Human CD4+ CD25+ Regulatory T Cells Effectively Control Xenogeneic-GvHD Induced by Autologous T Cells in Rag2−/− γc−/− Immune-Deficient Mice.

Abstract The curative Graft-versus-Leukemia (GvL) effect of allogeneic stem cell transplantation (SCT) and Donor Lymphocyte Infusions (DLI) is frequently complicated by Graft-versus-Host Disease (GvHD). To date, it is not possible to prevent GvHD without sacrificing the GvL effect. Recently, in a number of murine transplantation studies, administration of naturally occurring CD4+CD25+ regulatory T (Treg) cells in recipients of allogeneic bone marrow effectively prevented GvHD without abrogating GvL. If human (hu)CD4+CD25+ Treg cells also possess such properties, they may become new cellular immunotherapeutics for the prevention of GvHD. Therefore, we have started to investigate the impact of huTreg cells on GvHD in a recently developed, highly relevant xenogeneic(x)-GvHD model in immunodeficient Rag2−/− γc−/− mice. This model represents several features of human allo-GvHD, such as the involvement of both CD4 and CD8 T cells, the association of GvHD with a “cytokine storm” of several Th1/Th2 and inflammatory cytokines and the similarity of skin histopathology to the human allo-GvHD(1). As in this model the x-GvHD is induced by the i.v. injection of huPBMC and the severity of x-GvHD correlates with the number of T cells in the administered PBMC, we explored the impact of Treg cells on x-GvHD either by depletion of Treg cells from huPBMC at different administration doses of effector T cells (4-15 x106 CD25− T cells) and or co-injection of autologous Treg cells at high doses of effector T cells (12-15 x106 T cells). PBMC were isolated from the buffycoats of healthy blood bank donors. Part of the PBMC was used as effector cells, the remaining cells were fractionated into CD25+ and CD25− subsets, which contain Treg cells and conventional T cells, respectively. Different groups of mice were injected with low to high doses of Treg-cell-depleted-PBMC or with high doses PBMC supplemented with 4-6 x106 Treg cell-enriched CD25+ cells. Control mice received equivalent numbers of unmodified PBMC only. The development of x-GvHD was monitored weekly by determination of body weight, clinical scores (ruffled fur, alopecia, mobility) and survival. Peripheral blood obtained from orbital vein was analyzed for human T cell engraftment and expansion. In three independent experiments, depletion of Treg cells significantly exacerbated the x-GvHD signs and lethality. In striking contrast, the development of x-GvHD was significantly inhibited by the co-injection of Treg cell enriched cell fractions. In two independent experiments Treg cells completely protected mice from lethal x-GvHD. Phenotypical analyses of peripheral blood revealed that addition of Treg cells did not disturb huT cell engraftment, but inhibited the expansion of huT cells between 3-5 weeks of administration. These results demonstrate the effective control of x-GvHD in Rag2−/− γc−/− mice by huTreg cells. Studies are underway to reveal the mechanism of GvHD inhibition and the impact of huTreg cells on GvL. (1) R.S. van Rijn, E.R. Simonetti, M.C.H. Hogenes, G. Storm, A. Hagenbeek, H. Spits, K. Weijer, A. C. M. Martens, and S.B. Ebeling. A new in vivo model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2−/− γc−/− double mutant mice.

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The Equilibration of Peripheral Blood and Tissue CD3+ T Cell Engraftment Is Influenced by Graft Versus Host Disease Following Reduced Intensity Conditioning Stem Cell Transplantation

Blood ◽

10.1182/blood.v116.21.2546.2546 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2546-2546

Author(s):

Victoria Harries ◽

Rachel Dickinson ◽

Venetia Bigley ◽

Matthew Collin

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Graft Versus Host ◽

Post Transplant ◽

Cell Engraftment ◽

Donor T Cells ◽

Patients At Risk ◽

Reduced Intensity

Abstract Abstract 2546 Alemtuzumab-containing reduced intensity transplantation regimens frequently induce a state of partial T cell chimerism in the blood of the recipient. It has been widely shown that partial T cell chimerism is associated with freedom from graft versus host disease (GVHD) and that the occurrence of GVHD is often associated with rapidly rising donor T cell engraftment. The mechanism by which this occurs remains unknown and recipient cells may be killed, out-competed for homeostatic niches or simply diluted out by expanding donor T cells. The skin, a target organ of GVHD, normally contains T cells which enter from the blood in the steady state. Studies in mice have highlighted the gate-keeping function of inflammation in allowing trafficking of host-reactive donor T cells into tissues during conversion from mixed to full donor chimerism in blood. This implies that the equilibration of donor engraftment in the blood and tissue may occur more rapidly in patients at risk for GVHD. To test this hypothesis, we set out to define the relationship between skin and blood donor T cell engraftment in patients with and without GVHD. Methods: We studied a group of 51 patients receiving fludarabine melphalan (FM) conditioning with alemtuzumab 30mg for matched related donors and 60mg for matched unrelated donors. Skin biopsies were obtained at 28 and 100 days post transplant, dermal T cells isolated by migration and chimerism assessed in sex-mismatched transplants by combined immunofluorescence/in situ hybidization for XY chromosomes. Peripheral blood myeloid (CD15+) and T cell (CD3+) chimerism was determined by short tandem repeat amplification at monthly intervals after transplantation. All patients gave consent for clinical follow up and post transplant blood and skin sampling for research purposes, according to protocols approved by the local research ethics committee of Northumberland and North Tyneside. Results: All patients achieved >95% myeloid engraftment by day 100. Median (range) T cell engraftment was variable and significantly higher after MUD transplants: 70% (9-99%) than MRD transplants: 21% (5-85%; Mann Witney p <0.05). The incidence of acute GVHD was also greater after MUD transplantation at 47% (grade I or II) compared with 11% (grade I only) for MRD recipients. Overall a positive correlation was observed between donor T cell engraftment in skin and blood at all time points (r = 0.5792; P 0.0187) and at 100 days (r = 0.6570; P 0.0281). Analysis of the data with respect to GVHD showed a further interesting finding. Patients who developed GVHD had the closest correlation between blood and skin donor engraftment, even when they were in a state of partial T cell chimerism prior to the onset of GVHD. Patients who did not develop GVHD but nonetheless eventually achieved full donor engraftment in the blood tended to show lower levels of donor T cell engraftment in the dermis at day 100. Individual examples of patients who did not develop GVHD are: blood 77%, dermis 37%; blood 77%, dermis 6%; blood 92%, dermis 25%, compared with patients who did develop GVHD: blood 55%, dermis 56%; blood 90%, dermis 75%; blood 100%, dermis 100%. Conclusion: This analysis supports the hypothesis that the equilibration of blood and tissue donor T cells is influenced by GVHD and may offer a means to predict patients at risk of GVHD after withdrawal of immunosuppression or donor lymphocyte infusion. Disclosures: No relevant conflicts of interest to declare.

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T Regulatory Cells in Graft-Versus-Host Disease of the Skin

Blood ◽

10.1182/blood.v112.11.4354.4354 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4354-4354

Author(s):

Jose Azar ◽

Steven Billings ◽

Jennifer E. Schwartz ◽

Yunlong Liu ◽

Menggang Yu ◽

...

Keyword(s):

T Cells ◽

Clinical Outcomes ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Treg Cells ◽

P Value ◽

Host Disease ◽

Graft Versus Host

Abstract FoxP3+ CD25+ T regulatory (Treg) cells are known to be present in normal skin. Treg cell functional deficiency resulting in loss of suppression of activation, differentiation or expansion of effector T cells could conceivably contribute to the pathophysiology of graft-versus- host disease (GVHD). Rezvani et al. suggest that levels of peripheral blood Treg cells in donors and recipients may predict the risk of acute GVHD. Rieger et al. found a significantly lower number of Treg cells in human colonic biopsies with GVHD versus CMV colitis or normal samples. There are no reports that evaluate tissue Treg cells in human skin affected by GVHD. This study was conducted to evaluate the distribution of Treg cells in skin affected by acute GVHD versus chronic GVHD. Archived samples from patients previously reported by routine histopathological methods as acute or chronic GVHD were collected, coded and entered into a database. For immunostaining, 4-μm thick serial sections were cut and deparaffinized. Immunohistochemical stains for CD4 (Neomarkers, 1:20), CD8 (Dako, prediluted), CD25 (Dako, 1:100), and Foxp3 (Serotec, 1:250) were performed using standard techniques. The dermatopathologist evaluating the samples was blinded to the clinical outcomes. Results were scored as 0 (<10% of lymphocytes positive), 1+ (10–25% positive), 2+ (26–50% positive), and 3+ (≥50% positive). Fourteen patients with acute skin GVHD and seventeen with chronic GVHD were identified from a database of patients who had undergone nonmyeloablative allogeneic peripheral blood transplantation in the recent past. The average scores for each immunostain were calculated and are summarized in table 1. The average FoxP3 score in acute GVHD specimens was significantly lower than that in chronic GVHD specimens (average, 0.57 versus 1.41; p-value = 0.011). The average scores of CD3, CD4, CD8 and CD25 immunostains were not significantly different between acute and chronic GVHD biopsies. These findings represent the initial observation of a distinction between the distribution of regulatory T cells in acute and chronic GVHD of the skin. These observations should be confirmed in a larger sample, supported by functional assays of Treg cells, and correlated with clinical outcomes. Such studies may help to elucidate the role of Treg cells in acute and chronic skin GVHD. Table 1. Average score CD 3 CD 4 CD 8 CD 25 Fox P3 Acute GVHD 2.93 2.21 2.14 0.29 0.57 Chronic GVHD 2.82 2.44 2.24 0.71 1.41 p-value 1.00 0.42 0.43 0.34 0.011

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Tregs In Graft-Versus-Host Disease: A Task Force In Trouble?.

Blood ◽

10.1182/blood.v116.21.3748.3748 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3748-3748

Author(s):

Sya N. Ukena ◽

Jens Grosse ◽

Stefanie Buchholz ◽

Michael Stadler ◽

Arnold Ganser ◽

...

Keyword(s):

T Cells ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Peripheral Tolerance ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host ◽

The Impact

Abstract Abstract 3748 Graft-versus-host disease (GvHD) remains the major clinical complication in hematopoietic stem cell transplantation (SCT) resulting in severe morbidity and significant mortality. This alloreactive immune response is mainly induced by donor T cells transplanted with the graft. Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance. In addition, data from murine models have shown that Tregs can prevent GvHD while preserving the graft-versus-leukemia effect. In order to functionally and dynamically characterize human Tregs after allogeneic SCT, we analyzed CD4+CD25highCD127dim T cells isolated from the peripheral blood of more than 80 patients with hematological malignancies every 30 days over half a year following SCT. Patients were divided into the following clinical groups: (A) no signs of acute or chronic GvHD, (B) acute GvHD, (C) chronic GvHD and (D) acute GvHD passed into chronic GvHD. Human peripheral blood lymphocytes were separated by Ficoll gradient and CD4+CD14−CD25highCD127dim T cells were isolated by MoFlow cell sorting. Isolated RNA was pooled and microarray analysis was performed by using Affymetrix HG_U133_Plus2.0 Arrays. Results were verified by using quantitative realtime RT-PCR. Additionally, Tregs were phenotypically analyzed by FACS. We monitored a continous but slower recovery of Tregs in GvHD within the first 6 months following PBSCT. Manifestation of acute and chronic GvHD correlated with significantly reduced frequencies of peripheral Tregs in the first month after PBSCT compared to patients without GvHD. Microarray data revealed a high stability of the Treg transcriptome in the first half year representing the most sensitive time window for tolerance induction. Moreover, comparison of the Treg gene expression profiles from patients with and without GvHD point to a reduced suppressive function of Tregs with diminished migration capacity to the target organs likely contributing to the development of GvHD. Our findings corroborate the impact of human Tregs in the pathophysiology of GvHD and identify novel targets for the manipulation of Tregs to optimize strategies for prophylaxis and treatment of life-threatening GvHD. Disclosures: No relevant conflicts of interest to declare.

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Granzyme B is not required for regulatory T cell–mediated suppression of graft-versus-host disease

Blood ◽

10.1182/blood-2009-07-233676 ◽

2010 ◽

Vol 115 (9) ◽

pp. 1669-1677 ◽

Cited By ~ 19

Author(s):

Sheng F. Cai ◽

Xuefang Cao ◽

Anjum Hassan ◽

Todd A. Fehniger ◽

Timothy J. Ley

Keyword(s):

Immune Responses ◽

Treg Cell ◽

Graft Versus Host Disease ◽

Granzyme B ◽

Target Organ Damage ◽

Organ Damage ◽

Treg Cells ◽

Host Disease ◽

Graft Versus Host

Abstract Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B–deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

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FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽

10.1182/blood-2008-06-163048 ◽

2008 ◽

Vol 112 (13) ◽

pp. 4953-4960 ◽

Cited By ~ 98

Author(s):

Mojgan Ahmadzadeh ◽

Aloisio Felipe-Silva ◽

Bianca Heemskerk ◽

Daniel J. Powell ◽

John R. Wunderlich ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Metastatic Melanoma ◽

Treg Cell ◽

Peripheral Blood ◽

Ex Vivo ◽

Production Capacity ◽

Biological Marker ◽

Foxp3 Expression ◽

Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

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517 Regulatory T cell functional identity is sustained by a glucose:lactate axis that is exploited in the tumor microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0517 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A553-A553

Author(s):

McLane Watson ◽

Paolo Vignali ◽

Steven Mullet ◽

Abigail Overacre-Delgoffe ◽

Ronal Peralta ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Treg Cell ◽

High Glucose ◽

Cell Function ◽

Treg Cells ◽

Effector T Cells ◽

Flux Analysis ◽

Major Barrier ◽

Suppressive Function

BackgroundRegulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked.MethodsFlow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing.ResultsHere we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival.ConclusionsThese data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

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Graded RhoA GTPase Expression in Treg Cells Distinguishes Tumor Immunity From Autoimmunity

Frontiers in Immunology ◽

10.3389/fimmu.2021.726393 ◽

2021 ◽

Vol 12 ◽

Author(s):

Khalid W. Kalim ◽

Jun-Qi Yang ◽

Vishnu Modur ◽

Phuong Nguyen ◽

Yuan Li ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Treg Cell ◽

Tumor Immunity ◽

Th17 Cell ◽

Treg Cells ◽

Effector T Cells ◽

Conditional Knockout ◽

Cell Plasticity ◽

Cell Homeostasis

RhoA of the Rho GTPase family is prenylated at its C-terminus. Prenylation of RhoA has been shown to control T helper 17 (Th17) cell-mediated colitis. By characterizing T cell-specific RhoA conditional knockout mice, we have recently shown that RhoA is required for Th2 and Th17 cell differentiation and Th2/Th17 cell-mediated allergic airway inflammation. It remains unclear whether RhoA plays a cell-intrinsic role in regulatory T (Treg) cells that suppress effector T cells such as Th2/Th17 cells to maintain immune tolerance and to promote tumor immune evasion. Here we have generated Treg cell-specific RhoA-deficient mice. We found that homozygous RhoA deletion in Treg cells led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th17, Th1 and Th2 cells. The immune activation was due to impaired Treg cell homeostasis and increased Treg cell plasticity. Interestingly, heterozygous RhoA deletion in Treg cells did not affect Treg cell homeostasis nor cause systemic autoimmunity but induced Treg cell plasticity and an increase in effector T cells. Importantly, heterozygous RhoA deletion significantly inhibited tumor growth, which was associated with tumor-infiltrating Treg cell plasticity and increased tumor-infiltrating effector T cells. Collectively, our findings suggest that graded RhoA expression in Treg cells distinguishes tumor immunity from autoimmunity and that rational targeting of RhoA in Treg cells may trigger anti-tumor T cell immunity without causing autoimmune responses.

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Expression of the T-Cell Activation Antigen, OX-40, Identifies Alloreactive T Cells in Acute Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v89.12.4652 ◽

1997 ◽

Vol 89 (12) ◽

pp. 4652-4658 ◽

Cited By ~ 66

Author(s):

Thomas V. Tittle ◽

Andrew D. Weinberg ◽

Cara N. Steinkeler ◽

Richard T. Maziarz

Keyword(s):

T Cells ◽

T Cell Activation ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cell Activation ◽

Peripheral Blood Lymphocytes ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract The OX-40 molecule is expressed on the surface of recently activated T lymphocytes. The presence of OX-40 on CD4+ T cells was analyzed in a rat haplo-identical (parental → F1) bone marrow transplant model of acute graft-versus-host disease (aGVHD). Increased numbers of activated CD4+ T cells that expressed the OX-40 antigen were detected in peripheral blood soon after transplantation before the earliest sign of disease. The peak of OX-40 expression occurred 12 days posttransplantation with a range of 18% to 36% of circulating T cells and remained 10-fold above background, never returning to baseline. A slight increase in OX-40 expression (range, 1% to 6%) was also detected on peripheral blood lymphocytes from control syngeneic F1 → F1 recipients. OX-40+ T cells were isolated from spleen, skin, lymph node, and liver tissue of rats undergoing aGVHD, but not in syngeneic transplants. OX-40+ T cells isolated from these tissues were of donor origin and were shown to be allo-reactive. These data raise the possibility of using the OX-40 antibody to detect and deplete selectively the T cells that cause aGVHD.

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