Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4⁺ or CD8⁺ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4⁺ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4⁺ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8⁺ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4⁺ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4⁺ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4⁺ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.     

Rapamycin ameliorates the CTLA4‐Ig–mediated defect in CD8+ T cell immunity during gammaherpesvirus infection

Latent viral infections are a major concern among immunosuppressed transplant patients. During clinical trials with belatacept, a CTLA4‐Ig fusion protein, patients showed an increased risk of Epstein–Barr virus‐associated posttransplant lymphoproliferative disorder, thought to be due to a deficient primary CD8⁺ T cell response to the virus. Using a murine model of latent viral infection, we observed that rapamycin treatment alone led to a significant increase in virus‐specific CD8⁺ T cells, as well as increased functionality of these cells, including the ability to make multiple cytokines, while CTLA4‐Ig treatment alone significantly dampened the response and inhibited the generation of polyfunctional antigen‐specific CD8⁺ T cells. However, the addition of rapamycin to the CTLA4‐Ig regimen was able to quantitatively and qualitatively restore the antigen‐specific CD8⁺ T cell response to the virus. This improvement was physiologically relevant, in that CTLA4‐Ig treated animals exhibited a greater viral burden following infection that was reduced to levels observed in untreated immunocompetent animals by the addition of rapamycin. These results reveal that modulation of T cell differentiation though inhibition of mTOR signaling can restore virus‐specific immune competence even in the absence of CD28 costimulation, and have implications for improving protective immunity in transplant recipients.     

Uremia‐associated immunological aging is stably imprinted in the T‐cell system and not reversed by kidney transplantation

The uremia‐induced inflammatory environment in end‐stage renal disease (ESRD) patients is associated with premature T‐cell aging resulting in a defective T‐cell immunity. As kidney transplantation (KTx) reduces the pro‐inflammatory environment, we hypothesized that KTx would rejuvenate the aged T‐cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T‐cell receptor excision circle (TREC) content together with CD31⁺ naïve T‐cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre‐KTx, 3, 6 and 12 months post‐KTx. In addition, T‐cell function was determined by measuring the proliferative capacity and percentages of cytokine‐producing cells. Directly post‐KTx, memory T‐cell numbers were diminished but restored to pre‐KTx values at 12 months, except for CD4⁺EM T cells. The RTL of (memory) CD4⁺ and CD8⁺ T cells did not change. In contrast, TREC content and CD31⁺ naïve T‐cell numbers were stable post‐KTx although the RTL of naïve CD4⁺ and CD8⁺ T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T‐cell function was not improved post‐KTx. Our findings demonstrate that the uremia‐associated aged phenotype is stably imprinted in the T‐cell system and not reversed by KTx.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.     

Both CD45RA+ and CD45RO+ human CD4+ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells

Kim CH, Oh K, Kim D‐E, Lee SB, Yang JH, Lee G, Cho J, Lee D‐S. Both CD45RA⁺ and CD45RO⁺ human CD4⁺ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells. Xenotransplantation 2010; 17: 224–232. © 2010 John Wiley & Sons A/S. Abstract: Background: Xenogeneic cellular immune responses are mediated by either direct or indirect pathways depending on the participation of donor or host antigen presenting cells, respectively. The contribution of direct response of human T cells, especially memory T cells, to porcine antigen presenting cells is currently unknown. Here, we sought to determine whether human peripheral blood memory/activated phenotype T cells are directly responsive to porcine endothelial cells. Methods: Porcine aortic endothelial cells (PAECs) were prepared from Yorkshire or miniature pigs. Highly purified human T cells, including naïve and memory/activated phenotype cells, were incubated with PAECs with or without the addition of exogenous cytokines. T‐cell proliferation and T‐cell receptor (TCR) Vβ usage in response to PAECs were analyzed. Results: Both CD8⁺ and CD4⁺ T cells responded directly to PAECs and exhibited exclusive responsiveness to SLA class I and class II molecules, respectively. Naïve and memory/activated phenotype CD4⁺ T cells responded against PAECs, whereas only naïve phenotype CD8⁺ T cells contributed to such a response. In addition, both populations of xenogeneic human CD4⁺ T cells exhibited similar and diverse Vβ usage. Conclusion: Due to the considerable contribution of human CD45RO⁺CD4⁺ T cells to the xenoreactivity against PAECs, effective control of xenogeneic memory/activated T‐cell responses would significantly affect long‐term survival of transplanted grafts.     

Long‐term survival of pig‐to‐rhesus macaque renal xenografts is dependent on CD4 T cell depletion

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti‐pig antibodies significantly improved survival in a pig‐to–rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan–T cell depletion and an anti‐CD154–based maintenance regimen. Selective depletion of CD4⁺ T cells but not CD8⁺ T cells resulted in long‐term survival (median survival time >400 days vs 6 days). These studies suggested that CD4⁺ T cells may have a more prominent role in xenograft rejection compared with CD8⁺ T cells. Although animals that received selective depletion of CD8⁺ T cells showed signs of early cellular rejection (marked CD4⁺ infiltrates), animals receiving selective CD4⁺ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4⁺ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti‐pig antibody and CD4 depletion resulted in consistent, long‐term xenograft survival.     

CD4+ CD25+ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells

Nishimura T, Onda M, Takao S. CD4⁺ CD25⁺ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells. Xenotransplantation 2010; 17: 313–323. © 2010 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ regulatory T cells have been reported to suppress T cell‐mediated xenogeneic immune responses. Although the direct T cell response to xenogeneic cells is important, the indirect xenogeneic immune response mediated by dendritic cells (DCs) is also likely involved in rejection. We have generated an in vitro indirect immune reaction model and evaluated the effect of CD4⁺ CD25⁺ regulatory T cells on this system. Methods: Human DCs were generated from peripheral blood and cultured with X‐ray‐irradiated porcine kidney epithelial cells. Porcine cell‐pulsed DCs were mixed with autologous CD4⁺ T cells, CD4⁺ CD25^? T cells and/or CD4⁺ CD25⁺ T cells. After 7 days of culture, T cell proliferation was measured. Results: The co‐culture of human DCs and X‐ray‐irradiated porcine epithelial cells resulted in observable DC phagocytic activity within 2 days. These porcine cell–pulsed DCs stimulated CD4⁺ T cell proliferation much more potently than unpulsed DCs or porcine cells. This proliferation was blocked by CTLA4‐Ig or an anti‐HLA‐DR antibody. CD4⁺ CD25⁺ regulatory T cells also suppressed CD4⁺ CD25^? T cell proliferation in response to porcine cell‐pulsed DCs. Conclusions: An in vitro model of the indirect xenogeneic immune response was established. Porcine cell‐pulsed DCs stimulated CD4⁺ T cells, and CD4⁺ CD25⁺ regulatory T cells suppressed this response.     

Selective CD28 Blockade Results in Superior Inhibition of Donor‐Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4‐Ig

Donor‐specific antibodies (DSAs) are a barrier to improved long‐term outcomes after kidney transplantation. Costimulation blockade with CTLA4‐Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4⁺ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8⁺ T cells relative to CTLA4‐Ig, but the impact of CD28‐specific blockade on CD4⁺ Tfh cells is unknown. Thus, we identified and characterized donor‐reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti‐CD28 domain antibody (dAb) on the donor‐specific Tfh cell–mediated immune response. We observed that the anti‐CD28 dAb led to superior inhibition of donor‐reactive CXCR5⁺PD‐1^high Tfh cells, CD95⁺GL7⁺ germinal center B cells and DSA formation compared with CTLA4‐Ig. Interestingly, donor‐reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti‐CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.     

Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD‐ligand‐1 on porcine target cells

Cellular rejection is a relevant hurdle for successful pig‐to‐primate xenotransplantion. We have shown previously that the induction of a human anti‐pig T cell response (in vitro activation of CD4⁺ T cells) can be suppressed by the overexpression of human negative costimulatory ligands (e.g. programmed death receptor ligand, PD‐L1) on pig antigen presenting cells. Here, we asked whether PD‐L1 mediated enhancement of negative signaling might also be efficient during the effector phase of human anti‐pig cellular immune responses. The porcine B‐cell line L23 was transfected with human PD‐L1, and clones were selected stably expressing PD‐L1 with low, medium, or high density. Mock‐transfected L23 cells were effectively lysed by human cytotoxic effector cells (IL‐2 activated CD8⁺ T cells and CD56⁺ cells). The lytic potential of the effectors decreased with increasing levels of PD‐L1 and was reduced by about 50% in L23‐PD‐L^high targets. A proportion of activated CD8⁺ effector cells underwent apoptosis when exposed to PD‐L1 expressing L23 cells. These data suggest that the overexpression of PD‐L1 on target cells may (a) trigger negative signals in effector cells that prevent the release of cytolytic molecules and/or (b) induce apoptosis in the attacking effector cells thereby protecting targets from destruction.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

ATG‐Induced Accelerated Immune Senescence: Clinical Implications in Renal Transplant Recipients

Persistent ATG‐induced CD4⁺ T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one‐year later in 97 incident RTR ?62 patients receiving ATG and 35 receiving anti‐CD25 mAb (α‐CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34⁺ hematopoietic progenitor cells (CD34⁺HPC) and lymphoid (l‐HPC) and myeloid (m‐HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow‐up. Thymic output significantly decreased one‐year posttransplant in ATG‐treated patients. ATG was associated with a significant decrease in l‐HPC/m‐HPC ratio. Late stage differentiated CD57⁺/CD28^? T cells increased in ATG‐treated patients. One‐year posttransplant T cell RTL and RTA were consequently lower in ATG‐treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4⁺ T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG‐treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

     

A Novel Xenogeneic Graft‐Versus‐Host Disease Model for Investigating the Pathological Role of Human CD4+ or CD8+ T Cells Using Immunodeficient NOG Mice

Graft‐versus‐host disease (GVHD) is a major complication of allogenic bone marrow transplantation and involves the infiltration of donor CD4⁺ and/or CD8⁺ T cells into various organs of the recipient. The pathological role of human CD4⁺ and CD8⁺ T cells in GVHD remains controversial. In this study, we established two novel xenogeneic (xeno)‐GVHD models. Human CD4⁺ or CD8⁺ T cells were purified from peripheral blood and were transplanted into immunodeficient NOD/Shi‐scid IL2rg^null (NOG) mice. Human CD8⁺ T cells did not induce major GVHD symptoms in conventional NOG mice. However, CD8⁺ T cells immediately proliferated and induced severe GVHD when transferred into NOG mice together with at least 0.5 × 10⁶ CD4⁺ T cells or into NOG human interleukin (IL)‐2 transgenic mice. Human CD4⁺ T cell–transplanted NOG mice developed skin inflammations including alopecia, epidermal hyperplasia, and neutrophilia. Pathogenic T helper (Th)17 cells accumulated in the skin of CD4⁺ T cell–transplanted NOG mice. Further, an anti‐human IL‐17 antibody (secukinumab) significantly suppressed these skin pathologies. These results indicate that pathogenic human Th17 cells induce cutaneous GVHD via IL‐17–dependent pathways. This study provides fundamental insights into the pathogenesis of xeno‐GVHD, and these humanized mouse models may be useful as preclinical tools for the prevention of GVHD.     

Disengaging the IL‐2 Receptor with Daclizumab Enhances IL‐7–Mediated Proliferation of CD4+ and CD8+ T Cells

Allograft rejection is mainly driven by the production of IL‐2, which expands T cells by linking the IL‐2 receptor (IL‐2R) composed of three subunits: CD25, CD122 and CD132. Daclizumab, widely used in immunosuppression, is a humanized anti‐CD25 antibody that disrupts IL‐2 signaling by binding to CD25 and preventing the assembly of the high‐affinity IL‐2R. Here we show that Daclizumab, while blocking the T‐cell response to IL‐2, increases CD4⁺ and CD8⁺ T‐cell proliferative response to the homeostatic cytokine IL‐7. The IL‐7R shares CD132 with the IL‐2R and blocking of CD25 by Daclizumab results in the enhanced formation of the IL‐7R that in turn allows IL‐7 to bind more efficiently on the cell surface. The consequently increased IL‐7R signaling boosts intracellular phosphorylated STAT5 and T‐cell proliferation. In addition, treatment with Daclizumab delays the internalization of CD127 upon IL‐7 treatment, retaining T‐cell sensitivity to IL‐7 for a prolonged time. This effect of Daclizumab highlights the redundancy of the cytokine system, which may influence T‐cell proliferation in transplanted patients, and provides information to improve future immunosuppressive strategies.     

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

Immune activation‐ and regulation‐related patterns in stable hand transplant recipients

We assessed cell subsets and expression of a set of genes related to the T‐cell populations in peripheral blood mononuclear cells to elucidate whether immune status of stable hand transplant recipients (HTx) differs from stable kidney transplant recipients (KTx). The study was conducted on five HTx 4.8 ± 1.7 years after transplantation and 30 stable KTx 7.9 ± 2.4 years after transplantation as well as 18 healthy volunteers. The research involved PBMC gene expression analysis of CD4, CD8, CTLA4, GZMB, FOXP3, IL10, IL4, ILR2A, NOTCH, PDCD1, PRF1, TGF‐B, and TNF‐A genes on a custom‐designed low‐density array (TaqMan) as well as flow cytometry assessment of lymphocyte subpopulations. HTx presented significantly increased expression of immunomodulatory genes (TNF, IL10, GITR, and PDCD1) compared to KTx and controls. HTx revealed a proinflammatory molecular pattern with higher expression of NOTCH and CD8 compared to KTx and controls. KTx showed a reduced level of regulatory T cells compared to controls and HTx. Both HTx and KTx presented an increased number of CD8⁺ and CD8⁺CD28⁻ T cells compared to controls. Stable hand transplant recipients exhibit persistent immune activation with rejection‐related gene expression pattern counterbalanced by secondary induction of regulatory mechanisms.     

Healthy human second‐trimester fetal skin is deficient in leukocytes and associated homing chemokines

The lack of immune cells in mid‐gestational fetal skin is often mentioned as a key factor underlying scarless healing. However, the scarless healing ability is conserved until long after the immune system in the fetus is fully developed. Therefore, we studied human second‐trimester fetal skin and compared the numbers of immune cells and chemokine levels from fetal skin with adult skin. By using immunohistochemistry, we show that healthy fetal skin contains significant lower numbers of CD68⁺‐macrophages, Tryptase⁺‐mast cells, Langerin⁺‐Langerhans cells, CD1a⁺‐dendritic cells, and CD3⁺‐T cells compared to adult skin. Staining with an early lineage leukocyte marker, i.e., CD45, verified that the number of CD45⁺‐immune cells was indeed significantly lower in fetal skin but that sufficient numbers of immune cells were present in the fetal lymph node. No differences in the vascular network were observed between fetal and adult skin. Moreover, significant lower levels of lymphocyte chemokines CCL17, CCL21, and CCL27 were observed in fetal skin. However, levels of inflammatory interleukins such as IL‐6, IL‐8, and IL‐10 were undetectable and levels of CCL2 were similar in healthy fetal and adult skin. In conclusion, this study shows that second‐trimester fetal skin contains low levels of immune cells and leukocyte chemokines compared to adult skin. This immune cell deficiency includes CD45⁺ leukocytes, despite the abundant presence of these cells in the lymph node. The immune deficiency in healthy second‐trimester fetal skin may result in reduced inflammation during wound healing, and could underlie the scarless healing capacities of the fetal skin.     

Liver Transplantation in a Patient With CD40 Ligand Deficiency and Hyper‐IgM Syndrome: Clinical and Immunological Assessments

Monoclonal antibodies that disrupt CD40–CD40 ligand (CD40L) interactions are likely to have use in human transplantation. However, the extent of the immunosuppressive effects of CD40–CD40L blockade in humans is unknown. Hyper‐IgM syndrome (HIGM) is a rare primary immunodeficiency syndrome characterized by defects in the CD40–CD40L pathway, severe immune deficiency (IgG), and high or normal IgM levels. However, the effects of CD40L deficiency on T‐ and natural killer (NK)‐cell function is not established. Here, we present a patient with HIGM syndrome who underwent liver transplantation for hepatitis C virus infection. Posttransplantation, NK‐cell antibody‐dependent cytokine release (γ‐interferon) to alloantigens and T cell responses to viral antigens and mitogens were assessed and showed normal CD4⁺, CD8⁺, and NK‐cell responses. We also examined antibody‐dependent cellular cytotoxicity against a CD40⁺ and HLA‐expressing cell line. These experiments confirmed that the patient's NK cells were equivalent to those of normal subjects in mediating antibody‐dependent cellular cytotoxicity despite the absence of CD40–CD40L interactions. Mitogenic stimulation of the patient's peripheral blood mononuclear cells showed no expression of CD40L on T and NK cells compared with increased expression in normal subjects. Taken together, these data suggest that absence of CD40L expression is responsible for aberrant B cell immunity but had little impact on NK‐ and T cell immune responses in vitro.     

Antibody‐Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology

We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor‐specific antibody (DSA) response was monitored by flow cross‐match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti‐CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody‐mediated rejection (AMR). In two of three basiliximab‐injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27–44 days, with mixed AMR and ACR. Most T cell–depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20⁺CD27⁺IgD^?Ki67⁺), lymph node follicular helper T cells (ICOS⁺PD‐1^hiCXCR5⁺CD4⁺), and germinal center (GC) response. Depletion controlled cell‐mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.     

Anti‐TCRβ mAb Induces Long‐Term Allograft Survival by Reducing Antigen‐Reactive T Cells and Sparing Regulatory T Cells

TCR specific antibodies may modulate the TCR engagement with antigen–MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRβ (H57–597), TCRα or CD3 promptly reduced the number of CD4⁺ and CD8⁺ T cells in normal mice, but H57–597 mAb most potently increased the frequency of CD4⁺Foxp3⁺ Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57–597 mAb, the expansion of SEB‐reactive Vβ8⁺ T cells was completely abrogated while SEB‐nonreactive Vβ2⁺ T cells remained unaffected. More importantly, transient H57–597 mAb treatment exerted long‐lasting effect in preventing T cell responses to alloantigens, and produced long‐term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor‐specific long‐term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57–597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin‐sensitized recipients. Thus, transient modulation of the TCRβ chain by H57–597 mAb exhibits potent, long‐lasting therapeutic effects to control alloimmune responses.