Alopecia areata (AA) is a condition in which hair is lost in small regions or over the entire body. It has a prevalence of 1 in 1000 and has a great impact on psychological wellbeing. AA is generally ...considered an autoimmune disease in which a collapse of the immune privilege system of the hair follicle has shown to play an important role, potentially driven by interferon gamma (IFN-γ). The most prominent cells located in or around the hair follicle in AA are Langerhans cells, CD4
+
or CD8
+
T cells, macrophages and mast cells. Langerhans cells, specialized dendritic cells, are resident in the epidermis and are known to associate with hair follicles. Therefore, we aimed to develop in vitro generated Langerhans cells contributing as an in vitro model of disease. In vitro models provide insight into the behaviour of cells and are a valuable tool before being in need of an animal model or patient samples. For this, Langerhans-like cells were generated from CD14
+
monocytes in the presence of GM-CSF and TGF-β. After 10 days of cell culture, Langerhans-like cells express CD207 and CD1a but lack CD209 expression as well as Birbeck granules. Next, Langerhans-like cells were exposed to inflammatory conditions and the effect of different AA treatments was investigated. All treatments—diphencyprone contact immunotherapy, UV-B light therapy and JAK-STAT inhibition—affect the expression of costimulatory and skin-homing markers on Langerhans-like cells. Importantly, also the T cell stimulatory capacity of Langerhans-like cells was significantly reduced following treatment under inflammatory conditions. Noteworthy, JAK-STAT inhibition outperformed conventional AA treatments. In conclusion, our findings demonstrate that in vitro generated Langerhans-like cells can be used as a model of disease. Moreover, JAK-STAT inhibition may become a valuable new approach for the treatment of AA.
Antigen recognition through the T cell receptor (TCR) αβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to ...expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.
The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated ...antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms' tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen
mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards
mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.
Chimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of ...antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line.
Given our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated.
Co-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity.
Here, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.
Wilms’ tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we ...provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.
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•Robust fully autologous in vitro expansion of WT1-reactive T cells from healthy donors•Two consecutive one-week stimulations with autologous peptide-pulsed DCs and PBLs•Steps for tetramer staining, allowing WT137-45-specific T cell purification•Expanded T cells can serve for applications analyzing TCR repertoire or isolating TCRs
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Wilms’ tumor protein 1 (WT1) is a tumor-associated antigen (TAA) overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.
Dendritic cell (DC) vaccination can be an effective post-remission therapy for acute myeloid leukemia (AML). Yet, current DC vaccines do not encompass the ideal stimulatory triggers for innate gamma ...delta (γδ) T cell anti-tumor activity. Promoting type 1 cytotoxic γδ T cells in patients with AML is, however, most interesting, considering these unconventional T cells are primed for rapid function and exert meaningful control over AML. In this work, we demonstrate that interleukin (IL)-15 DCs have the capacity to enhance the anti-tumoral functions of γδ T cells. IL-15 DCs of healthy donors and of AML patients in remission induce the upregulation of cytotoxicity-associated and co-stimulatory molecules on the γδ T cell surface, but not of co-inhibitory molecules, incite γδ T cell proliferation and stimulate their interferon-γ production in the presence of blood cancer cells and phosphoantigens. Moreover, the innate cytotoxic capacity of γδ T cells is significantly enhanced upon interaction with IL-15 DCs, both towards leukemic cell lines and allogeneic primary AML blasts. Finally, we address soluble IL-15 secreted by IL-15 DCs as the main mechanism behind the IL-15 DC-mediated γδ T cell activation. These results indicate that the application of IL-15-secreting DC subsets could render DC-based anti-cancer vaccines more effective through, among others, the involvement of γδ T cells in the anti-leukemic immune response.
Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value ...has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by
gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with
checkpoint inhibition supports further clinical translation.
Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens ...is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes.
In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG
-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D
-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load.
Treatment of MOG
-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG
-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG
-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score.
Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.
Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies ...are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8
T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific
mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and
mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8
T cell activation and killing activity. Altogether, DsiRNA and
mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.
Background
We previously engineered mice to accumulate elevated levels of age‐related, antigen‐specific memory CD8 T cells as in humans. These mice spontaneously develop all major hallmarks of AD ...with aging. Analogous T cells reactive to an epitope on Amyloid Precursor Protein (APP) were found in aging humans, accumulated in AD brain, and decreased in AD blood. Here, we examine whether their levels are associated with established AD biomarkers in CSF.
Method
Antigen‐specific CD8 T cells in blood were quantified by flow cytometric analysis after staining with anti‐CD8, anti‐KLRG1 and APP peptide‐HLA‐A2 multimers in normal aging, MCI with or without AD biomarkers, and confirmed late‐onset AD patient cohorts (n = 50). Aβ1‐42, total Tau, and pTau181 were quantified in CSF, and cognitive performance assessed by MMSE.
Result
Percentage of APP‐specific memory CD8 T cells was significantly decreased with dementia (0.68 + 0.29% for MMSE <25; 1.63 + 0.32% for MMSE >24), reaching minimal levels in AD blood, which precluded meaningful correlations in AD patients. Nevertheless, increasing levels of APP‐specific memory CD8 T cells in blood from all subjects combined corresponded with increased CSF Aβ1‐42 (r = 0.373, P < 0.0004) and decreased total Tau but not pTau181 or MMSE (r = ‐0.234, P < 0.0292; r = ‐0.208, P < 0.0531; and r = ‐0.009, P < 0.9410, respectively). Decreases in the larger parental (KLRG1+) memory CD8 T cell population also correlated with decreased CSF Aβ1‐42 in AD patients alone (r = 0.511, P = 0.003 for % KLRG1+ of CD8 T cells; r = 0.682, P < 0.00009 for % KLRG1+CD8+ cells; n = 31).
Conclusion
Our results reveal that age‐related memory CD8 T cells in blood decrease with dementia, and in proportion to decreased CSF Aβ1‐42 and total Tau in all patients, while their parental KLRG1+ population is directly proportional to Aβ1‐42 in AD patients only. This validates our previous findings that loss of APP‐specific memory CD8 T cells from blood accurately tracks the AD continuum, and support the notion that antigen‐specific memory CD8 T cells are associated with the earliest detectable pathologic changes in AD.
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