The Proportion of CD45RA+CD62L+ (Quiescent-Phenotype) T Cells within the CD8+ Subset Increases in Advanced Weight Loss in the Protein- or Energy-Deficient Weanling Mouse

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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Weight Loss in Obesity Reduces Colorectal Inflammation With Upregulation of FOXP3 Regulatory T Cells

Gastroenterology ◽

10.1016/s0016-5085(11)60519-2 ◽

2011 ◽

Vol 140 (5) ◽

pp. S-128

Author(s):

Swaroop Pendyala ◽

Michaela M. Ennis ◽

Judilyn Fuentes-Duculan ◽

Peter R. Holt

Keyword(s):

Weight Loss ◽

T Cells ◽

Regulatory T Cells

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Homeostatic Expansion of Donor FoxP3+ CD25+CD4+ T Cells in Recipients of CD4+ Cells Depleted Allogeneic Hematopoietic Graft.

Blood ◽

10.1182/blood.v108.11.3207.3207 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3207-3207

Author(s):

Mohammad S. Hossain ◽

Cynthia R. Giver ◽

Ned Waller

Keyword(s):

Weight Loss ◽

T Cells ◽

Stem Cell ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

De Novo ◽

Clinical Signs ◽

Post Transplant ◽

Allogeneic Bmt ◽

Homeostatic Expansion

Abstract Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects. Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS. Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p<0.005). Conclusion: Adoptive immunotherapy with the CD4-depleted hematopoietic graft results better immune reconstitution, caused extensive homeostatic expansion of donor stem cell-derived CD4+ T cells including significantly increased levels of FoxP3+CD25+ CD4+ regulatory T cells derived from de novo thymopoiesis without GvHD. The presence of donor FoxP3+CD25+ CD4+ regulatory T cells in the hematopoietic graft are not necessary for post-transplant expansion of donor stem-cell-derived regulatory T-cells via thymopoiesis.

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Total Body Irradiation Creates a Proinflammatory Milieu That Is Required for Minor, but Not Major, Mismatch Graft-Versus-Host-Disease.

Blood ◽

10.1182/blood.v116.21.3736.3736 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3736-3736

Author(s):

Sam C Nalle ◽

Peter A Savage ◽

Jerrold R Turner

Keyword(s):

Bone Marrow ◽

Weight Loss ◽

T Cells ◽

Graft Versus Host Disease ◽

Clinical Symptoms ◽

Conditioning Regimen ◽

Target Organ ◽

Host Disease ◽

Graft Versus Host ◽

Donor Cells

Abstract Abstract 3736 Background Graft-versus-host disease (GVHD) is a potentially fatal complication following allogeneic bone marrow transplantation (BMT). GVHD is characterized by three phases: (1) recipient tissue injury mediated by the conditioning regimen of irradiation and/or chemotherapy; (2) donor cell priming and activation; and (3) effector destruction of target tissue such as the intestine, liver, skin, and lung. There is some evidence to suggest that the second and third phases are dependent on the first, however, this has not been rigorously tested. Therefore, we developed major and minor mismatch models of GVHD and used immunodeficient recipients that readily accept allografts to analyze the requirement of irradiation conditioning on GVHD initiation and development. Experimental Design Wild-type (WT) B6 (H-2b) recipients received major mismatch (Balb/c, H-2d), minor mismatch (129, H-2b), or syngeneic (B6) BMTs. Recipients were lethally irradiated, and 24 hours later received an intravenous infusion of 5 × 106 bone marrow cells and 30 × 106 splenocytes, as a source of mature T cells. In adoptive transfer (AT) experiments, B6 Rag1−/− or B6 Rag2−/−/Pfp−/− (perforin deficient) recipients received 30 × 106 splenocytes. Data are represented as mean ± SEM. Results In lethally irradiated WT recipients, Balb/c→B6 caused severe GVHD that was fatal in all recipients by 24 days after BMT. 129→B6 led to a milder GVHD, where 50% of recipients survived to 35 days after BMT, and a few survived long-term (>100 days). Both major and minor mismatch GVHD were characterized by 15–25% weight loss, clinical symptoms including decreased activity, hunched posture, ruffled fur, and hair loss, and target organ histopathology. To test the requirement of irradiation conditioning in GVHD, we transferred Balb/c splenocytes into unconditioned B6 Rag1−/− recipients. No signs of GVHD developed. However, donor T cells were virtually undetectable 5 weeks after AT, indicating graft rejection. To test if this was due to recipient natural killer (NK) cells, a major mismatch AT was performed into B6 Rag2−/−/Pfp−/− recipients, which lack fully functional NKs. Balb/c→B6 Rag2−/−/Pfp−/− resulted in GVHD, including 10–15% weight loss, clinical symptoms, and target organ histopathology, although the disease was not as severe as that following lethal irradiation of WT recipients, consistent with a facilitative role for conditioning in disease progression. In contrast to the above, a 129 splenocyte AT did not cause GVHD in Rag1−/− or Rag2−/−/Pfp−/− recipients, despite donor T cell engraftment. This suggested that conditioning was required for minor mismatch GVHD. To test this, we sublethally irradiated Rag1−/− recipients prior to 129 AT. This resulted in GVHD, with 10–15% weight loss, clinical symptoms, and histopathology. At day 7 after AT, serum IFNγ, TNF, and IL-6 were significantly greater in mice that received irradiation and AT (compared to AT alone, p<0.05). Donor cells within spleens of mice that received irradiation and AT had a reduced fraction of Foxp3+/CD4+ (9.5%±2.3) compared to AT alone (16.8%±1.6, p=0.06), and increased intracellular CD8+ IFNγ expression (%IFNγ+:47.7±2.4 vs. 41.6±1.8, p<0.05; MFI:15722±2003 vs. MFI:8025±319, p<0.05). To determine whether more alloreactive donor cells were primed after irradiation, an in vivo killing assay was performed with recipient-specific targets. Combined irradiation and AT resulted in 66.3±13.5% killing efficiency while AT only had an 18.9±11.7% killing efficiency. Conclusions We conclude that irradiation conditioning is required for minor, but not major mismatch GVHD. The conditioning regimen creates the necessary proinflammatory milieu to prime sufficient numbers of alloreactive cells for GVHD. While this milieu can enhance development of major mismatch GVHD, it is not required for development of disease. Disclosures: No relevant conflicts of interest to declare.

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Presence of Vasoactive Intestinal Polypeptide Signaling in Plasmacytoid Dendritic Cells Improves Survival in a Murine Allo-BMT Model

Blood ◽

10.1182/blood.v128.22.2154.2154 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2154-2154

Author(s):

Jing-Xia Li ◽

Jian-Ming Li ◽

Edmund K Waller

Keyword(s):

Bone Marrow ◽

Weight Loss ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Expansion ◽

Cell Activation ◽

Post Transplant ◽

Cytokine Analysis

Abstract Introduction: Pre-clinical murine experiments and clinical data from allogeneic bone marrow transplantation (allo-BMT) have shown that increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft results in better clinical outcomes with less severe graft-versus-host disease (GvHD) and improved survival. The mechanism by which donor pDC modulate GvHD is unknown. Knowing that vasoactive intestinal polypeptide (VIP) is an immunosuppressive peptide , we reasoned that VIP signaling might play a role in regulating T-cell activation and expansion, and the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-BMT. We have tested the hypothesis that VIP synthesis by donor pDCs can modulate T cell alloreactivity. Methods: To explore the mechanisms by which pDC and VIP signaling regulate T cell activation in murine allo-BMT, we prepared B6-background donor cell grafts and transplanted them into lethally irradiated B10.BR recipients. In experiment 1, recipients were transplanted with grafts containing the combination of 5 x 103 VIP-GFP hematopoietic stem cells (HSC) and 3 x 106 VIP-wild type (VIP-WT) or VIP-knock out (VIP-KO) splenocytes. At day 7, splenocytes were isolated for flow cytometric analysis looking for GFP signal, which represents VIP-promotor activity. Experiment 2 used combinations of 5 x 103 VIP-WT HSC, 1 x 106 luciferase+ T cells, and 50 x 103 VIP-WT or VIP-KO pDC from B6 as donor grafts. Recipients were monitored for survival and GvHD based on fur texture, posture, activity, skin integrity and weight loss. T cell expansion was measured by bioluminescent imaging (BLI). Serum cytokines from bleeds at day 3 and day 8 post-transplant were analyzed using a Luminex 38 plex panel. Some recipients were euthanized on day 3 for intracellular cytokine analysis of splenic T cells. Results: In experiment 1, 7 days post-transplant, analysis of splenocytes from all mice showed increased activity of the VIP gene promoter in donor pDC that were derived from HSC, compared to other cell types. The VIP promoter signal was also stronger in donor HSC-derived pDCs, if originally transplanted with VIP-KO splenocytes. In experiment 2 over 70% of mice receiving HSC+T+VIP-WT pDC in the BM graft survived to day 100 post-transplant, while those getting VIP-KO pDC instead only had 30% survival (Fig 1A). All surviving recipients were fully engrafted by day 30. BLI revealed greater total T-cell proliferation (measured as radiance) in recipients of VIP-KO pDC (Fig 1B). Furthermore, recipients of VIP-KO pDC had more severe acute GvHD, with increased weight loss and GvHD clinical scores (Fig 1C, 1D). Some recipients were euthanized and their serum were collected for cytokine analysis on day 8 post-transplant, which showed up-regulation of pro-inflammatory or chemotactic cytokines MCP1, IL-1, IL-12, IL-17 in T cells co-transplanted with VIP-KO pDC compared to WT pDC. Conclusion: The present findings show that: 1) VIP is produced by donor pDC early after allo-BMT; 2) absence of VIP production by donor pDC leads to increased T-cell expansion in a murine allo-BMT model. Thus the pDC-T cell VIP signaling pathway is a critical element in controlling donor T cell alloreactivity after allo-BMT. Future studies will include VIP qPCR to confirm VIP production in donor pDC post-transplant, and determine the mechanism by which VIP production by pDC regulates T cell activity and modulates GvHD. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Tnfaip6 Secreted by Bone Marrow–Derived Mesenchymal Stem Cells Attenuates TNBS-Induced Colitis by Modulating Follicular Helper T Cells and Follicular Regulatory T Cells Balance in Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2021.734040 ◽

2021 ◽

Vol 12 ◽

Author(s):

Guangli Gu ◽

Xiaodan Lv ◽

Gengfeng Liu ◽

Ruizhi Zeng ◽

Shiquan Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Weight Loss ◽

T Cells ◽

Mesenchymal Stem Cells ◽

Tumor Necrosis ◽

Helper T Cells ◽

Follicular Helper ◽

Tnf Α ◽

Necrosis Factor

Objective: To investigate the immunological mechanism of bone marrow–derived mesenchymal stem cells (BM-MSCs) in inflammatory bowel disease (IBD).Methods: Mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)–induced colitis were intraperitoneally injected with phosphate-buffered saline, BM-MSCs, BM-MSCs with tumor necrosis factor–induced protein 6 (Tnfaip6) knockdown mediated by RNA interference recombinant adenovirus, and BM-MSCs–infected with control adenovirus or recombinant mouse Tnfaip6. The disease activity index, weight loss, and histological scores were recorded. Serum levels of Tnfaip6 and pro- and anti-inflammatory cytokines, including interleukin (IL)-21, tumor necrosis factor-alpha (TNF-α), IL-10 were measured by enzyme-linked immunosorbent assay. The relative expression levels of these cytokines, B-cell lymphoma 6 (BCL-6) and fork-like transcription factor p3 (Foxp3) in the colon were determined by real-time quantitative PCR (RT-qPCR). BCL-6 and Foxp3 are the master regulators of follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr), respectively. The infiltration of Tfh and Tfr in mesenteric lymph nodes (MLNs) and spleens was analyzed by flow cytometry.Results: Compared to the normal control group, the expression levels of BCL-6 and IL-21 in the colon, Tfh infiltration, and ratios of Tfh/Tfr in the MLNs and spleen, and the serum concentrations of IL-21 and TNF-α increased significantly in the colitis model group (p < 0.05). Intraperitoneal injection of BM-MSCs or Tnfaip6 ameliorated weight loss and clinical and histological severity of colitis, downregulated the expression of BCL-6, IL-21, and TNF-α, upregulated the expression of Foxp3, IL-10, and Tnfaip6 (p < 0.05), increased Tfr and reduced the infiltration of Tfh in the MLNs and spleen, and downregulated the Tfh/Tfr ratio (p < 0.05). On the other hand, BM-MSCs lost the therapeutic effect and immune regulatory functions on Tfh and Tfr after Tnfaip6 knockdown.Conclusion: Tfh increase in the inflamed colon, Tfh decrease and Tfr increase during the colitis remission phase, and the imbalance of the Tfh/Tfr ratio is closely related to the progression of IBD. Tnfaip6 secreted by BM-MSCs alleviates IBD by inhibiting Tfh differentiation, promoting Tfr differentiation, and improving the imbalance of Tfh/Tfr in mice.

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CD4+CD25+ T Cells Can Inhibit CD8 T Cell Mediated GVHD: Requirement for In Vivo Recognition of Allogeneic Host MHC Class II Antigens.

Blood ◽

10.1182/blood.v106.11.1307.1307 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1307-1307

Author(s):

Robert B. Levy ◽

Angela Jones

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

T Regulatory Cells ◽

Cd8 T Cell ◽

Class Ii ◽

Regulatory Cells

Abstract CD4 regulatory T (Treg) cells have shown promise in the transplantation mileu including the ability to inhibit the development of graft vs host disease (GVHD) following allogeneic hematopoietic stem cell transplants (HCT). The antigen specificity of the Treg population(s) involved is not yet clear nor is the role of their activation following transplant. We are interested in determining the requirement for recognition of host MHC antigens following infusion of CD4+CD25+ T cells in an experimental model of GVHD. To clearly distinguish the requirements of regulatory vs GVH reactive cells, a model of CD8 T cell mediated GVHD was developed using highly purified BALB/c (H2d) donor CD8+ T cells (Miltenyi column, 95-98%). CD8 T cells were transplanted together with T cell depleted (TCD) BALB/c BMC into 12.0 GY (6.0 Gy split dose) TBI conditioned C57BL/6 (B6, H2b) recipients. To support development of GVHD by these cells, resistance was inhibited by treatment of recipients with anti-NK1.1mab (PK136) at Days -1, 0 and +7. BALB/c CD8+ T cells at doses of 5.0x106 but not 2.5x106 induced weight loss and some lethality in B6 recipients. 5x106 CD8+ T cells were then transplanted into B6-MHC class II−/ − recipients. GVHD symptoms including weight loss and lethality were readily apparent in these mice post-transplant. Interestingly, GVHD was consistently more severe with respect to the induction of weight loss and lethality in MHC Class II−/ − vs B6-wt recipients. Highly enriched BALB/c CD4+CD25+ T cells (> 95%) were produced from spleen and lymph node cells following negative (B-cells, CD8 and NK) and positive (CD25) selection using Miltenyi magnetic bead columns. Co-transplant of 1x106 CD4+CD25+ T cells together with BALB/c CD8+ T cells into B6 recipients inhibited GVHD as assessed by the absence of weight loss and lethality compared to B6 recipients of CD8+ T cells alone. In contrast, BALB/c CD4+CD25+ T cells failed to protect B6-MHC class II−/ − recipients from severe CD8+ T cell mediated GVHD. These findings demonstrate that donor CD4+ T regulatory cells can suppress GVHD inducing CD8+ T cells after the former recognize host class II alloantigen following transplant. We hypothesize that activated CD4+CD25+ T regulatory cells inhibit GVH reactive T cells at the host APC interface. Future studies in this model can be designed to examine ex-vivo activated and expanded CD4+CD25+ T regulatory populations. Transplant of such cells will enable us to address questions regarding the importance of in vivo recognition of host class II in the regulation of GVHD by these cells.

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Donor T Cell Production of IL-21 Accelerates Graft-Versus-Host Disease Lethality.

Blood ◽

10.1182/blood.v112.11.2342.2342 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2342-2342

Author(s):

Christoph Bucher ◽

Christine Vogtenhuber ◽

Lisa Jasperson ◽

Angela Panoskaltsis-Mortari ◽

Emily Goren ◽

...

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Histopathological Examination ◽

Treg Cells ◽

Gamma Chain ◽

Term Survival ◽

Mean Values

Abstract IL-21, produced by T-cells, binds to the common gamma chain family member, IL21R, expressed on immune and colonic epithelial cells. IL-21 signaling results in the maturation, activation and proliferation of T, B, NK-cells and DCs. IL-21 has been implicated in Th17 generation/amplification and also modulating Treg differentiation. However, the relevance of this in disease is unclear. Therefore, we wanted to study the effects of IL-21 depletion and the role of Th17 and Treg cells in the context of GVHD. A lethally irradiated, complete MHC disparate model (B6 to B10.BR) using donor bone marrow cells alone or with the addition of wild-type (wt) CD4+CD25- Effector T-cells and irrelevant or anti-IL-21 Ab from days 0 to 25 twice per week. The administration of anti-IL-21Ab lead to a significant delay of weight loss and mortality (P<0.0001). To determine whether IL-21 deficiency accelerates or inhibits GVHD, studies were performed comparing GVHD-inducing ability of wt or IL-21 knockout (IL21−/−) donor CD4+CD25- T cells. A striking survival advantage of recipients of IL-21−/− vs wt CD4+CD25- T cells was observed (100% vs 0% surviving, P < 0.0001). Subsequent studies were performed using whole T cells, a more clinically relevant donor T cell graft source. Whereas recipients of wt T-cells all died of GvHD within 50 days of GvHD, recipients of IL-21 −/− T-cells showed significantly less weight loss and superior long-term survival (P < 0.0001). Histopathological examination of recipients of CD25-depleted T-cells on d14 revealed significantly reduced GvHD scores of the colonic mucosa and a trend towards lower GvHD scores in the small intestine, liver and the spleen in recipients of IL-21−/− cells. Although flow cytometry analysis of mononuclear cells showed no changes in the frequency of IL-17 producing cells in spleen, liver and colon, the frequency of interferon gamma producing CD4+ T- (Th1) cells was significantly lower in the spleen and the colon of recipients of IL-21−/− vs. wt T-cells. Moreover, increased Treg frequencies were seen in the colon of IL-21−/− vs wt CD25-depleted T cells (mean values: 7.5% vs. 2.1%; P < 0.003). We conclude that IL-21 production by either CD25- depleted T-cells or CD4+CD25- T cells is sufficient to increase GVHD mortality and that Treg inhibition, rather than Th17 generation or amplification, are likely contributing to GVHD lethality acceleration. We also conclude that IL-21 neutralization represents a novel approach for GVHD inhibition that warrants further investigation.

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Blockade of Interleukin 6 Signaling Attenuates Graft Versus Host Disease by Augmenting Regulatory T Cell Reconstitution

Blood ◽

10.1182/blood.v112.11.811.811 ◽

2008 ◽

Vol 112 (11) ◽

pp. 811-811

Author(s):

Xiao Chen ◽

Rupali Das ◽

Richard Komorowski ◽

Masahiko Mihara ◽

William Drobyski

Keyword(s):

Immune Response ◽

Weight Loss ◽

T Cells ◽

Graft Versus Host Disease ◽

Interleukin 6 ◽

Absolute Number ◽

Graft Versus Host ◽

Donor T Cells ◽

Peripheral Conversion

Abstract Graft-versus-host disease (GVHD) is characterized by a proinflammatory milieu that is attributable to conditioning regimen-induced host tissue damage as well as secretion of inflammatory cytokines by alloactivated donor T cells and other effector cell populations. Interleukin 6 (IL-6) is one of the cytokines that is a component of this proinflammatory environment and is of particular interest with respect to GVHD biology since presence or absence of IL-6 plays a pivotal role in determining the fate of naive T cells as they differentiate to become either proinflammatory or regulatory T cells, respectively. How IL-6 contributes to the pathophysiology of GVHD, however, is not well understood. To address this question, we employed a monoclonal antibody (Ab), MR16-1, that binds to both the soluble and membrane components of the IL-6 receptor to determine whether inhibition of IL-6 signaling affected GVHD severity and what impact antibody blockade had on the reconstitution and expansion of regulatory and effector T cells post transplantation. To identify regulatory T cells (Tregs) in vivo, we used transgenic donor mice in which the foxp3 gene is coupled to the enhanced green fluorescent protein (eGFP) so that eGFP would serve as a surrogate marker for Tregs. In initial studies, lethally irradiated Balb/c (H-2d) mice were transplanted with T cell depleted (TCD) B6 eGFP-foxp3 (H-2b) bone marrow plus splenocytes to induce GVHD. Cohorts of animals were then administered anti-IL-6R or isotype control Ab (0.5 mg/dose) once weekly. Mice treated with anti-IL-6R Ab had significantly less weight loss and reduced pathological damage in the liver, lung and colon when assessed 3–4 weeks post-BMT, demonstrating that protection was generalized and not restricted to a single target organ. Furthermore, antibody-treated animals also had a 12-fold increase in the number of donor-derived Tregs in the spleen compared to control mice (mean 9.5 × 104 versus 0.8 × 104, p=0.0001), indicating that blockade of IL-6 signaling was associated with increased Treg reconstitution. To determine if the increase in Tregs was operative under conditions where thymic function was absent, similar experiments were performed in thymectomized animals. These studies revealed that administration of anti-IL-6R Ab significantly reduced pathological damage in GVHD target organs and increased the absolute number of splenic Tregs. Since one of the mechanisms by which Tregs can be generated in the periphery in the absence of a thymus is through the peripheral conversion of CD4+ foxp3− to CD4+ foxp3+ Tregs, we examined whether blockade of IL-6 enhanced Treg conversion during GVHD. Lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4+ eGFP− T cells so that peripheral conversion could be determined by expression of eGFP. Anti-IL-6R antibody-treated mice had significantly reduced GVHD pathology that was associated with increased absolute numbers of converted Tregs in the spleen. Notably, we also observed a marked reduction in both TH1 and TH17 cells in these same animals, suggesting that IL-6 blockade might also affect expansion of alloreactive donor T cells. To address this question, we employed a murine model (B6® Balb.B) in which donor and recipient animals differ at multiple defined minor histocompatibility antigens. One of these minor antigens, termed H60, is preferentially recognized by donor CD8+ T cells during a GVH reaction. Mice administered anti-IL-6R antibody had a significant reduction in GVHD as assessed by weight loss and overall pathological score when compared to isotype-treated controls. Coincident with the reduction in pathology, there was also a significant decrease in the absolute number of CD8+ H60+ T cells, demonstrating that blockade of IL-6 signaling decreased the expansion of GVH-reactive donor T cells. In summary, we conclude that IL-6 plays a pivotal role in the pathophysiology of GVHD by driving the immune response in a proinflammatory direction. Conversely, blockade of IL-6/IL-6R interactions significantly attenuates GVHD by augmenting Treg reconstitution and decreasing expansion of alloreactive donor T cells which serves to re-balance the immune response. Given the challenges associated with the ex vivo expansion of Tregs for therapy in humans, these results also suggest that anti-IL-6R antibody treatment might represent a novel approach for the expansion of these cells in vivo.

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Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,

Blood ◽

10.1182/blood.v118.21.4015.4015 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4015-4015

Author(s):

Atsushi Satake ◽

Norifumi Sawamukai ◽

Taku Kambayashi

Keyword(s):

Weight Loss ◽

T Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Receptor Expression ◽

Host Disease ◽

Graft Versus Host ◽

Tgfβ Receptor

Abstract Abstract 4015 FoxP3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3+ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs. Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4+ T cells in the spleen were FoxP3+. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8+FoxP3+ T cells (1–3% of all CD8+ T cells) in the spleen and in the lymph nodes. These CD8+FoxP3+ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation. In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8+Foxp3+T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8+ and CD4+ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD. Disclosures: No relevant conflicts of interest to declare.

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