STAT3-dependent IL-21 production from T helper cells regulates hematopoietic progenitor cell homeostasis

Abstract The contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. We have previously identified T cell–dependent regulation of hematopoietic progenitor cell (HPC) numbers and cycling. In this report, we demonstrated that HPC activity is decreased in mice with STAT3-deficient T cells, a phenotype that is not because of decreased expression of IL-17 or RORγt. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets, and neutralization of IL-21 resulted in decreased HPC activity identical to that in mice with STAT3-deficient T cells. Importantly, injection of IL-21 rescued HPC activity in mice with STAT3-deficient T cells. Thus, STAT3-dependent IL-21 production in T cells is required for HPC homeostasis.

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Prognostic Analysis of Pre-Transplant CD26-Positive Lymphocytes and CD26bright/CD45RO-Positive Memory T-Cell Levels in Patients Receiving an Autologous Hematopoietic Progenitor-Cell Transplant.

Blood ◽

10.1182/blood.v110.11.1653.1653 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1653-1653

Author(s):

Martin Hildebrandt ◽

Hella Gollasch ◽

Kirstin Rautenberg ◽

Bernd Doerken ◽

Wolf-Dieter Ludwig

Keyword(s):

T Cells ◽

T Cell ◽

Progenitor Cell ◽

Memory T Cells ◽

Cell Subset ◽

Hematopoietic Progenitor Cell ◽

Cell Transplant ◽

Hematopoietic Progenitor ◽

Memory T Cell ◽

Kinetics Of

Abstract The lymphocyte surface glycoprotein CD26 is involved in an array of diverse signalling pathways and costimulatory events. CD26positive cells possess dipeptidylpeptidase IV (DPP IV) activity, a serine protease known to inactivate SDF-1/CXCL12, a key mediator of stem cell homing and engraftment. Furthermore, CD26 modulates surface expression of CTLA-4 and contributes to T-cell migration through endothelial layers. CD26 has been attributed with a central role in alloantigen-mediated immune pathways and memory T-cell responses. The purpose of this study was to evaluate the levels of a distinct memory T-cell subset, CD26bright/CD45RO-positive, in autologous hematopoietic progenitor-cell transplant (HPCT) recipients. Between 2003 and 2006 we enrolled 42 patients scheduled to undergo high-dose chemotherapy and autologous HPCT (multiple myeloma, n=31; Hodgkin’s Disease, n=3; NHL, n=6; PNET, n=1; AML, n=1). Levels of memory and naïve CD26, CD34, CD4, CD8, as well as co-expression of CD4 or CD8 among CD26bright/CD45RO-positive cells were analyzed before autologous HPCT. In addition, the number of memory and naïve CD26-positive T cells transfused per kg body weight in the progenitor cell harvest were determined. The subsets of CD26-positive cells were correlated with kinetics of engraftment and with the occurrence of disease progression or relapse after autologous transplantation. With regard to kinetics of engraftment, only the number of CD34-positive cells transfused was associated with rapid engraftment (P=0.001). However, CD26-positive cells were of predicitve value for the occurrence of disease progression or relapse. Pre-transplant CD26-positive T-cell levels correlated with progression-free survival (PFS) (P=0.022). Specifically, the number of CD26bright/CD45RO-positive memory T-cells in the autograft correlated with PFS and, in regression analyses, emerged as the only variable predictive for the occurrence of diease progression or relapse (P=0.006). The prognostic effects of pre-transplant CD26bright/CD45RO-positive memory T-cells were independent of the type of disease and of the conditioning regimen applied. The analysis of antigens CD4 and CD8, respecively, on CD26bright/CD45RO-positive T-cells yielded no additional information. Our results suggest that pre-transplant levels of CD26-positive T cells, specifically a memory cell subset of CD26bright cells coexpressing CD45RO, may yield information with regard to outcome in autologous HPCT recipients. These observations may contribute to a prospective identification of those patients at higher risk of relapse, based on their immune status.

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Predictors of More Rapid Lymphoid Reconstitution after Allogeneic Transplantation.

Blood ◽

10.1182/blood.v104.11.3159.3159 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3159-3159

Author(s):

Martha L. Arellano ◽

Susan G. Moore ◽

Ragini Kudchadkar ◽

Christopher R. Flowers ◽

Amelia A. Langston ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Progenitor Cell ◽

T Cell Subsets ◽

Hematopoietic Progenitor Cell ◽

Gamma Delta ◽

Median Dose ◽

Hematopoietic Progenitor ◽

Post Transplant ◽

Cd34 Cells

Abstract Introduction: Aberrant post-transplant immune reconstitution contributes to the major risks of graft versus host disease (GVHD), infection, and relapse following allogeneic hematopoietic progenitor cell transplantation. Faster lymphocyte engraftment has been found to predict better clinical outcomes in patients after autologous and allogeneic bone marrow transplantation. The purpose of our study was to investigate factors that may contribute to more rapid lymphocyte engraftment in patients who underwent transplantation using grafts procured from HLA-matched volunteer unrelated donors. Methods:We analyzed 47 recipients of BM (n=13) and G-CSF mobilized blood hematopoietic progenitor cell grafts (n=34) for post-transplant lymphoid reconstitution, and constituents of the graft that predicted early lymphoid recovery. Diagnoses included 28 patients with AML/MDS/MPD, 6 with lymphoma/CLL, 6 with ALL, 3 with CML, 2 with MM and 2 with AA, with a median age of 43 years (range 17–68). 34 patients received TBI or busulfan-based myeloablative regimens; 13 patients received fludarabine and low-dose TBI. Numbers of T-cell, NK, B-cell, and dendritic cell subsets in the grafts were enumerated by flow cytometry. Our primary end point was lymphocyte engraftment at day+30 (ALC > 500 x 2 days). Secondary analyses included: univariate and multivariate models exploring the relationship between graft constituents and early lymphoid recovery. Results: With a median follow-up of 1 year (range 1–32 months), 11 (23%) patients died prior to lymphocyte engraftment; 2 patients failed to achieve ALC > 500; and 20 (43%) patients were alive with stable lymphocyte engraftment. The median dose of CD34+ cells administered was 5.35 x 10E6 cells/kg; and the median dose of CD3+ cells was 211 x 10E6 cells/kg.The mean absolute lymphocyte count (ALC) on day +30 for the entire group was 714 ± 125 cells per mcL, with a median time to achieve an ALC > 500/mcL for 2 consecutive days of 23 days (range 0–111 days). In univariate analyses, higher doses of the following T-cell subsets in the graft were predictive of faster lymphocyte engraftment (all x 10E6 cells/kg): CD3+ > 296 (p=0.01), CD4+ > 172 (p=0.04), CD123+11c- (DC2) > 3.38 (p=0.03), and gamma-delta T- cells > 5.50 (p=0.01). The graft content of CD34+ cells and other graft constituents were not significantly associated with faster lymphoid engraftment. Stepwise multivariate Cox regression models including all graft constituents, conditioning regimen and patient characteristics determined that gamma-delta T-cell dose in the graft was the only factor associated with faster lymphocyte engraftment (p < 0.01). Faster lymphocyte engraftment was not significantly associated with CMV reactivation, or the incidence of either acute or chronic GVHD. Conclusions: The kinetics of lymhoid reconstitution following allogeneic volunteer unrelated donor transplantation are associated with the content of donor T-cells and DC2 in the graft. Faster lymphoid reconstitution was not associated with less CMV infection nor improved survival in this relatively small population. Ongoing studies are examining the role of lymphoid reconstitution in post-transplant outcomes in a larger data base of allogeneic transplant recipients.

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PTEN loss correlates with T cell exclusion across human cancers

BMC Cancer ◽

10.1186/s12885-021-08114-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ziying Lin ◽

Lixia Huang ◽

Shao Li Li ◽

Jincui Gu ◽

Xiaoxian Cui ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Helper Cells ◽

Pi3k Pathway ◽

T Helper ◽

Immune Microenvironment ◽

Pten Loss ◽

Pathway Activation ◽

Tumor Types ◽

T Cell Exclusion

Abstract Background Recent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood. Methods Utilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation. Results We reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy. Conclusion These data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

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Neonatal lymphocyte subpopulations analysis and maternal preterm premature rupture of membranes: a pilot study

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2021-0375 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Margherita Amadi ◽

Silvia Visentin ◽

Francesca Tosato ◽

Paola Fogar ◽

Giulia Giacomini ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

T Helper Cells ◽

Lymphocyte Subpopulations ◽

T Helper ◽

Premature Rupture Of Membranes ◽

Premature Rupture ◽

Preterm Premature Rupture ◽

Helper Cells

Abstract Objectives Preterm premature rupture of membranes (pPROM) causes preterm delivery, and increases maternal T-cell response against the fetus. Fetal inflammatory response prompts maturation of the newborn’s immunocompetent cells, and could be associated with unfavorable neonatal outcome. The aims were to examine the effects of pPROM (Mercer BM. Preterm premature rupture of the membranes: current approaches to evaluation and management. Obstet Gynecol Clin N Am 2005;32:411) on the newborn’s and mother’s immune system and (Test G, Levy A, Wiznitzer A, Mazor M, Holcberg G, Zlotnik A, et al. Factors affecting the latency period in patients with preterm premature rupture of membranes (pPROM). Arch Gynecol Obstet 2011;283:707–10) to assess the predictive value of immune system changes in neonatal morbidity. Methods Mother-newborn pairs (18 mothers and 23 newborns) who experienced pPROM and controls (11 mothers and 14 newborns), were enrolled. Maternal and neonatal whole blood samples underwent flow cytometry to measure lymphocyte subpopulations. Results pPROM-newborns had fewer naïve CD4 T-cells, and more memory CD4 T-cells than control newborns. The effect was the same for increasing pPROM latency times before delivery. Gestational age and birth weight influenced maturation of the newborns’ lymphocyte subpopulations and white blood cells, notably cytotoxic T-cells, regulatory T-cells, T-helper cells (absolute count), and CD4/CD8 ratio. Among morbidities, fewer naïve CD8 T-cells were found in bronchopulmonary dysplasia (BPD) (p=0.0009), and more T-helper cells in early onset sepsis (p=0.04). Conclusions pPROM prompts maturation of the newborn’s T-cell immune system secondary to antigenic stimulation, which correlates with pPROM latency. Maternal immunity to inflammatory conditions is associated with a decrease in non-major histocompatibility complex (MHC)-restricted cytotoxic cells.

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Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽

10.1182/blood.v94.12.4060 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4060-4066 ◽

Cited By ~ 39

Author(s):

Maria Fiammetta Romano ◽

Annalisa Lamberti ◽

Rita Bisogni ◽

Corrado Garbi ◽

Antonio M. Pagnano ◽

...

Keyword(s):

Transcription Factors ◽

Cell Apoptosis ◽

Progenitor Cell ◽

Cell Types ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Mobility Shift ◽

Human Cord Blood ◽

Mobility Shift Assay ◽

Induced Apoptosis

Abstract We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

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Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽

10.1182/blood.v66.4.1002.bloodjournal6641002 ◽

1985 ◽

Vol 66 (4) ◽

pp. 1002-1005 ◽

Cited By ~ 4

Author(s):

J Cashman ◽

AC Eaves ◽

CJ Eaves

Keyword(s):

Growth Medium ◽

Specific Activity ◽

High Specific Activity ◽

Cell Types ◽

Hematopoietic Progenitor Cell ◽

Specific Cell ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Marrow Cultures

We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

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Keratinocyte growth factor augments immune reconstitution after autologous hematopoietic progenitor cell transplantation in rhesus macaques.

Blood ◽

10.1182/blood-2006-12-065623 ◽

2007 ◽

Vol 110 (1) ◽

pp. 441-449 ◽

Cited By ~ 75

Author(s):

Ruth Seggewiss ◽

Karin Loré ◽

F. Javier Guenaga ◽

Stefania Pittaluga ◽

Joseph Mattapallil ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Growth Factor ◽

Immune Reconstitution ◽

Progenitor Cell ◽

Rhesus Macaques ◽

Keratinocyte Growth Factor ◽

Hematopoietic Progenitor Cell ◽

Thymic Epithelial Cells ◽

T Cell Reconstitution

Opportunistic infections contribute to morbidity and mortality after peripheral blood progenitor cell (PBPC) transplantation and are related to a deficient T-cell compartment. Accelerated T-cell reconstitution may therefore be clinically beneficent. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells in mice. Here, we evaluated immune reconstitution after autologous CD34+ PBPC transplantation in rhesus macaques conditioned with myeloablative total body irradiation in the absence or presence of single pretotal body irradiation or repeated peritransplant KGF administration. All KGF-treated animals exhibited a well-preserved thymic architecture 12 months after graft. In contrast, thymic atrophy was observed in the majority of animals in the control group. The KGF-treated animals showed higher frequencies of naive T cells in lymph nodes after transplantation compared with the control animals. The animals given repeated doses of KGF showed the highest levels of T-cell receptor excision circles (TRECs) and the lowest frequencies of Ki67+ T cells, which suggest increased thymic-dependent reconstitution in these animals. Of note, the humoral response to a T-cell–dependent neo-antigen was significantly higher in the KGF-treated animals compared with the control animals. Thus, our findings suggest that KGF may be a useful adjuvant therapy to augment T-cell reconstitution after human PBPC transplantation.

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Hematopoietic Progenitor Cell Transplantation from Parental Donors for Children with Sickle Cell Anemia and Stroke.

Blood ◽

10.1182/blood.v110.11.3393.3393 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3393-3393

Author(s):

Paul Woodard ◽

Gregory Hale ◽

Wing Leung ◽

Raymond Barfield ◽

Kimberly Kasow ◽

...

Keyword(s):

T Cell ◽

Cell Transplantation ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Progenitor Cell ◽

Chronic Gvhd ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Pilot Studies ◽

Cd34 Cells

Although allogeneic hematopoietic progenitor cell transplantation (HPCT) can be curative for sickle cell anemia (SCA), most patients lack an HLA matched sibling donor or matched unrelated donor. A 2002 multidisciplinary conference held at St. Jude Children’s Research Hospital reached consensus that pilot studies using parental donors were reasonable and ethical. Subsequently, eight children with a history of clinically overt stroke were transplanted on two sequential pilot studies. Peripheral blood progenitor cells were obtained from parents with sickle cell trait. Conditioning was i.v. busulfan (targeted to Css 900 ng/ml) q 6 hours x 4 days, fludarabine 150–200 mg/m2, and OKT3/methylprednisolone and infusion of CD34+ HPCT for 3 patients. Five patients received i.v. busulfan (targeted to Css 900 ng/ml) x 4 days, cyclophosphamide 200 mg/kg, thiotepa 10 mg/kg, OKT3/methylprednisolone, and infusion of both CD34+ cells and CD3+ cells with a fixed T-cell addback of 1.0–1.5 x 105 CD3+ cells/kg. Six children received pre-transplant immunosuppression with hydroxyurea and azathioprine. The median follow-up of eight patients is 1.4 years (range 2 months–4 years). Five of eight had durable donor engraftment,4 of whom are alive and free of SCA post-HPCT. Rejection occurred in 4 patients and was successfully reversed with additional CD34+ cells in one of three patients. GVHD occurred in patients receiving a fixed T-cell addback or DLI: two patients had grade II acute graft-versus-host disease (aGVHD), one grade III aGVHD, and three patients developed chronic GVHD, including a fatal case of bronchiolitis obliterans organizing pneumonia (BOOP) and fungal sepsis. One engrafted patient developed medulloblastoma 2 years post-HPCT and is in remission after treatment. Further investigation revealed that this child inherited a novel germline p53 mutation. In this preliminary experience, haploidentical HPCT for children with SCA and stroke was associated with significant graft rejection and chronic GVHD. The addition of pre-transplant hydroxyurea and azathioprine, increased intensity of conditioning, and the use of T-cell addback to the graft did not improve engraftment in the second cohort. While offering the possibility of cure, haploidentical HPCT for SCA as performed in this experience is associated with significant toxicity and should only be pursued in the context of a rigorously designed and controlled prospective clinical trial.

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Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽

10.1182/blood.v94.12.4060.424k23_4060_4066 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4060-4066 ◽

Cited By ~ 2

Author(s):

Maria Fiammetta Romano ◽

Annalisa Lamberti ◽

Rita Bisogni ◽

Corrado Garbi ◽

Antonio M. Pagnano ◽

...

Keyword(s):

Transcription Factors ◽

Cell Apoptosis ◽

Progenitor Cell ◽

Cell Types ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Mobility Shift ◽

Human Cord Blood ◽

Mobility Shift Assay ◽

Induced Apoptosis

We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

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T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

eLife ◽

10.7554/elife.32417 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Tobias X Dong ◽

Shivashankar Othy ◽

Amit Jairaman ◽

Jonathan Skupsky ◽

Angel Zavala ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Tracking ◽

Cell Types ◽

Cell Receptor ◽

Receptor Activation ◽

Specific Cell ◽

Transgenic Expression ◽

Reporter Mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of ‘Salsa6f’, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients (‘sparkles’) as cells migrate.

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