CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.     

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (～50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.     

T-cell-depleted HLA non-identical bone marrow transplantation in the child: prevention of graft-versus-host reaction by administration of donor T lymphocytes alloreactive against the recipient

The success of HSCT from HLA partially disparate donors depends on the development of new strategies able to efficiently prevent GVHD and to protect patients from infections and relapse. Using an immunotoxin (IT) directed against the alpha-chain (p55) of the human IL-2r (RFT5-SMPT-dgA), we have previously shown that it is possible to kill mature T cells activated towards a specific HLA complex by a one-way MLR. We designed a clinical trial assessing the effect of infusing increasing doses of T lymphocytes in the setting of children recipients of non HLA genetically identical HSCT. Thirteen patients have been enrolled from September 1998 to April 2000 and fourteen HSCT have been realized in 13 patients (pts). Donors were MUD in 3 cases and familial HLA partially disparate in the remaining cases. Allodepleted donor T cells were injected between day +14 and day +30 provided that ATG was undetectable in the serum and blood PMN counts was > 500/microliter. The mean age of these patients was 17 months (range 1 to 42). Diagnosis included immune deficient and malignant hemopathies. Three patients received 1 x 10(5) allodepleted T cell/kg, 7 patients received 4 x 10(5)/kg and 4 patients received 6 x 10(5)/kg allodepleted T cells. Full inhibition of MLR was achieved in 12 out of 14 cases. In two cases, a residual T cell reactivity to the recipient was observed (4 to 5%) and patients developed grade II aGVHD. aGVHD occurred in 4 out of 11 grafted patients (all grade II). No chronic GVHD has developed, so far. Three patients died from severe VOD or PHT at day +34, day 51 and day +166, while one infected patient by VZV, CMV and EBV before HSCT died 6 months after transplantation from meningoencephalitis and another patient died from relapse at day +291. The patient for which there was no engraftment died at day +48 from staphylococcus infection. Overall survival is 54%, with a median follow up of 8 months; the mean time to reach a blood lymphocyte count > 500 was 41 days, to reach a CD3 count > 300 microliters 63 days (20-111), CD4 > 200 microliters 97 days and positive mitogen-induced proliferation 90 days. In three patients, a tetanus-toxoid positive proliferation was detected before immunization. From this intermediate analysis, we conclude that 1) specific allodepletion is an effective approach to prevent aGVHD in a haploincompatible setting, 2) data on immunological reconstitution suggest that infused T cells do survive and expand. A higher number of patients must be enrolled to determine the optimal number of T cells to infuse.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions

Skin lesions in the allergic form of atopic dermatitis (AD) are induced by allergen-specific T cells that infiltrate the skin at the site of allergen exposure. Although Th2-type CD4+ T cells appear to be crucial in AD pathophysiology, little is known about the contribution of CD8+ T cells in the development of the allergic skin inflammation. In the present study, we have analyzed the respective role of CD8+ and CD4+ T cells in the development of AD skin lesions in a mouse model of allergen-induced AD. In sensitized mice, CD8+ T cells are rapidly and transiently recruited to the allergen-exposed site and initiate the inflammatory process leading to skin infiltration with eosinophils and Th1/Th2-producing cells. CD8+ T cell-depleted mice show no inflammation, demonstrating that these cells are mandatory for the development of AD. In contrast, CD4+ T cell-depleted mice develop a severe form of eczema. Furthermore, adoptive transfer of CD8+ T cells from sensitized mice into naive recipient mice leads to skin inflammation soon after allergen exposure. These data indicate that allergen-primed CD8+ T cells are required for the development of AD-like lesions in mice.     

T cell subsets mediating lethal graft versus host disease: demonstration that synergy between CD4+ and CD8+ T cells is the predominant mechanism in low responder rat strains

T cell subsets from rat strains that have been characterized as high and low responders to alloantigen were examined for their capacity to mediate lethal graft versus host disease (GVHD) across strain combinations incompatible for class I, class II, and non-MHC antigens. Inocula of 5 X 10(7) lymph node and spleen cells (LC) from low responder DA (RT1a) and high responder W/F (RT1u) strains caused lethal GVHD in (W/F X DA)F1 hybrids given 6 Gy whole body irradiation. W/F CD4+ (W3/25+) cells (2 X 10(7], equal to the number in 5 X 10(7) LC mediated lethal GVHD but 10(8) DA CD4+ cells were required to cause lethal GVHD. CD8+ (MRC OX8+) cells (5 X 10(7] from W/F rats alone caused lethal GVHD but those from DA rats could not. Mixtures of CD4+ and CD8+ DA T cells, equivalent to the number in 5 X 10(7) LC, did mediate lethal GVHD, demonstrating that synergy between the subsets was the predominant mechanism with DA cells. These results suggest that differences in alloreactivity between the strains tested may be due to alternate requirements for the alloactivation of T cell subsets; the high responder subsets being self-sufficient and the low responder subsets being dependent upon each other.     

Alloantigen affinity and CD4 help determine severity of graft-versus-host disease mediated by CD8 donor T cells

TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.     

Gene expression profile of cytokines in patients with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with reduced conditioning

There are no reliable markers useful to predict the onset or the evolution of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT), although several candidate biomarkers have been identified from limited hypothesis-driven studies. In this study we evaluated 14 patients who received a reduced intensity conditioning HSCT. Seven patients had cGVHD, whereas 7 never developed cGVHD during the period of observation. The expression of 114 cytokines in immunoselected cell populations was explored by microarray analysis and 11 cytokines were selected for further evaluation by real-time PCR. Differential gene expression measurements showed a significant up-regulation for INFγ (interferon, gamma) in CD8+ and for TNFSF3 (tumor necrosis factor superfamily, member 3) and for TNFSF10 (tumor necrosis factor superfamily, member 10) in CD14+ cell population when comparing cGVHD with control samples. The expression levels were significantly decreased for TNFSF10 in CD8+ cell population and for TNFSF12 (tumor necrosis factor superfamily, member 12) and for PDGFβ (platelet-derived growth factor, beta) in CD4+. Our data seem to suggest that different immune populations can play a role in cGVHD pathogenesis and the early detection of gene expression profile in these patients could be useful in the monitoring of GVHD. We hypothesized that PDGFβ down-regulation could represent a negative feedback to compensate for enhanced expression of its receptor recently reported.     

Anti-CD95 (APO-1/Fas) autoantibodies and T cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected children

Advanced stages of HIV-1-infection are characterized by progressive CD4+ T cell depletion. Peripheral T cells from HIV-1+ donors show accelerated apoptosis in vitro. The CD95 (APO-1/Fas) receptor/ligand system is involved in this process. To further study deregulation of the CD95 system in peripheral T cells during HIV-1-infection, we measured CD95-expression on CD4+ and CD8+ T cells together with serum levels of soluble CD95 (sCD95) and anti-CD95 autoantibodies in HIV-1+ children and healthy controls. Anti-CD95 levels in HIV-1+ children were significantly elevated when compared to uninfected controls, whereas serum levels of sCD95 were not different. In HIV-1+ children, CD95-expression on CD4+ and CD8+ T cells increased with age. A strong correlation between depletion of CD4+ cells in vivo and increase in CD95-expression on CD4+ T cells was observed. In contrast, such a correlation was not found for CD8+ T cells. A negative correlation between anti-CD95 autoantibody levels and CD4+ T cell counts, that was predicted by multiple linear regression analysis of pooled data, was found in individual patients observed longitudinally by repeated measurements. Since anti-CD95 autoantibodies isolated from HIV-infected adults have previously been shown to induce apoptosis of sensitive target cells in vitro, we speculate that the interaction of these antibodies with CD95-positive and CD95-sensitive T cells in vivo might be involved in progressive T cell loss during HIV-1-infection.     

Breeding and preliminarily phenotyping of a congenic mouse model with alopecia areata

In the current study, the alopecia areata gene was introduced into the C57BL/6(B6) mouse through repeated backcrossing/intercrossing, and the allelic homozygosity of congenic AAtjmice(named B6.KM-AA) was verified using microsatellites. The gross appearance, growth characteristics, pathological changes in skin, and major organs of B6.KM-AA mice were observed. Counts and proportions of CD4+ and CD8+ T lymphocytes in peripheral blood were determined by flow cytometry. Results show that congenic B6.KM-AA mice were obtained after 10 generations of backcrossing/intercrossing. B6.KM-AA mice grew slower than B6 control mice and AA skin lesions were developed by four weeks of age. The number of hair follicles was reduced, but hair structures were normal. Loss of hair during disease progression was associated with CD4+ and CD8+ T lymphocytes infiltration peri- and intrahair follicles. No pathological changes were found in other organs except for the skin. In the peripheral blood of B6.KM-AA mice, the percentage of CD4+ T cells was lower and percentage of CD8+ T cells higher than in control mice. These findings indicate that B6.KM-AA mice are characterized by a dysfunctional immune system, retarded development and T-cell infiltration mediated hair loss, making them a promising new animal model for human alopecia areata.     

An imbalance of T cell subgroups exists in children with sepsis

The purpose of this study is to explore the role of different T cell subgroups in the pathogenesis of sepsis in children. Flow cytometry was used to detect the changes in the activation status and the number of T cell subgroups in the peripheral blood of children with sepsis; healthy children were selected as the control group. Compared with healthy children, the number of CD4+ T cells in the peripheral blood of children with sepsis did not change significantly (Z = 1.945, P = 0.052); though the ratio decreased and the median level dropped from 34.6% to 30.7% (Z = 2.257, P = 0.024). However, the number of CD8+ T cells in the blood of children with sepsis increased, and the median level also increased from 0.2 × 10⁹/L to 0.4 × 10⁹/L (Z = ?2.404, P = 0.016). In addition, CD3+CD8+HLA-DR + cell level significantly increased, and the median level increased from 4.2% to 24.3% (Z = ?5.370, P = 0.000). There was a large heterogeneity in the hospitalization time of sepsis in clinical patients. Compared to patients with a mean hospital stay of 6 days, patients with a median hospital stay of 13 days had a lower CD3+CD4+CD25 + cells percentage, while the percentage of CD3+CD8+HLA-DR+ was higher, resulting in a more apparent increase of CD3+ CD8+HLA-DR+/CD3+CD4+CD25+. Therefore, the failure of CD4+ T cell activation and proliferation, and the excessive activation and proliferation of CD8+ T cells play an important role in the pathogenesis of sepsis. The increase of CD3+CD8+HLA-DR+/CD3+CD4+CD25 + ratio was associated with the extended course of sepsis.     

CTL-promoting effects of CD40 stimulation outweigh B cell-stimulatory effects resulting in B cell elimination and disease improvement in a murine model of lupus

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA-->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.     

In vivo CD86 blockade inhibits CD4+ T cell activation, whereas CD80 blockade potentiates CD8+ T cell activation and CTL effector function

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F(1) graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F(1) mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F(1) recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4(+) T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8(+) T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8(+) T cell activation. In contrast, CD86 is critical for the activation of naive CD4(+) T cells in either a Th1 or a Th2 cytokine-mediated response.     

Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR beta-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed alphabetaTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+alphabetaTCR+ cells consisted of two subpopulations: a CD4+CD8- subpopulation and a CD4-CD8- subpopulation. Together, these results show that thymus-derived, Thy1+alphabetaTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.     

Foxp3-expressing T regulatory cells and mast cells in acute graft-versus-host disease of the skin

Acute graft-versus-host disease (aGVHD) limits the effectiveness of allogeneic hematopoietic stem cell transplantation. Foxp3 is required for the development and function of CD4+/CD25+ regulatory T cells (T-regs). Foxp3-expressing T-regs are thought to protect against GVHD. Mast cells are thought to be essential in CD4+/CD25+ regulatory T cell-dependent peripheral tolerance. Twenty biopsies of skin with grades I-III aGVHD were stained for Foxp3 and CD117. Inflammation was quantified by a 4 point scale, 0=no inflammation, 1=50%. T-regs and mast cells were quantified by a 4 point scale, 0=no cells per 20x field, 1=10 cells. T-regs were positively correlated with both inflammation and aGVHD grade. Twelve cases with low T-regs had mild inflammation and lower grades of aGVHD and 6 cases with high T-regs had dense inflammatory infiltrate and higher grades of aGVHD. The number of T-regs, mast cells and density of the inflammatory infiltrate were positively correlated only in cases with mild inflammation. In aGVHD of the skin, T-regs increased with the degree of inflammation and GVHD grade. Mast cells were present at the same density whether aGVHD was of lower or higher grade.     

同种异基因骨髓移植GVHD动物模型的建立及其病理生理学机制初探

目的：建立稳定的异基因骨髓移植GVHD（移植物抗宿主病）动物模型,并初步了解其病理生理学机制。方法：以BALB／c（H-2^d）雌性小鼠为受者,接受8Gy致死剂量的。Co全身照射后,输注雄性C57BL／6（H-2^b）供鼠的脾细胞和骨髓细胞,观察受鼠的体征、造血功能恢复及生存时间的变化,并进行病理学、嵌合体和T细胞亚群及相关细胞因子的检查。结果：模型组小鼠在移植后出现了典型的GVHD症状;肠、肝、脾、皮肤的病理学分析均属于Ⅳ度GVHD;嵌合体植入成功;以7、14和21d为检测时间点,发现模型组鼠体内T细胞亚群移植后较移植前CD3^＋CD4^＋T细胞数量减少,CD3＋CD8＋T细胞显著升高,CD4^＋和CD8^＋T细胞比例严重倒置,随着时间变化比值会逐渐升高,但仍然处于较显著的倒置水平;血清中IFN-γ、TNF-α在移植后＋7d表达显著增高,尤其是IFN-γ的表达在＋7d达峰值;IL-4和IL-10的水平在移植前后几乎没有变化。结论：建立了稳定的GVHD动物模型;此模型发病过程中,CD8^＋T细胞介导的CTL细胞毒性作用可能要大于CD4＋Th介导的细胞因子效应;IFN-γ、TNF-α炎症因子在GVHD的早期发挥重要作用;IL-4、IL-10的低水平分泌与急性GVHD的高发病率有关。     

Differential kinetics of antigen-specific CD4+ and CD8+ T cell responses in the regression of retrovirus-induced sarcomas

Despite the accepted role for CD4+ T cells in immune control, little is known about the development of Ag-specific CD4+ T cell immunity upon primary infection. Here we use MHC class II tetramer technology to directly visualize the Ag-specific CD4+ T cell response upon infection of mice with Moloney murine sarcoma and leukemia virus complex (MoMSV). Significant numbers of Ag-specific CD4+ T cells are detected both in lymphoid organs and in retrovirus-induced lesions early during infection, and they express the 1B11-reactive activation-induced isoform of CD43 that was recently shown to define effector CD8+ T cell populations. Comparison of the kinetics of the MoMSV-specific CD4+ and CD8+ T cell responses reveals a pronounced shift toward CD8+ T cell immunity at the site of MoMSV infection during progression of the immune response. Consistent with an important early role of Ag-specific CD4+ T cell immunity during MoMSV infection, CD4+ T cells contribute to the generation of virus-specific CD8+ T cell immunity within the lymphoid organs and are required to promote an inflammatory environment within the virus-infected tissue.     

Unexpected role of B and T lymphocyte attenuator in sustaining cell survival during chronic allostimulation

B and T lymphocyte attenuator (BTLA; CD272) can deliver inhibitory signals to B and T cells upon binding its ligand herpesvirus entry mediator. Because CD28, CTLA-4, programmed death-1, and ICOS regulate the development of acute graft-vs-host disease (GVHD), we wished to assess if BTLA also played a role in this T cell-mediated response. In the nonirradiated parental-into-F1 model of acute GVHD, BTLA+/+ and BTLA-/- donor lymphocytes showed equivalent engraftment and expansion during the first week of the alloresponse. Unexpectedly, BTLA-/- donor T cells failed to sustain GVHD, showing a decline in surviving donor cell numbers beginning at day 9 and greatly reduced by day 11. Similarly, inhibition of BTLA-herpesvirus entry mediator engagement by in vivo administration of a blocking anti-BTLA Ab also caused reduced survival of donor cells. Microarray analysis revealed several genes that were differentially expressed by BTLA-/- and BTLA+/+ donor CD4+ T cells preceding the decline in BTLA-/- donor T cells. Several genes influencing Th cell polarization were differentially expressed by BTLA+/+ and BTLA-/- donor cells. Additionally, the re-expression of the IL-7Ralpha subunit that occurs in BTLA+/+ donor cells after 1 wk of in vivo allostimulation was not observed in BTLA-/- donor CD4+ cells. The striking loss of BTLA-/- T cells in this model indicates a role for BTLA activity in sustaining CD4+ T cell survival under the conditions of chronic stimulation in the nonirradiated parental-into-F1 GVHD.     

Induction of epithelial migration of lymphocytes by intercellular adhesion molecule-1 in a rat model of oral mucosal graft-versus-host disease

To elucidate the involvement of intercellular adhesion molecule-1 (ICAM-1) in the migration of lymphocytes to the oral mucosal epithelium in a rat model of acute graft-versus-host disease (AGVHD), we investigated (1) ICAM-1 and major histocompatibility complex (MHC) class II expression by keratinocytes (KCs) and their role in the epithelial infiltration of CD8+ cells, (2) the tissue expression of interferon-γ (IFN-γ) mRNA and expression of IFN-γ receptor by KCs, and (3) the ability of KCs to direct CD8+ cells into the epithelial layers. We classified the oral mucosal lesions into three consecutive temporal phases on the basis of increased epithelial ICAM-1 expression: basal- (phase I), parabasal- (phase II), and pan-epithelial except for the cornified cell layer (phase III). Basal ICAM-1 expression by KCs preceded that of MHC class II molecules, infiltration of CD8+ cells and epithelial histological changes. Tissue expression of IFN-γ mRNA and expression of IFN-γ receptor on KCs evidenced by immunohistochemistry were detected in early lesions (phase I), indicating that locally produced IFN-γ induced ICAM-1 expression by KCs. CD8+ cells were bound to KCs in frozen sections of epithelial lesions, whereas no lymphocyte attachment was observed in normal KC. Adherence could be inhibited by pretreating CD8+ cells with lymphocyte function-associated antigen-1 (LFA-1) antibody and/or by pretreating sections with ICAM-1 antibody. Our data suggest that in the early phase of acute oral mucosal GVHD, the induction of ICAM-1 expression on KCs leads to the migration of CD8+ cells into the epithelium and that this is mediated in part by the ICAM-1/LFA-1 pathway.