Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.     

The influence of allo-class II MHC-specific Th2 cells on the generation of CD4 and CD8 cytotoxic T cells to associated class I and class II MHC alloantigen

There is considerable interest in whether CD4 T cell function can affect the outcome of allogeneic transplants. In mice tolerant to an isolated class II MHC disparity, the normal Th1 activity in vitro associated with graft rejection is switched to Th2 in tolerant animals. Because clinical transplants involve multiple class I and II MHC disparities we tested how the switch to Th2 activity of tolerant mice would affect the generation of CD4 and CD8 cytotoxic T cells (CTL) against MHC alloantigens to which the mice were not tolerant. A.TH mice (K^kI^sD^d) were rendered neonatally tolerant of A.TL (K^kI^kD^d) and the generation of CD4 or CD8 CTL measured in a mixed lymphocyte reaction (MLR) against (A.TLxB6)F₁ stimulators. Normal mice generated CD4 CTL against both A.TL and B6 (K^bI^bD^b), but tolerant mice were unable to generate cytotoxicity against either A.TL or B6. However, tolerant cells were able to generate CD8 CTL against B6. IL-4 inhibited the generation of CD4, but not CD8, CTL by normal cells and anti-IL-4 antibody was shown to increase the generation of CD4 CTL against B6 in F₁ stimulated cultures. Overall the results showed that a Th2 response could inhibit the generation of CD4 CTL against concomitant alloantigen in a process at least partially involving IL-4, but that, conversely, tolerant Th2 cells could help in the generation of CD8 CTL. The results suggest that with whole MHC disparities a simple change of CD4 T cells to Th2 would not be enough to procure graft acceptance.     

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.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

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.     

Evidence for Th2 cell-mediated suppression of antibody responses in transgenic,beef insulin-tolerant mice

Clonal deletion, anergy and suppression have all been considered mechanisms of immunological tolerance. Although adoptive transfer of immunosuppression has been shown to occur in the periphery, particularly for transplantation tolerance, it has proven difficult to characterize this phenomenon further, due to the lack of suppressor T cell clones. To characterize tolerance towards a physiological soluble antigen, we constructed beef insulin (BI) transgenic (Tg) BALB/c (H-2^d) mice, in which the BI transgene is expressed in pancreatic β cells. These Tg mice were tolerant to BI immunization at the level of both humoral and cell-mediated immune responses. Adoptive transfer of splenocytes from Tg mice into normal syngeneic BALB/c mice demonstrated that the reduction in antibody production is regulated by transferred T cells. The cytokine profile of T cell clones obtained after selection in vitro demonstrated dominant Th1 clones from normal non-Tg mice and dominant Th2 clones from Tg mice. Some Th2 clones (CD4⁺) from Tg mice produced significant suppression of antibody production after adoptive transfer into normal syngeneic BALB/c mice. These data confirm the existence of Th2 regulatory T cells in vivo in a model of peripheral tolerance to a physiological soluble antigen as a potential mechanism for self tolerance.     

Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells

We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host–graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II major histocompatibility complex (MHC) molecule (I-A^d) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with anti-I-A^d antibodies than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on anti-I-A^d antibody-loaded membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in allogeneic transplant models.     

Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells

IDDM is characterized by leukocyte invasion to the pancreatic tissues followed by immune destruction of the islets. Despite the important function of Th17 cells in other autoimmune disease models, their function in IDDM is relatively unclear. In this study, we found association of elevated Th17 cytokine expression with diabetes in NOD mice. To understand the function of Th17 cells in IDDM, we differentiated islet‐reactive BDC2.5 TcR transgenic CD4⁺ cells in vitro into Th17 cells and transferred them into NOD.scid and neonate NOD mice. NOD.scid recipient mice developed rapid onset of diabetes with extensive insulitic lesions, whereas in newborn NOD mice, despite extensive insulitis, most recipient mice did not develop diabetes. Surprisingly, BDC2.5⁺ cells recovered from diabetic NOD.scid mice, in comparison with those from neonate NOD mice, showed predominant IFN‐γ over IL‐17 expression, indicating conversion of donor cells into Th1 cells. Moreover, diabetes progression in NOD.scid recipients was dependent on IFN‐γ while anti‐IL‐17 treatment reduced insulitic inflammation. These results indicate that islet‐reactive Th17 cells promote pancreatic inflammation, but only induce IDDM upon conversion into IFN‐γ producers.     

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4⁺ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4⁺ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4⁺ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.     

Amelioration of Skewed Th1/Th2 Balance in Tumor-Bearing and Asthma-Induced Mice by Oral Administration of Agaricus blazei Extracts

We showed in a previous study that hot-water extracts of Agaricus blazei (Agaricus extracts) had anti-tumor activity to Meth A fibrosarcoma, but it remains unclear whether the Agaricus extracts ameliorate the skewed balance of type-1 T helper (Th1) and type-2 T helper (Th2) cells. We examined whether Agaricus extracts effect the skewed Th1/Th2 balance in tumor-bearing and asthma-induced mice. When Meth A-bearing mice were given orally either Agaricus extracts or water once a day starting 5 days after tumor implantation, spleen T cells, prepared from tumor-bearing mice treated with Agaricus extracts, in response to anti-CD3 monoclonal antibody produced significantly higher levels of interferon γ (IFN-γ) than that of controls. The mRNA expression of IFN-γ-inducing protein 10 and the frequency of CD69⁺ or CD49d⁺ cells, among activated T cells infiltrated into tumors, significantly increased in Agaricus-treated mice, compared with those of tumor-controls. In asthma-induced mice, treatment with the Agaricus extracts caused significant downregulation of OVA-specific antibody responses of IgG1 and IgE but not of IgG2a, and significantly decreased total cell numbers, levels of interleukin 5, and eosinophil numbers in bronchial alveolar lavage fluids. IFN-γ production by anti-CD3-stimulated spleen cells, obtained from Agaricus-treated mice, significantly increased. Our results strongly suggest that oral administration of Agaricus extracts ameliorates the Th1/Th2 balance from the Th2-skewed conditions.     

Gut-homing CD4+ T cell receptor αβ+ T cells in the pathogenesis of murine inflammatory bowel disease

We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2^d (BALB/c, BALB/c^dm2, C. B-17+/+) mice. No antigen receptor-expressing lymphoid cells were found in GALT of congenic C. B-17 scid/scid (scid) mice. The heterotopic transplantation of a full-thickness gut wall graft from the ileum or colon of immunocompetent (C. B-17+/+, BALB/c^dm2) donor mice onto immunodeficient scid mice selectively reconstituted a CD3⁺ T cell receptor αβ⁺ CD4⁺ T cell subset. CD4⁺ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4⁺ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial layer and lamina propria of the small and large intestine, but not in peripheral LN. Scid mice heterotopically transplanted with gut from a congenic, immunocompetent donor developed clinical and histological signs of inflammatory bowel disease (IBD). Hence, the selective repopulation of GALT compartments with CD4⁺ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.     

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES:

FTY720 modulates CD4⁺T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment.

METHODS:

F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis.

RESULTS:

The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4⁺ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft.

CONCLUSIONS:

Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long‐term renal allograft outcome

The chemokine (C‐C motif) receptor 5 (CCR5) has been implicated in experimental and clinical allograft rejection. To dissect the function of CCR5 in acute and chronic renal allograft rejection, bilaterally nephrectomized WT and Ccr5^?/? C57BL/6 mice were used as recipients of WT BALB/c renal allografts and analyzed 7 and 42 days after transplantation. Lesion scores (glomerular damage, vascular rejection, tubulointerstitial inflammation) and numbers of CD4⁺, CD8⁺, CD11c⁺ and alpha smooth muscle actin (αSMA)⁺ cells were reduced in allografts from Ccr5^?/? recipients during the chronic phase. Increasing creatinine levels indicated deterioration of allograft function over time. While mRNA expression of Th1‐associated markers decreased between 7 and 42 days, Th2‐associated markers increased. Markers for alternatively activated macrophages (arginase 1, chitinase 3‐like 3, resistin‐like α, mannose receptor, C type 1), were strongly upregulated (mRNA and/or protein level) only in allografts from Ccr5^?/? recipients at 42 days. Ccr5 deficiency shifted intragraft immune responses during the chronic phase towards the Th2 type and led to accumulation of alternatively activated macrophages. Additionally, splenocytes from unchallenged Ccr5^?/? mice showed significantly increased arginase 1 and mannose receptor 1 mRNA levels, suggesting constitutive alternative activation of splenic macrophages. We conclude that Ccr5 deficiency favors alternative macrophage activation. This finding may be relevant for other inflammatory diseases that involve macrophage activation and may also influence future therapeutic strategies targeting CCR5.     

Modeling Chronic Graft-versus-Host Disease in MHC-Matched Mouse Strains: Genetics,Graft Composition,and Tissue Targets

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen–mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naïve CD4⁺ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4⁺ or CD8⁺ cells. Furthermore, co-transferred naïve and effector memory CD4⁺ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4⁺ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period.     

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4⁺ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4⁺ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35^?/? and IL-12p40^?/?) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4⁺ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-γ (IFN-γ) and interleukin-4 (IL-4) in purified CD4⁺ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-γ and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-γ mRNA in CD4⁺ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-γ in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.     

IL-2协同抗CD45RB抗体调节Treg/Th17细胞的比例并延长小鼠移植皮肤存活时间

目的:研究IL-2在抗CD45RB抗体诱导免疫耐受中对Treg/Th17细胞分化的影响,进一步阐明抗CD45RB抗体诱导免疫耐受的机制。方法:用免疫磁珠分选C57BL/6小鼠脾脏中的CD4+T细胞,在抗CD45RB抗体与IL-2的作用下培养72小时后,流式检测Treg/Th17细胞的变化。以BALB/c小鼠为供体,C57BL/6小鼠为受体建立同种异基因皮肤移植模型,分别给予抗CD45RB抗体及IL-2等治疗,术后1、3、5、7、9天取受体鼠脾细胞,动态检测Treg/Th17细胞的变化;术后第9天取移植皮肤HE染色观察炎性细胞的浸润情况;观察并记录移植皮肤的存活时间。结果:CD4+T细胞在IL-2联合抗CD45RB抗体的作用下培养72小时后,Treg比例升高,Th17细胞比例下降;IL-2联合抗CD45RB抗体治疗后明显延长小鼠移植皮肤的存活时间。结论:IL-2可以明显增强抗CD45RB抗体诱导免疫耐受的形成,使Treg细胞上调,下调Th17细胞,有利于免疫耐受的形成。     

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: A role for alloantibody

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8⁺ T cells and the mechanism whereby CD4⁺ T cells are able to independently mediate rejection. In this study of rejection of RT1A^a class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1^u recipients, we show that elimination of CD8⁺ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4⁺ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1^u athymic nude rats reconstituted with CD8⁺ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4⁺ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4⁺ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1^u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4⁺ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4⁺ T cell-dependent alloantibody plays a decisive role in effecting rejection.     

New Production of Eosinophils and the Corresponding Th1/Th2 Balance in the Lungs after Allergen Exposure in BALB/c and C57BL/6 Mice

Allergic asthma is associated with eosinophilic inflammation in the airways. Animal models commonly used to elucidate allergic inflammation mechanisms include BALB/c and C57BL/6 mice. Our aim was to evaluate lung eosinophilia and the corresponding Th1/Th2 balance in the two strains after allergen exposure. BALB/c and C57BL/6 mice were subjected to ovalbumin‐induced allergic airway inflammation using BrdU to label newly produced cells. The numbers of new eosinophils were evaluated by differential cell count and immunocytochemistry (MBP⁺BrdU⁺). Proliferation rate of lung eosinophils was measured by analysis of CD45⁺CCR3⁺BrdU⁺ cells by FACS. Distribution of newly produced eosinophils in the lung and the Th1/Th2 (CD4⁺T‐bet⁺/CD4⁺GATA‐3⁺) balance was evaluated by immunohistochemistry. Allergen challenge with ovalbumin induced comparable eosinophilia in bone marrow (BM), blood and lung tissue in both strains of mice compared to phosphate‐buffered saline controls, which was confirmed by immunocytochemistry. There was a small increase in the number of lung MBP⁺BrdU^? eosinophils in C57BL/6 mice compared to BALB/c mice, which suggests a basal increase in this strain following sensitization. While there was no difference in eosinophilic proliferation in the lung, the distribution of the newly produced eosinophils differs between the two strains. BALB/c mice showed staining primarily around vessels and airways, whereas C57BL/6 mice showed a more even distribution in the lung tissue. No difference in the Th1/Th2 balance was observed between two strains. This study shows that there is a difference in the distribution of eosinophils in the lung between the C57BL/6 and BALB/c mice, but no difference in eosinophil production or Th1/Th2 balance.     

Selective engraftment of memory CD4+ T cells with an unusual recirculation pattern and a diverse T cell receptor-Vβ repertoire into scid mice

Young (H-2^d, L^d+) severe combined immunodeficiency (scid) mice were injected intravenously with 10⁵ CD4+CD8^? T cells purified from spleen, thymus or lymph nodes (LN) of dm2 (H-2^d, L^d ) donor mice. In the immunodeficient recipients, the lymphoid compartment in the splenic white pulp was repopulated with donor-type T cells and cellularity in the red pulp was increased. In addition, donor-type CD4⁺ T cells repopulated the peritoneal cavity, mesenteric LN and the lamina propria of the small intestine of scid mice, but were undetectable in thymus and peripheral (inguinal, axillary) LN. Histological examination of repopulated mesenteric LN showed expanded subcapsular sinuses, repopulated cortical areas, but poorly developed high endothelial venules (HEV) indicating deficient blood-LN lymphocyte recirculation. The engrafted CD4⁺ T cell population had the surface phenotype of memory T cells (CD44/Pgp-l^high CD45RB^low) and expressed the Peyer's patch HEV-specific homing receptor CD49d (LPAM-1), but not the LN HEV-specific homing receptor LECAM-l. The CD4+ T cell population in spleen and mesenteric LN of transplanted scid mice displayed a diverse T cell receptor-V(3 repertoire. Transfer of titrated numbers (10³, 10⁴, 10⁵ cells per mouse) of CD4⁺ T cells into scid mice established donor-type T cell populations with this unusual homing pattern in all recipients. Repeated serial transfers of dm2 CD4⁺ T cells through young scid mice revealed an extensive in vivo expansion potential of transferred cells for > 18 months. The experimental system described represents an in vivo model to study the functional competence and the differentiation potential of a murine memory CD4⁺ T cell subset.