CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

Adoptive transfer: The role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo

ABSTRACT: The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3⁺ CD8⁺ T cells, but not CD4⁺ T or NK1.1⁺ cells, completely inhibited lysis of human tumor cells, suggesting that CD3⁺ CD8⁺ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8⁺ cytotoxic T lymphocytes to eradicate established xenografts.     

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor-Specific CD8+ Memory T Cells

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8⁺ T-cell responses in mice that lack the IFN-I receptor (IFN-IR^−/−). We found that IFN-IR^−/− mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR^−/− male skin was transplanted to syngeneic IFN-IR^−/− female mice. Quantitation of the male (H-Y)-specific CD8⁺ T-cell response in these mice revealed normal generation of donor-specific CD8⁺ effector T cells but fourfold reduction in CD8⁺ memory T cells. Memory CD8⁺ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR^−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8⁺ T cells into long-lived, functional memory T cells.     

CD4+CD25+FOXP3+ Regulatory T Cells Increase De Novo in Kidney Transplant Patients After Immunodepletion with Campath-1H

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3⁺ regulatory T (Treg) cells. Flow cytometry demonstrated that CD4⁺CD25⁺FOXP3⁺ lymphocytes increased significantly within the CD4⁺ T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4⁺CD25⁺FOXP3⁺ cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo .     

Successive emergence of two CD 8 subsets in primary CMV infection of allograft recipients

Abstract Allograft recipients with cytomegalovirus (CMV) infection develop increased proportions of circulating CD8 lymphocytes. A longitudinal study of 11 kidney and 5 liver allograft recipients with primary CMV infection but no other aetiological factor to explain graft dysfunction revealed selective imbalances in peripheral blood CD8' T cell subsets. Initially, CMV viraemia was associated with elevated CD8+bright' T cell numbers and T cell activation. Activation markers fell to normal when viral cultures became negative (before the end of the 1st month). During the 2nd-6th months, most (12/16) patients continued to have high CD8⁺ T cell counts (1050–2900 CD8⁺ cells/mm3), comprising an uncommon CD8⁺ T cell subset, as 45–73% of CD8^{+ bright} lymphocytes were CD3⁺ and TCRαβ⁺ but were not stained by anti-CD28, CD11b, CD16, CD56 and CD57 antibody. Unexpectedly, CD 8⁺ CD 57⁺ T cells, a hallmark of CMV infection, did not appear until the 2nd-6th months of primary CMV infection, and their numbers increased progressively thereafter. They became the predominant CD8⁺ T cell subset after about 6 months of infection and their persistence for several (up to 4) years was strongly correlated ( r = 0.87) with expansion of CD8⁺ cells. Persistence of CD 8 lymphocytosis was, thus, directly related to the rate of expansion of an uncommon CD 8⁺CD 57^- subset and its progressive replacement by CD 8⁺CD 57 ⁺ T cells that were chronically elicited by CMV.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

An Anti-CD103 Immunotoxin Promotes Long-Term Survival of Pancreatic Islet Allografts

Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103⁺ cells from wild type hosts. To circumvent this problem, we conjugated the nondepleting anti-CD103 monoclonal antibody, M290, to the toxin, saporin, to produce an immunotoxin (M290-SAP) that efficiently depletes CD103⁺ cells in vivo . Herein, we show that M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in the blood, spleen, mesenteric lymph nodes and intestinal epithelium of treated mice. We further demonstrate that M290-SAP promotes indefinite islet allograft survival in a fully MHC mismatched mouse model. The prolonged islet allograft survival resulting from M290-SAP treatment was associated with multiple effects in the host immune system including not only depletion of CD103-expressing leukocytes, but also an increase in CD4⁺CD25⁺FoxP3⁺ T regulatory cells and a predominance of effector-memory CD8 T cells. Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in allograft rejection.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with rejection.     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

Epidural anaesthesia blocks changes in peripheral lymphocytes subpopulation during gastrectomy for stomach cancer

To examine the effects of surgery and anaesthesia on cell-mediated and humoral immunity, we investigated changes in subpopulations of peripheral T cells and B cells during gastrectomy. Twenty-one patients with gastric cancers who underwent total or subtotal gastrectomy were randomly assigned to receive thiopental/N₂O general anaesthesia supplemented with either epidural (epidural group, n =12) or enflurane(enflurane group, n =9). The changes in peripheral lymphocyte subpopulations were detected and quantified using single and double label analysis of monoclonal antibodies against lymphocyte membrane surface markers (Leu series). Subpopulations were measured before induction, and 1 and 2 hours after skin incision. In the enflurane group, B cells (Leu12⁺), total T cells (Leu4⁺), inducer T cells (CD4⁺, Leu8⁺) and the CD4/CD8 ratio decreased and suppressor T cells (CD8⁺, Leu15⁺) increased significantly after skin incision. Whereas, the epidural group showed no change in the number of B cells and each T cell subpopulation during the study. These data suggest that epidural anaesthesia blocks the effect of stress induced by major surgery on fluctuation of peripheral lymphocyte subpopulations which may be associated with suppression of immunity.     

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4⁺ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8⁺ T cells participate in rejection. To address this, we placed MHC class I-deficient D^bK^b knockout skin grafts onto allogeneic H-2 ^k SCID recipients followed by adoptive transfer of purified H-2 ^k CD8⁺ T cells. The MHC class I-deficient grafts were rejected and only CD8⁺ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8⁺ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8⁺ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.     

Circulating monocytes have the capacity to be transdifferentiated into keratinocyte-like cells

Transdifferentiation is a process in which the original commitment of a cell is changed to give rise to unexpected peripheral mature cells. Our previous report showed that circulating stem cells can generate keratinocyte-like cells (KLCs). However, it remains to be determined whether or not other peripheral blood mononuclear cells (PBMC) subsets have the potential to follow the same cell fate. In this study, the cell transdifferentiation of circulating CD14⁺ monocytes into KLCs and their regulatory effect on matrix metalloproteinase-1 (MMP-1) expression in dermal fibroblasts were evaluated. The results showed that monocytes isolated from peripheral blood mononuclear cells have the capacity to generate KLCs. These transdifferentiated cells exhibited, along with a keratinocyte-like morphology, a characteristic profile consisting in stratifin⁺, cytokeratins⁺ (types I and II), CD14^low, and involucrin⁺ on day 21 in culture. Similar to keratinocyte-conditioned media, KLC-derived conditioned media were able to induce an increase in the MMP-1 expression in dermal fibroblasts. This effect was significantly reduced by using 14-3-3 protein-depleted KLC-conditioned media. Our findings show the potential transdifferentiation of circulating CD14⁺ monocytes into KLCs and their regulatory effect on MMP-1 expression in dermal fibroblasts.     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

Selective Immunomodulation: Utilization of CD29/VLA Molecules

Abstract: Accumulating evidence suggests that the VLA/ CD29 molecule plays an important role in T-cell costim-ulation, and CD4 ⁺ CD29/VLA ⁺ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bi-specific antibody-toxin conjugate designed to target CD4 ⁺ CD29 ⁺ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50, and 45 kilodalton proteins. In addition, we identified the ppl40 protein as PLCγ, ppl20 protein as ppl25^FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59^fyn and pp56^lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that ppl25^FAK is directly associated with paxillin. The paxillin binding domain of ppl25^FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between ppl25^FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation. Key Words: Immunomodulation–Graft rejection– Transplantation.