Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes,and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease

Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4⁺ and CD8⁺ T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg^null mice. CD4⁺ T cells and, to a lesser extent, CD8⁺ T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4⁺ T cells also supported the activation and proliferation of CD8⁺ T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4⁺ T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4⁺ T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC)^+/+ mice but not in MHC^?/? mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD.     

Allogeneic Th1 Cells Home to Host Bone Marrow and Spleen and Mediate IFNγ-Dependent Aplasia

Bone marrow graft failure and poor graft function are frequent complications after hematopoietic stem cell transplantation and result in significant morbidity and mortality. Both conditions are associated with graft-versus-host disease (GVHD), although the mechanism remains undefined. Here we show, in 2 distinct murine models of GVHD (complete MHC- and class II-disparate) that mimic human peripheral blood stem cell transplantation, that Th1 CD4⁺ cells induce bone marrow failure in allogeneic recipients. Bone marrow failure after transplantation of allogeneic naïve CD4⁺ T cells was associated with increased CD4⁺ Th1 cell development within bone marrow and lymphoid tissues. Using IFNγ-reporter mice, we found that Th1 cells generated during GVHD induced bone marrow failure after transfers into secondary recipients. Homing studies demonstrated that transferred Th1 cells express CXCR4, which was associated with accumulation within bone marrow and spleen. Allogeneic Th1 cells were activated by radiation-resistant host bone marrow cells and induced bone marrow failure through an IFNγ-dependent mechanism. Thus, allogeneic Th1 CD4⁺ cells generated during GVHD traffic to hematopoietic sites and induce bone marrow failure via IFNγ-mediated toxicity. These results have important implications for prevention and treatment of bone marrow graft failure after hematopoietic stem cell transplantation.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Genetically modified human CD4+ T cells can be evaluated in vivo without lethal graft‐versus‐host disease

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti‐tumour T‐cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T‐cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD‐scid IL2rγ^null (NSG) mice accept the engraftment of mature human T cells; however, long‐term evaluation of transferred cells has been hampered by the xenogeneic graft‐versus‐host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4⁺ T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell‐derived peptide in the context of HLA‐DR4. The TCR‐transduced cells were transferred to NSG mice engineered to express HLA‐DR4 and to be deficient for murine class II MHC molecules. CD4⁺ T‐cell‐depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long‐term survival (up to 3 months post‐transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre‐transfer T‐cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T‐cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.     

Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b⁺Gr-1^high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1^high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b⁺Gr-1⁺ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.     

人源化NOD/SCID小鼠免疫细胞的动态变化与鉴定

目的： 比较脐血干细胞与单个核细胞移植NOD/SCID鼠所建立的人源化SCID模型，分析人源化淋巴细胞重建。方法： 磁珠分选法分离脐血中CD34⁺细胞，淋巴细胞分层液分离脐血单个核细胞，分别经尾静脉输入NOD/SCID小鼠。每隔2周采血至10周，流式细胞术动态检测人源淋巴细胞CD45、CD19、CD3抗原。第10周处死小鼠收集外周血、骨髓、胸腺组织，RT-PCR检测模型鼠组织中人β₂M基因及RAG2基因。结果： 两种类型细胞移植均可重建人源免疫细胞，人源淋巴细胞表达水平均在第8周达高峰。骨髓中人源淋巴细胞表达水平明显高于外周血。RT-PCR在外周血与骨髓检测到人β₂M基因及RAG2基因标志。结论： CD34⁺细胞移植重建人源化NOD/SCID免疫系统模型效果要好于脐血单个核细胞。人源T淋巴细胞在模型鼠骨髓中分化成熟。     

Improved Engraftment of Human Spleen Cells in NOD/LtSz-scid/scid Mice as Compared with C.B-17-scid/scid Mice

T and B lymphocyte-deficient mice homozygous for the severe combined immunodeficiency (SCID) mutation can be immunologically engrafted with human lymphocytes. However, low levels of human peripheral blood mononuclear cell engraftment are commonly observed, impeding full use of this model We now demonstrate that strain background in mice homozygous for the scid mutation is a strong determinant of levels of human lymphocyte engraftment. NOD/LtSz-scid/scid mice support higher levels of engraftment of both human spleen and peripheral blood mononuclear cells than do C.B-17-scid/scid mice. We observed, using human spleen cell injected scid mice, 1), high levels of engraftment of the host peripheral lymphoid tissues with human CD45⁺ (leukocytes), CD3⁺ (T cells), CD4⁺ (helper/inducer), and CD8⁺ (suppressor/cytotoxic) lymphoid cells for up to 24 weeks in NOD/LtSz-scid/scid mice; 2), migration of high numbers of human lymphocytes to peripheral lymphoid and nonlymphoid organs in NOD/LtSz-scid/scid, but not in C.B-17-scid/scid mice; 3), higher levels of serum immunoglobulin of human origin in NOD/LtSz-scid/scid mice than in C.B-17-scid/scid mice; 4), histological lesions character-istic of human anti-mouse xenoreactivity in NOD/LtSz-scid/scid mice; and 5), human origin antibodies against filarial antigens after engraftment with naive human spleen cells. The use of NOD/LtSz-scid/scid mice as recipients to achieve significantly enhanced human lymphopoietic cell engraftment will now enable human immunity to be more easily studied in animal models.     

Therapeutic Benefit of Bortezomib on Acute Graft-versus-Host Disease Is Tissue Specific and Is Associated with Interleukin-6 Levels

Bortezomib, a proteasome inhibitor capable of direct antitumor effects, has been shown to prevent acute graft-versus-host disease (GVHD) when administered in a short course immediately after bone marrow transplantation (BMT) in mice. However, when bortezomib is given continuously, CD4⁺ T cell–mediated gastrointestinal tract damage increases GVHD mortality. To investigate the protective effects of bortezomib on other organs, we used a CD8-dependent acute GVHD (aGVHD) model of C3H.SW donor T cells engrafted into irradiated C57BL/6 recipients (minor MHC mismatch), which lack significant gut GVHD. Our data in this model show that bortezomib can be given continuously to prevent and treat aGVHD mediated by CD8⁺ T cells, but this effect is organ specific, such that only skin, and not liver, protection was observed. Despite the lack of hepatic protection, bortezomib still significantly improved survival, primarily because of its skin protection. Reduced skin GVHD by bortezomib was correlated with reduced serum and skin IL-6 levels. Administration of a blocking IL-6 antibody in this model also resulted in similar cutaneous GVHD protection. These results indicate that bortezomib or blockade of IL-6 may prevent CD8⁺ T cell–mediated cutaneous acute GVHD.     

IL-7 Is the Limiting Homeostatic Factor that Constrains Homeostatic Proliferation of CD8+ T Cells after Allogeneic Stem Cell Transplantation and Graft-versus-Host Disease

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4⁺ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4⁺ T cell niche also contribute to impair CD8⁺ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8⁺ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8⁺ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naïve CD8⁺ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8⁺ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naïve CD8⁺ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 ⁺ T cell HP in the absence of DCs.     

Human immune system development and survival of non‐obese diabetic (NOD)‐scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells

Immunodeficient mice bearing targeted mutations in the IL2rg gene and engrafted with human immune systems are effective tools for the study of human haematopoiesis, immunity, infectious disease and transplantation biology. The most robust human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver haematopoietic stem cells [often referred to as the BLT (bone marrow, liver, thymus) model]. To evaluate the non‐obese diabetic (NOD)‐scid IL2rγ^null (NSG)–BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG–BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG–BLT mice develop a fatal graft‐versus‐host disease (GVHD)‐like syndrome, which correlates with the activation of human T cells and increased levels of human immunoglobulin (Ig). Onset of GVHD was not delayed in NSG mice lacking murine major histocompatibility complex (MHC) classes I or II and was not associated with a loss of human regulatory T cells or absence of intrathymic cells of mouse origin (mouse CD45⁺). Our findings demonstrate that NSG–BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD‐like pathological changes.     

Recipient-Derived Allo-iTregs Induced by Donor DCs Effectively Inhibit the Proliferation of Donor T Cells and Reduce GVHD

To compare the potency of recipient-derived, antigen-specific regulatory T cells induced by different dendritic cells (DCs; iTregs) and freshly isolated natural regulatory T cells (nTregs) in preventing mouse graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). CD4⁺ T cells from recipient BALB/c mice were stimulated with DCs from recipient BALB/c (syn-DCs), donor B6 (allo-DCs), and third-party C3H (third-party-DCs) mice to induce different iTregs. In parallel, nTregs were isolated from spleen cells of recipient BALB/c (syn-nTregs) and donor B6 (allo-nTregs) mice using magnetic-activated cell sorting. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the suppressive ability of these various regulatory T cells (Tregs). Both the iTregs and nTregs were transfused to GVHD mice on Days 0, 1, 3, and 5. Body weight, GVHD score, and survival time were monitored. Peripheral Tregs were subsequently examined on Days 7, 14, 21, and 28 after BMT, while chimerism was evaluated on Days 14 and 60. Histopathology of colon, liver, and spleen were also performed. DCs markedly induced CD25⁺ and Foxp3⁺ expression on CD4⁺ T cells. The allo-DC-induced Tregs (allo-iTregs) suppressed the proliferation of alloreactive T cells better than the other iTregs/nTregs in MLR assays (P < 0.05). Meanwhile, transfusion of the allo-iTregs reduced the severity of GVHD (P < 0.05), increased survival time compared with the GVHD group (P < 0.05), and enhanced the chimerism proportion. On Day 28 after BMT, the allo-iTregs group had the highest frequency of peripheral Tregs (P < 0.05). Recipient-derived allo-iTregs induced by donor DCs included predominant clones that specifically recognized donor antigens. These allo-iTregs not only prevented GVHD by suppressing the proliferation of donor-alloreactive T cells, but also promoted engraftment, and prolonged the survival of GVHD mice. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:825–836, 2019. © 2018 Wiley Periodicals, Inc.     

Host-Derived CD8+ Dendritic Cells Protect Against Acute Graft-versus-Host Disease after Experimental Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a frequent life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT) and induced by donor-derived T cells that become activated by host antigen-presenting cells. To address the relevance of host dendritic cell (DC) populations in this disease, we used mouse strains deficient in CD11c⁺ or CD8α⁺ DC populations in a model of acute GVHD where bone marrow and T cells from BALB/c donors were transplanted into C57BL/6 hosts. Surprisingly, a strong increase in GVHD-related mortality was observed in the absence of CD11c⁺ cells. Likewise, Batf3-deficient (Batf3^-/-) mice that lack CD8α⁺ DCs also displayed a strongly increased GVHD-related mortality. In the absence of CD8α⁺ DCs, we detected an increased activation of the remaining DC populations after HSCT, leading to an enhanced priming of allogeneic T cells. Importantly, this was associated with reduced numbers of regulatory T cells and transforming growth factor-β levels, indicating an aggravated failure of peripheral tolerance mechanisms after HSCT in the absence of CD8α⁺ DCs. In summary, our results indicate a critical role of CD8α⁺ DCs as important inducers of regulatory T cell–mediated tolerance to control DC activation and T cell priming in the initiation phase of GVHD.     

A role for Th2 cytokines in the suppression of CD8+ T cell-mediated graft rejection

A major histocompatibility complex (MHC) class I-specific T cell receptor (TCR)-transgenic mouse was used to study classical-type transplantation tolerance in the adult. Engraftment of MHC class I-incompatible bone marrow and tolerance to donor-type skin grafts were obtained using dimethylmyeleran (DMM) as a myeloablative agent and a non-depleting anti-CD8 monoclonal antibody (mAb) as the sole immunosuppressant. Surprisingly, bone marrow engraftment was facilitated by host CD4⁺ T cells, a subset normally considered unable to reject class I MHC-incompatible grafts. A combination of mAb to interleukins (IL)?4 and ?10 antagonized the “permissive” effects of host CD4⁺ T cells, indicating a possible role for Th2-type immunoregulation that can act on CD8⁺ T cells in this form of transplantation tolerance. The fate of graft-reactive T cells was monitored using anti-clonotypic antibodies. It was observed that bone marrow engraftment then led to peripheral deletion of mAb-blockaded, clonotype⁺ CD8⁺ T cells.     

Dipeptide γ-d-Glu-d-Trp (thymodepressin) inhibits migration of Cd34<Superscript>+</Superscript> cells from the bone marrow into peripheral blood during tumor growth

We studied the effect of dipeptide γ-d-Glu-d-Trp (thymodepressin) on migration of CD34⁺ hemopoietic precursors and their direct adhesion to fibronectin in tumor-bearing mice on days 8, 11, 15, and 17 of tumor growth and on expression of CXCR-4 (CD184⁺) to SDF-1 and integrin β₁ (CD29⁺) by bone marrow cells. In tumor-bearing mice treated with γ-d-Glu-d-Trp, the percent of CD34⁺ hemopoietic precursors in the peripheral blood considerably decreased throughout the observation period; the content of CD34⁺ hemopoietic precursors in the tumor tissue was 2–3-fold below the control against the background of increased content of CD34⁺ cells in the bone marrow. In animals treated with the peptide, the content of cells expressing CXCR-4 in the peripheral blood, bone marrow, and tumor tissue significantly decreased, while the percent of cells expressing integrin β₁ receptor (CD29⁺) in the bone marrow increased 2-fold, which was paralleled by an almost 2-fold increase in the percent of cells binding to fibronectin. We hypothesized that dipeptide γ-d-Glu-d-Trp suppressed mobilization/migration of CD34⁺ hemopoietic precursor cells from the bone marrow to the peripheral blood of tumor-bearing mice. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 105–108, July, 2008     

Prevention of Acute Graft-versus-Host Disease in a Xenogeneic SCID Mouse Model by the Humanized Anti-CD74 Antagonistic Antibody Milatuzumab

Prevention and treatment of graft-versus-host disease (GVHD) remains a major challenge, given that current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, often leading to severe infections and disease relapse. Thus, there is a critical need for novel anti-GVHD agents that can spare protective T-cell memory. Here we show that milatuzumab (hLL1), a humanized anti-CD74 antagonist monoclonal antibody, can moderately reduce the numbers of CD74-expressing B cells and myeloid dendritic cells, but has no effect on the survival of T cells that are CD74⁻. Consequently, milatuzumab inhibits allogeneic T-cell proliferation in mixed leukocyte reactions. In a human/mouse xenogeneic SCID mouse model in which GVHD is induced and mediated by engrafted human CD4⁺ T cells and dendritic cells, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses serum levels of human IFN-γ and IL-5, eliminates the infiltration of human lymphocytes into GVHD target organs (ie, lung, liver, and spleen), and significantly promotes survival (90% versus 20% for controls; P = .0012). Importantly, exposure to milatuzumab does not affect the number of cytomegalovirus-specific, IFN-γ–producing human CD8⁺ T cells in allogeneic mixed leukocyte reactions. These encouraging results warrant further exploration of milatuzumab as a possible new therapeutic agent for GVHD.     

Delayed onset of graft‐versus‐host disease in immunodeficent human leucocyte antigen‐DQ8 transgenic,murine major histocompatibility complex class II‐deficient mice repopulated by human peripheral blood mononuclear cells

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non‐obese diabetic (NOD)‐Rag1^–/– mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD‐Rag1^–/– γc^–/– mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft‐versus‐host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)‐DQ8] on a mouse class II‐deficient background (Aβ^–/–). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype‐matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3⁺ T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8‐expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.     

The immunosuppressive properties of non-cultured dermal-derived mesenchymal stromal cells and the control of graft-versus-host disease

Mesenchymal stromal cells (MSCs) have been developed for the prevention and treatment of graft-versus-host disease (GVHD). Non-cultured natural MSCs are considered ideal, as they better maintain their biological and therapeutic properties. The skin is the largest organ in the body and constitutes an interesting alternative to bone marrow for the generation of MSCs. Large numbers of dermal-derived-MSCs (DMSCs) can be easily generated without culturing in vitro, but their therapeutic effects still remain unclear. In this study, we described for the first time the use of non-cultured DMSCs for controlling GVHD in an MHC-mismatched mouse model and investigated their immunomodulatory effects. Our results showed that non-cultured mouse DMSCs decreased the incidence and severity of acute GVHD during MHC-mismatched stem cell transplantation in mice. This effect was mediated by the inhibition of splenic cell (SPC) proliferation and the enhancement of Treg cells. Consistent with the results in vivo, the results in vitro showed that human DMSCs inhibited the proliferation of peripheral blood mononuclear cells (PBMCs) by inhibiting the proliferation of CD3⁺ T cells. hDMSCs prevented PBMCs from entering S phase, suppressed the activation of CD3⁺ T cells and increased Treg proportions. In conclusion, DMSCs should be considered as a novel MSC source for the control of refractory GVHD.     

Adoptive Transfer of Low Numbers of CD4+ T Cells into SCID Mice chronically treated with Soluble IL-4 Receptor does not prevent Engraftment of IL-4-Producing T Cells

After intravenous injection of 10⁵ purified, lymph node (LN)-derived dm2 (H-2^d/L^d) CD4⁺ T cells into young C.B-17 scidjscid (severe combined immunodeficiency, SCID) mice (H-2^d/L^d+), the transplanted L^d- T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4⁺ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin-2 (IL-2) and interleukin-4 (IL-4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL-4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL-4 by chronic treatment of SCID mice with high doses of recombinant soluble IL-4 receptor (sIL-4R) changes the IL-4 or IL-2 expression pattern of CD4⁺ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL-4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL-4R protein (which does not bind murine IL-4), treated with the anti-murine IL-4 monoclonal antibody (MoAb) 11B11, or non-treated. Transplanted SCID mice treated with the recombinant murine sIL-4R protein preparations displayed detectable sIL-4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti-IL-4 activity in SCID mice. By contrast, no serum sIL-4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non-treated, treated with the MoAb 11B11, or treated with the recombinant human sIL-4R protein. The efficiency and the pattern of CD4⁺ T-cell engraftment, and the lymphokine-producing phenotype of the engrafted dm2 CD4⁺ cells, was not affected by the continuous IL-4-neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL-4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL-4 on the lymphokine-producing phenotype of CD4⁺ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL-4 activity (by either different sIL4-R protein constructs, or by the anti-IL-4 MoAb 11B11) did not lead to preferential engraftment of Th1-type CD4⁺ T cells after adoptive transfer of CD4⁺ T-cell populations into an immunodeficient recipient.     

Importance of Interleukin-7 in the Development of Experimental Graft-Versus-Host Disease

Interleukin (IL)-7 promotes both thymopoiesis and mature T lymphocyte survival and proliferation in experimental murine models of hematopoietic stem cell (HSC) transplantation. Because HSC products for transplantation also may contain IL-7–responsive mature T lymphocytes, we examined whether IL-7 is necessary for the induction of GVHD after allogeneic bone marrow transplantation (BMT). Lethally irradiated C57BL6J (B6) and B6.IL-7^-/- (both H2K^b) recipient mice were co-transplanted with T cell–depleted (TCD) bone marrow cells and lymph nodes (LNs) from either congenic B6.SJL (CD45.1⁺) or allogeneic BALB/c (H2K^d) donor mice. After transplantation, the recipient mice were subcutaneously injected with either human recombinant IL-7 or phosphate-buffered saline (PBS) for 60 days. No evidence of GVHD was detected in the congenic recipients or in the allogeneic B6/IL-7^-/- recipients treated with PBS; in contrast, significantly increased rates of GVHD-related mortality and morbidity were found in the allogeneic B6.IL-7^-/- recipients treated with IL-7. The proliferation and number of donor T cells were significantly lower at day 30 post-BMT in the PBS-treated B6.IL-7^-/- recipients compared with the IL-7–treated B6.IL-7^-/- mice. These experiments demonstrate that IL-7 is an important factor in the development of GVHD, presumably by supporting the survival, proliferation, and possibly activation of alloreactive donor-derived T cells in the recipients.