeutic options in systemic sclerosis (SSc) are limited mainly to the management of complications, and decelerating fibrosis and preventing disease progression are still great challenges. Extracorporeal photopheresis (ECP) is one of the promising therapeutic strategies in SSc; nevertheless, there is no consensus on the ideal timing and frequency of treatment cycles. In the present study, we evaluated the long-term effects of consecutive ECP treatments, and the stability of clinical and laboratory improvements. We enrolled nine patients with diffuse cutaneous SSc and performed 12 ECP cycles (24 ECP treatments) per patient in total. ECP cycles were carried out once in every 6 weeks, and each cycle consisted of two procedures. Sixteen healthy individuals served as controls for laboratory assessment. Following the sixth ECP cycle, we observed further improvement in skin score, which was confirmed by high-resolution ultrasonography as well. After the second ECP cycle, values of Tr1 and CD4+CD25(bright) Treg cells increased; however, Tr1 cells remained under control values until the 10th cycle. Suppressor activity of CD4+CD25+ Treg cells improved, while percentages of Th17 cells decreased. At the end of 12-month follow-up, we did not observe significant deterioration in skin involvement; however, improvement in laboratory parameters diminished after 12 months. If the first six ECP cycles are effective, uninterrupted continuation of treatment should be considered, which may lead to the normalization of Tr1 cell values along with further clinical improvement. Our laboratory observations indicate that immunomodulatory effect of ECP treatments lasts for 1 year only.