CD8+ Treg - From Mouse To Man

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3474-3474 ◽  
Author(s):  
Tobias A.W. Holderried ◽  
Hye-Jung Kim ◽  
Philipp A Lang ◽  
Harvey Cantor
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Human Homologue ◽  
Cd8 Cells ◽  
Tfh Cells ◽  
Self Tolerance

Abstract T cell-mediated regulation of the immune response to self and foreign antigens is essential to maintain immune homeostasis and prevent autoimmune tissue destruction. The majority of studies addressing this issue, both in mice and humans, have focused on the contribution of CD4+CD25+FOXP3+ regulatory T cells. The contribution of regulatory CD8+ T cells has not been appreciated until recently. Recent studies have identified a small subset of IL-15 dependent CD8+ regulatory T cells (Treg) that is essential for maintenance of self- tolerance and prevention of autoimmune disease in mice. Expression of a triad of cell surface markers – CD44+CD122+Ly49+ – has been used to distinguish and purify CD8+Treg (Kim et al., Nature 2010; Kim et al., PNAS 2011). Here we have defined the human homologue of CD8+ Treg. The Ly49 receptor, identified as a stable surface marker on CD8+ Treg, is a member of a multigenic/multiallelic receptor family recognizing classical MHC class I molecules. The functional homologue of murine Ly49 is the killer cell immunoglobulin-like receptor (KIR). Our analysis has revealed that, while expression of KIR subtype combinations appears to be stochastic and co-expression of these KIR receptors is random, the inhibitory KIR2DL2/3 and KIR3DL1 subtypes are dominantly expressed by human CD3+CD8+ T cells. Similar to murine CD8+ Treg, CD8+ T cells that express KIR2DL2/3 or KIR3DL1 also express CD44 and CD122. Moreover, consistent with murine CD8+ Treg, incubation with IL-15 results in activation and proliferation of KIR+CD8+ T cells that maintain a stable surface phenotype. Gene array analyses in mice has indicated that Helios, a highly conserved zinc finger transcription factor and member of the Ikaros family of transcription factors, is expressed by CD44+CD122+Ly49+ CD8+ Treg. Helios is involved in T cell development and expressed by ∼70% of FoxP3+CD4+ Treg but not by mature B cells, dendritic cells or myeloid cells. Our analyses identified a Helios+ subset in the CD8+ Treg population, which (compared to the Helios- subset) embodies many of the functional characteristics of CD8+ Treg in mice. Co-expression of Helios is also apparent in some KIR+CD8+ T cells in human samples. In mice, we have shown that CD8+ Treg target CD4 TFH cells and thus maintain self-tolerance. In vitro suppression assays revealed that KIR+CD8+ cells but not KIR–CD8+ cells confer inhibitory activity on CD4+CXCR5+ TFHtarget cells in humans. Taken together, our findings implicate KIR+CD8+ cells as the human homologue of murine CD8+ Treg, including expression of transcription factor Helios, responsiveness to IL-15 and suppression of CD4+ TFH cells. Understanding the genetic and biological features of this CD8+ T cell subset in humans opens the possibility of exploiting their regulatory activity for the development of immunotherapy in the context of autoimmune disease and cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3490-3490 ◽  
Author(s):  
Yuchi Honaker ◽  
Yufei Xiang ◽  
Logan Fisher ◽  
Karen Sommer ◽  
Troy R. Torgerson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Gene Editing ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Self Tolerance ◽  
Equity Ownership

Abstract Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4+ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3+ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease. Disclosures Scharenberg: Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership.

scholarly journals Alterations in regulatory T cells and immune checkpoint molecules in pancreatic cancer patients receiving FOLFIRINOX or gemcitabine plus nab-paclitaxel

2021 ◽  
Author(s):  
L. Sams ◽  
S. Kruger ◽  
V. Heinemann ◽  
D. Bararia ◽  
S. Haebe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Progression Free Survival ◽  
Ductal Adenocarcinoma ◽  
Immune Checkpoints ◽  
Prognostic Information ◽  
Peripheral Blood Mononuclear

Abstract Purpose This pilot study aimed on generating insight on alterations in circulating immune cells during the use of FOLFIRINOX and gemcitabine/nab-paclitaxel in pancreatic ductal adenocarcinoma (PDAC). Patients and methods Peripheral blood mononuclear cells were isolated before and 30 days after initiation of chemotherapy from 20 patients with advanced PDAC. Regulatory T cells (FoxP3+) and immune checkpoints (PD-1 and TIM-3) were analyzed by flow cytometry and immunological changes were correlated with clinical outcome. Results Heterogeneous changes during chemotherapy were observed in circulating T-cell subpopulations with a pronounced effect on PD-1+ CD4+/CD8+ T cells. An increase in FoxP3+ or PD-1+ T cells had no significant effect on survival. An increase in TIM3+/CD8+ (but not TIM3+/CD4+) T cells was associated with a significant inferior outcome: median progression-free survival in the subgroup with an increase of TIM-3+/CD8+ T cells was 6.0 compared to 14.0 months in patients with a decrease/no change (p = 0.026); corresponding median overall survival was 13.0 and 20.0 months (p = 0.011), respectively. Conclusions Chemotherapy with FOLFIRNOX or gemcitabine/nab-paclitaxel induces variable changes in circulating T-cell populations that may provide prognostic information in PDAC.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

scholarly journals Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells

2018 ◽  
Vol 215 (9) ◽  
pp. 2265-2278 ◽  
Author(s):  
Colleen M. Lau ◽  
Ioanna Tiniakou ◽  
Oriana A. Perez ◽  
Margaret E. Kirkling ◽  
George S. Yap ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd8 T Cells ◽  
Functional Differentiation ◽  
Specific Gene ◽  
Hematopoietic Stem

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen–specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

scholarly journals CXCR5 and ICOS expression identifies a CD8 T-cell subset with TFH features in Hodgkin lymphomas

2018 ◽  
Vol 2 (15) ◽  
pp. 1889-1900 ◽  
Author(s):  
Kieu-Suong Le ◽  
Patricia Amé-Thomas ◽  
Karin Tarte ◽  
Françoise Gondois-Rey ◽  
Samuel Granjeaud ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Subset ◽  
Cd8 T Cell ◽  
T Cell Subset ◽  
Tfh Cells ◽  
Key Points ◽  
Functional Features

Key Points A subset of CD8 T cells in some Hodgkin lymphomas shares phenotypic and functional features with CD4 TFH cells.

Thymomas alter the T-cell subset composition in the blood: a potential mechanism for thymoma-associated autoimmune disease

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3872-3879 ◽  
Author(s):  
Viola Hoffacker ◽  
Anja Schultz ◽  
James J. Tiesinga ◽  
Ralf Gold ◽  
Berthold Schalke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Disease ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Cell Subset ◽  
Cell Repertoire ◽  
T Cell Subset ◽  
Functional Studies ◽  
Circulating T Cells

Abstract Thymomas are the only tumors that are proven to generate mature T cells from immature precursors. It is unknown, however, whether intratumorous thymopoiesis has an impact on the peripheral T-cell pool and might thus be related to the high frequency of thymoma-associated myasthenia gravis. This study shows, using fluorescence-activated cell sorting-based analyses and T-cell proliferation assays, that thymopoiesis and T-cell function in thymomas correspond with immunologic alterations in the blood. Specifically, the proportion of circulating CD45RA+CD8+ T cells is significantly increased in patients with thymoma compared with normal controls, in accordance with intratumorous T-cell development that is abnormally skewed toward the CD8+ phenotype. Moreover, it is primarily the proportion of circulating CD45RA+CD8+ T cells that decreases after thymectomy. The results also demonstrate that T cells reactive toward recombinant autoantigens are distributed equally between thymomas and blood, whereas T-cell responses to foreign antigen (ie, tetanus toxoid) are seen only among circulating T cells and not among thymoma-derived T cells. These functional studies support the hypothesis that thymopoiesis occurring within thymomas alters the peripheral T-cell repertoire. Because many thymomas are enriched with autoantigen-specific T cells, a disturbance of circulating T-cell subset composition by export of intratumorous T cells may contribute to paraneoplastic autoimmune disease arising in patients with thymoma.

scholarly journals A Critical Role for Cd40–Cd40 Ligand Interactions in Amplification of the Mucosal Cd8 T Cell Response

1999 ◽  
Vol 190 (9) ◽  
pp. 1275-1284 ◽  
Author(s):  
Leo Lefrançois ◽  
Sara Olson ◽  
David Masopust
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Cd40 Ligand ◽  
Interleukin 12 ◽  
Cd8 Cells

The role of CD40 ligand (CD40L) in CD8 T cell activation was assessed by tracking antigen-specific T cells in vivo using both adoptive transfer of T cell receptor transgenic T cells and major histocompatibility complex (MHC) class I tetramers. Soluble antigen immunization induced entry of CD8 cells into the intestinal mucosa and cytotoxic T lymphocyte (CTL) differentiation, whereas CD8 cells in secondary lymphoid tissue proliferated but were not cytolytic. Immunization concurrent with CD40L blockade or in the absence of CD40 demonstrated that accumulation of CD8 T cells in the mucosa was CD40L dependent. Furthermore, activation was mediated through CD40L expressed by the CD8 cells, since inhibition by anti-CD40L monoclonal antibodies occurred after adoptive transfer to CD40L-deficient mice. However, mucosal CD8 T cells in normal and CD40−/− mice were equivalent killers, indicating that CD40L was not required for CTL differentiation. Appearance of virus-specific mucosal, but not splenic, CD8 cells also relied heavily on CD40–CD40L interactions. The mucosal CTL response of transferred CD8 T cells was MHC class II and interleukin 12 independent. The results established a novel pathway of direct CD40L-mediated CD8 T cell activation.

Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3188-3188
Author(s):  
Denise E. Sabatino ◽  
Federico Mingozzi ◽  
Haifeng Chen ◽  
Peter Colosi ◽  
Hildegund C.J. Ertl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Capsid Protein ◽  
Cd8 T Cells ◽  
Cell Epitope ◽  
T Cell Response ◽  
Cd8 Cells ◽  
Media Control ◽  
Ifn Γ

Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.

Effects of Amotosalen Hydrochloride and Ultraviolet a Light on CD4 and CD8 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4981-4981
Author(s):  
Catherine T. Jordan ◽  
James C. Zimring ◽  
John D. Roback
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Differential Sensitivity ◽  
Ultraviolet A ◽  
Differential Effects ◽  
Cd8 Cells ◽  
Uva Light ◽  
In Vivo Effects

Abstract Background: Graft versus host disease (GvHD) and infections by opportunistic pathogens, such as cytomegalovirus (CMV), are causes of significant morbidity and mortality in recipients of allogeneic bone marrow transplants (BMT). Thus, there is a need for methods of graft engineering that inhibit the alloreactive T cells responsible for lethal GvHD without compromising the activity of antiviral T cells. In a murine MHC-mismatched BMT model, we have previously demonstrated that adoptive transfer of polyclonal T cells from donors immunized to murine CMV (MCMV) can decrease viral load but cause lethal GvHD. However, pretreatment of these cells with the psoralen, amotosalen hydrochloride, and ultraviolet A (UVA) light prevents GvHD without compromising antiviral response. We have previously hypothesized that these effects were due to differential sensitivity of naïve and memory T cells to amotosalen/UVA. Recent investigations have demonstrated that CD4 T cells are most directly responsible for lethal GvHD in this model. This observation suggested an alternative hypothesis, equally consistent with previous data, that the observed in vivo effects of amotosalen/UVA treatment are due to differential effects on CD4 and CD8 T cells. The assessment of this new hypothesis is the focus of the current studies. Methods: Two models of T cell activation/proliferation were utilized to test the effects of amotosalen/UVA treatment on CD4 and CD8 cells: stimulation of B6.PL (H-2b) cells with concavalinA, and primary mixed lymphocyte reaction (MLR) between MHC-mismatched B6.PL (H-2b) and BALB/c (H-2d). Responder cells were pretreated with 2nM amotosalen and varying doses of UVA light (0–5 minutes). Proliferation of CD4 and CD8 cells was measured by flow cytometric analysis of CFSE-labeled responder cells. Results: In both systems, CD4 proliferation was effectively eliminated by immediately prior treatment with amotosalen and UVA doses of 1 minute or higher. CD8 proliferation was eliminated by amotosalen and UVA doses of 2 minutes and higher. Both the amount of division on a per cell basis and the overall number of cells that initiated division followed similar trends. Conclusions: These data demonstrate that both CD4 and CD8 T cells are sensitive to treatment with amotosalen/UVA and suggest a subtle difference in sensitivity of CD4 and CD8 populations. Since division of both CD4 or CD8 cells is inhibited at doses of amotosalen/UVA that prevent GvHD but allow anti-viral activity in vivo, it is unlikely that the observed differential sensitivity of T-cell subsets is sufficient to explain the in vivo effects of amotosalen/UVA treatment in this model. Using similar methodologies, ongoing studies are assessing the hypothesis that amotosalen/UVA has differential effects on naïve and mature T cells.

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