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.     

CMV‐specific T‐cell immunity,viral load,and clinical outcome in seropositive renal transplant recipients: a pilot study

Sund F, Lidehäll A‐K, Claesson K, Foss A, Tötterman TH, Korsgren O, Eriksson B‐M. CMV‐specific T‐cell immunity, viral load, and clinical outcome in seropositive renal transplant recipients: a pilot study.
Clin Transplant 2010: 24: 401–409. © 2009 Wiley Periodicals, Inc. Abstract: Background: Cytomegalovirus (CMV) infection is still the leading opportunistic infection following solid organ transplantation. The aim of this prospective study of renal transplant recipients was to evaluate the dynamics of CMV‐specific T‐cells, viral load, and clinical symptoms of CMV infection. Methods: Levels of tetramer‐selected CD8⁺ T‐cells (TetraCD8), CMV‐specific interferon‐γ producing CD8⁺ T‐cells (IFNγCD8), and CD4⁺ T‐cells (IFNγCD4), measured using major histocompatibility complex‐tetramer and cytokine flow cytometry techniques, and CMV DNA were monitored monthly in 17 CMV‐seropositive patients up to one yr (median 12 months, range 3–12) after transplantation and correlated to clinical outcome. Results: CMV DNAemia was detected in 94% of the patients, but only one patient developed CMV disease. CMV DNAemia >1 million copies/mL was seen in asymptomatic patients. CMV‐specific T‐cells decreased rapidly after transplantation. TetraCD8 and IFNγCD8 regenerated within three months, whereas IFNγCD4 recovery was impaired up to one yr after transplantation. The proportion of IFNγCD4 at two months post‐transplantation as compared with baseline, correlated strongly with the magnitude of the CMV DNAemia. Conclusions: Monitoring the reduction of IFNγCD4 compared with baseline during the first months after transplantation could be considered in predicting risk for high‐grade CMV DNAemia and in deciding strategic approaches for pre‐emptive and prophylactic therapy.     

Association of high IFN‐γ plasma levels with low B‐cell counts in renal transplant recipients with stable long‐term graft function

Daniel V, Naujokat C, Sadeghi M, Renner FC, Weimer R, Opelz G. Association of high IFN‐γ plasma levels with low B‐cell counts in renal transplant recipients with stable long‐term graft function.
Clin Transplant 2009 DOI: 10.1111/j.1399‐0012.2009.01067.x
© 2009 John Wiley & Sons A/S. Abstract: Recently, we reported that patients with long‐term stable good graft function had higher interferon‐gamma (IFN‐γ) and lower IL‐4 plasma levels late as compared with early post‐transplant. These patients had more often detectable CD3⁺CD4⁺CD25⁺IFN‐γ⁺Foxp3⁺ peripheral blood lymphocytes (PBL) late post‐transplant than patients with impaired graft function. We therefore speculated that high plasma IFN‐γ late post‐transplant might contribute to the maintenance of graft acceptance. Using ELISA and four‐color flow cytometry, plasma cytokines and PBL subpopulations were measured in 65 renal transplant recipients with stable graft function late post‐transplant. High IFN‐γ plasma levels were associated with low CD19⁺ B PBL (r = ?0.329; p = 0.009) and low activated CD3⁺CD8⁺DR⁺ T PBL (r = ?0.266; p = 0.035). Plasma IFN‐γ increased with time post‐transplant (r = 0.288; p = 0.022) and was not associated with the dose of immunosuppressive drugs (p = n.s.). High plasma IFN‐γ was not associated with serum creatinine (r = 0.038; p = 0.765). Five patients showed evidence of chronic allograft nephropathy in previous biopsies and none of them exhibited increased plasma IFN‐γ. In patients with good long‐term graft function, high IFN‐γ plasma levels were associated with low numbers of B PBL and activated CD8⁺ T PBL. High IFN‐γ plasma levels might prevent the development of an immunological alloresponse and thereby contribute to the maintenance of graft acceptance.     

Potential role of CC chemokine receptor 6 in prediction of late‐onset cytomegalovirus infection following solid organ transplant

CCR6 is a chemokine receptor involved in homing memory T cells, particularly Th17 cells, to sites of mucosal inflammation. Despite the critical role of memory T cells in long‐term protective immunity against cytomegalovirus (CMV), a virus that reactivates at multiple mucosal sites, the ability of CCR6 or other Th17 marker expression to predict CMV reactivation following transplantation is not clear. Using 11‐color flow cytometry, in this prospective single‐center pilot study, we measured the expression of CCR6 and other markers of T‐cell function in peripheral blood samples obtained from 21 SOT recipients at the time of discontinuation of anti‐CMV prophylaxis. CMV viremia was monitored on a monthly basis after discontinuation of prophylaxis. Eleven patients (52%) developed CMV viremia during the six‐month follow‐up period. Late‐onset CMV infection was preceded by an immune phenotype characterized by increased CCR6 expression on bulk CD4⁺ T cells and a reduced number of circulating CMV IE‐1‐specific Th1 (CD4⁺ IFN‐γ⁺) cells. Among the markers evaluated, CCR6 was the best single predictor of late‐onset CMV infection. Our results suggest that CCR6 expression at the time of discontinuation of antiviral prophylaxis might be a useful predictor of late‐onset CMV reactivation and provide the basis for future larger prospective studies.     

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.     

Interferon Gamma and Contact‐dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell–Mediated Antibody‐dependent Chronic Rejection

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor‐specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody‐mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN‐γ) versus the contact‐dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune‐deficient C57BL/6 rag^?/?γc^?/? recipients, who also received monoclonal anti–major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild‐type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN‐γ–deficient NK cells or host IFN‐γ neutralization led to amelioration of these lesions. Use of either perforin‐deficient NK cells or CD95 (Fas)–deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell–derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN‐γ production and contact‐dependent cytotoxic activity are rate‐limiting effector pathways that contribute to this form of antibody‐induced chronic allograft vasculopathy.     

The role of interleukin‐17 in murine cytomegalovirus interstitial pneumonia in mice with skin transplants

We hypothesized that the T helper (Th)17 response plays an important role in murine cytomegalovirus (MCMV) interstitial pneumonia. BALB/c mice with skin grafts from C57/BJ6 mice were intranasally inoculated with 1.0 × 10⁵ PFU MCMV. Lung tissues and skin grafts were histologically evaluated and expression of interleukins (IL)‐17, ‐6 and ‐8, monocyte chemotactic protein (MCP)‐1 and interferon (IFN)‐γ in serum and bronchoalveolar lavage (BAL) fluid, intracellular IL‐4, ‐17, and IFN‐γ, in spleen lymphocytes were analysed. The levels of IL‐17 in the serum and BAL fluid were significantly higher in MCMV‐infected mice versus not‐infected mice (P = 0.0286 and P = 0.007, respectively) and the BAL levels of IL‐17 peaked in 9 days (P = 0.001). The IL‐17 level in the BAL was correlated with the grade of lung interstitial inflammation (r = 0.554, P = 0.0144). Serum IFN‐γ levels were also higher after infection than that in the not‐infected mice (P = 0.0286). IL‐17 production increases locally and systemically during MCMV interstitial pneumonia. Neutralization of IL‐17 significantly suppressed lung inflammation at day14 as assessed by histology. These findings suggest that IL‐17 is important in the pathology of MCMV interstitial pneumonia.     

PD‐1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation‐Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD‐1) on cytomegalovirus (CMV)‐specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28^?CD27^? double negativity is known as a typical phenotype of CMV‐specific CD4 T cells. Therefore, the co‐expression of inhibitory receptors on CD28^?CD27^? CD4 T cells was assessed as a rapid, stimulation‐independent parameter for monitoring CMV complications after transplantation. Ninety‐three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross‐sectional and longitudinal manner. CMV‐specific CD4 T cell levels quantified after stimulation were compared to levels of CD28^?CD27^? CD4 T cells. PD‐1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) expression on CD28^?CD27^? CD4 T cells were related to viremia. A percentage of ≥0.44% CD28^?CD27^? CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV‐specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD‐1 expression levels on CD28^?CD27^? CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA‐4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD‐1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD‐1 expression on CD28^?CD27^? CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation‐independent parameter to guide duration of antiviral therapy.

     

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid‐Derived Suppressor Cells

Increasing evidence from small animal models shows that myeloid‐derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3^?CD20^?HLA‐DR^?CD14⁺CD33⁺CD11b⁺ cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin^?HLA‐DR^? peripheral blood mononuclear cells. Administration of granulocyte‐macrophage colony‐stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10⁶ (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti‐CD3– and anti‐CD28–stimulated autologous T cells markedly suppressed CD4⁺ and CD8⁺ T cell proliferation and cytokine secretion (interferon γ, IL‐17A). Moreover, these MDSCs enhanced CD4⁺CD25^hiFoxp3⁺ regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase‐1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8⁺ T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

     

The effect of Gal expression on pig cells on the human T-cell xenoresponse

Wilhite T, Ezzelarab C, Hara H, Long C, Ayares D, Cooper DKC, Ezzelarab M. The effect of Gal expression on pig cells on the human T cell xenoresponse. Xenotransplantation 2012; 19: 56–63. © 2012 John Wiley & Sons A/S. Abstract: Background: Lack of Gal expression on pig cells is associated with a reduced primate humoral immune response as well as a reduction in cytokine production by human cells in vitro. We investigated whether lack of Gal expression is associated with reduced human T‐cell response in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy humans and naïve baboons. Human CD4⁺ and CD8⁺ T cells were isolated. Porcine aortic endothelial cells (pAECs) were isolated from wild‐type (WT) and α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs. WT pAECs were treated with α‐galactosidase, reducing Gal expression. Swine leukocyte antigen (SLA) class I and II expression on pAECs was measured, as was T‐cell proliferation and cytokine production in response to pAECs. Results: Reduced Gal expression on WT pAECs after α‐galactosidase treatment was associated with reduced human PBMC proliferation (P < 0.005). SLA class I and II expression on WT and GTKO pAECs was comparable. Human CD4⁺ and CD8⁺ T‐cell proliferation was less against GTKO pAECs before (P < 0.001) and after (P < 0.01 and P < 0.05, respectively) activation. Human and baboon PBMC proliferation was less against GTKO pAECs before (P < 0.05) and after (P < 0.01 and P < 0.05, respectively) activation. Human PBMCs produced a comparable cytokine/chemokine response to WT and GTKO pAECs. However, there was less production of IFN‐γ/TNF‐α by CD4⁺ and IFN‐γ/granzyme B/IP‐10 by CD8⁺ T cells in response to GTKO pAECs. Conclusions: The absence of Gal on pig cells is associated with reduced human T‐cell proliferation (and possibly selected cytokine production). Adaptive primate T‐cell responses are likely to be reduced in GTKO xenograft recipients.     

Belatacept‐Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28‐CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept‐ or a tacrolimus‐based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28⁺CD8⁺T_EMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28⁺CD8⁺T_EMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28^?. These data suggest that CD28⁺ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28⁺ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation‐independent rejection.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

Cytomegalovirus‐Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8⁺ T cell responses to CMV polypeptides immediate‐early‐1 and pp65 were analyzed in 16 CMV‐seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell–depleting induction therapy. The frequency of CMV‐responsive CD8⁺ T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV‐responsive T cell population posttransplantation.     

Allo‐HLA Cross‐Reactivities of Cytomegalovirus‐, Influenza‐, and Varicella Zoster Virus–Specific Memory T Cells Are Shared by Different Healthy Individuals

Virus‐specific T cells can recognize allogeneic HLA (allo‐HLA) through TCR cross‐reactivity. The allospecificity often differs by individual (private cross‐reactivity) but also can be shared by multiple individuals (public cross‐reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross‐reactivities of human virus‐specific CD8⁺ T cells directed against allo‐HLA by assessing their reactivity in mixed‐lymphocyte reactions. Further characterization was done by studying TCR usage with primer‐based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with ⁵¹chromium‐release assays. We identified three novel public allo‐HLA cross‐reactivities of human virus‐specific CD8⁺ T cells. CMV B35/IPS CD8⁺ T cells cross‐reacted with HLA‐B51 and/or HLA‐B58/B57 (23% of tetramer‐positive individuals), FLU A2/GIL (influenza IMP[58‐66] HLA‐A*02:01/GILGFVFTL) CD8⁺ T cells with HLA‐B38 (90% of tetramer‐positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593‐601] HLA‐A*02:01/ALWALPHAA) CD8⁺ T cells with HLA‐B55 (two unrelated individuals). Cross‐reactivity was tested against different cell types including endothelial and epithelial cells. All cross‐reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo‐HLA cross‐reactivity of virus‐specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.     

Memory T cell–mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells

Donor‐reactive memory T cells generated via heterologous immunity represent a potent barrier to long‐term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor‐specific effector/memory CD8⁺ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8⁺ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB‐expressing CD8⁺ donor‐reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b^?/? CD8⁺ T cells resulted in an accumulation of donor‐specific CD8⁺ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8⁺ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor‐specific CD8⁺ memory T cells precipitated costimulation blockade‐resistant rejection. These data therefore identify a novel cell‐intrinsic inhibitory pathway that functions to limit the risk of memory T cell–mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.     

CMV Infection in the Donor and Increased Kidney Graft Loss: Impact of Full HLA‐I Mismatch and Posttransplantation CD8+ Cell Reduction

Despite a large body of literature, the impact of chronic cytomegalovirus (CMV) infection in donor on long‐term graft survival remains unclear, and factors modulating the effect of CMV infection on graft survival are presently unknown. In this retrospective study of 1279 kidney transplant patients, we analyzed long‐term graft survival and evolution of CD8⁺ cell population in donors and recipients by CMV serology and antigenemia status. A positive CMV serology in the donor was an independent risk factor for graft loss, especially among CMV‐positive recipients (R⁺). Antigenemia was not a risk factor for graft loss and kidneys from CMV‐positive donors remained associated with poor graft survival among antigenemia‐free recipients. Detrimental impact of donor's CMV seropositivity on graft survival was restricted to patients with full HLA‐I mismatch, suggesting a role of CD8⁺ cells. In R⁺ patients with positive CMV antigenemia during the first year, CD8⁺ cell count did not increase at 2 years posttransplantation, in contrast to R^? recipients. In addition, marked CD8⁺‐cell decrease was a risk factor of graft failure in these patients. This study identifies HLA‐I full mismatch and a decrease of CD8⁺ cell count at 2 years as important determinants of CMV‐associated graft loss.

     

Primary response against cytomegalovirus during antiviral prophylaxis with valganciclovir,in solid organ transplant recipients

Antiviral prophylaxis has proved successful for prevention of cytomegalovirus (CMV) disease in solid organ transplant (SOT) patients; though emerging data suggest that antiviral agents interfere with immunity, and may inhibit immune priming. In this context, we investigated levels and phenotype of primary CMV‐specific immune responses that developed during antiviral prophylaxis in a cohort of CMV seronegative recipients (R^?) of a SOT from a seropositive donor (D⁺). We longitudinally monitored CMV viral load, antibodies and levels of the negative immuno‐modulator IL‐10. PBMC were stimulated with CMV‐specific peptide libraries to measure CD137 activation marker on CMV‐specific T‐cells and levels of PD‐1 receptor, which is over expressed on exhausted T‐cells. Unexpectedly, the majority (13/18) of D⁺R^? patients who developed a primary CMV response showed early post‐transplant CMV‐specific responses, though levels of PD‐1 on CMV‐specific T‐cells remained elevated throughout prophylaxis. A strong inverse association was found between levels of plasma IL‐10 and CMV‐specific cellular immune responses. Our study suggests that during prophylaxis, subclinical CMV infection might have occurred in the D⁺R^? patients, and primary CMV‐specific responses were detected early post‐transplant when levels of plasma IL‐10 were low. Extended prophylaxis or antiviral treatment did not appear to suppress CMV‐specific antibodies or T‐cells, which, however, showed exhaustion phenotypes.     

Pretransplant Immune‐Regulation Predicts Allograft Tolerance

CD4⁺ Tregs specific for noninherited maternal antigens (NIMA^d) are detectable in some but not all B6 × BDF₁ backcross, H‐2^b homozygous offspring, and their presence is strongly correlated with extent of maternal (BDF₁) microchimerism. We hypothesized that the level of pretransplant donor antigen‐specific Tregs could predict allograft tolerance. To test this idea, mice were screened for bystander suppression in a DTH assay, followed 1 week later by DBA/2 heterotopic heart transplantation. NIMA^d‐exposed, H‐2^b offspring that failed to suppress DTH uniformly rejected heart allografts (12/12) by d15. In contrast, 5/6 NIMA^d‐exposed DTH ‘regulators’ accepted their allografts >100 days. The defect in ‘nonregulator“ offspring could be corrected by transfer of CD4⁺CD25⁺, but not CD4⁺CD25^neg or CD8⁺T cells from transplant acceptor mice. In conclusion, donor‐specific T reg screening of F1 backcross offspring correctly predicted which recipients would accept a heart allograft. If translated to the clinic, similar pretransplant Treg screening could greatly enhance the effectiveness of tolerance as a clinical strategy in transplantation between family members.     

Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo

Osteocytes are the most abundant cells in bone yet are the most challenging to study because they are embedded in a mineralized matrix. We generated a clonal cell line called IDG‐SW3 (for Immortomouse/Dmp1‐GFP‐SW3) from long‐bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T antigen regulated by interferon γ (IFN‐γ). Cells from these mice can be expanded at 33 °C in the presence of IFN‐γ and then allowed to resume their original phenotype at 37 °C in the absence of IFN‐γ. IDG‐SW3 cells are Dmp1‐GFP^? and T antigen⁺ under immortalizing conditions but Dmp1‐GFP⁺ and T antigen^? under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type 1 collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and fibroblast growth factor 23 (FGF‐23), regulated by parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D₃. When cultured on 3D matrices, they express Dmp1‐GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast‐to‐osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF‐23. © 2011 American Society for Bone and Mineral Research     

Primary Cytomegalovirus Infection Significantly Impacts Circulating T Cells in Kidney Transplant Recipients

Cytomegalovirus (CMV) infection profoundly affects the T cell compartment and is associated with alterations in T cell aging parameters and generation of cytotoxic CD4⁺CD28null T cells. Hence, the effect of a primary CMV infection post–kidney transplantation (KT) on the peripheral T cell compartment was examined. As aging parameters, we determined the T cell differentiation status, T cell receptor excision circle (TREC) content, CD31⁺ naïve T cell numbers and relative telomere length (RTL) pre‐KT and 12 months post‐KT. CMV‐seronegative KT recipients, receiving a kidney from a CMV‐seropositive donor (D+/R?) were compared to D+/R+ KT recipients. Eleven out of the 22 D+/R? KT recipients had CMV viremia post‐KT. They developed CMV‐specific CD4⁺ and CD8⁺ T cells and their T cell compartment shifted towards a more differentiated memory phenotype with expansion of CD4⁺CD28null and CD8⁺CD28null cells. One year post‐KT, the CD8⁺ T cell count was almost doubled compared to nonviremic D+/R? and D+/R+ KT recipients. In addition, the RTL of the CD8⁺ T cell was significantly lower and both the TREC content and CD31⁺ naïve T cell numbers significantly decreased. Moreover, primary CMV infection was associated with a negative impact on glomerular filtration rate. In conclusion, primary CMV infection has a substantial impact on the number and phenotype of peripheral T cells and may negatively affect renal allograft function.