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.

     

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.     

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV‐Naïve Recipients of Infected Donor Allograft Hearts

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV‐accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV‐naïve or H₂O₂‐inactivated RCMV‐vaccinated Lewis recipients. Recipients of RCMV‐infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection‐POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T‐cell and B‐cell arms of the adaptive immune response provide protection against CMV‐accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti‐viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.     

Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.     

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.     

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.     

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.     

Peritransplant VLA‐4 blockade inhibits endogenous memory CD8 T cell infiltration into high‐risk cardiac allografts and CTLA‐4Ig resistant rejection

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC‐mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen‐4 (VLA‐4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti‐VLA‐4 mAb given to C57BL6 (H‐2^b) recipients of AJ (H‐2^a) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra‐allograft IFN‐γ‐induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA‐4Ig treated recipients. Anti‐VLA‐4 mAb also inhibited priming of donor‐specific T cells producing IFN‐γ until at least day 7 posttransplant. Peritransplant anti‐VLA plus anti‐CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti‐VLA‐4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.     

Graft‐Versus‐Host Disease Following Liver Transplantation: Development of a High‐Incidence Rat Model and a Selective Prevention Method

Graft‐versus‐host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT‐GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti‐CD8α mAb treatment, which invariably resulted in acute LT‐GvHD. Compared with those in the peripheral counterpart, graft CD4⁺CD25^? passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4⁺ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti‐TCRαβ mAb. T cell–depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT‐GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT‐GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high‐risk donor–recipient combinations and for analyzing immunoregulatory mechanisms of the liver.     

Easier Control of Late‐Onset Cytomegalovirus Disease Following Universal Prophylaxis Through an Early Antiviral Immune Response in Donor‐Positive,Recipient‐Negative Kidney Transplants

Universal prophylaxis for cytomegalovirus (CMV) prevention is viable but, compared with a preemptive strategy, leads to higher incidence of late‐onset disease (LOD) associated with poor patient and graft survival. The purpose of this study was to compare LOD with early onset disease (EOD), with a focus on the highest risk kidney transplant recipients (KTRs): CMV seronegative recipients transplanted from seropositive donors (D+R?). Since CMV control depends on both antiviral treatment and specific immune response, we also compared Vδ2‐negative (Vδ2^neg) γδ T cell expansion involved in CMV infection resolution. EOD was defined as occurring <3 mo and LOD as occurring >3 mo after transplantation. Depending on the period, universal prophylaxis or preemptive treatment was used. Overall, 168 D+R? KTRs were included between 2003 and 2011. LOD was associated with a lower peak DNAemia (p = 0.04), fewer recurrences (odds ratio 0.16; 95% confidence interval 0.05–0.55; p = 0.01) and shorter anti‐CMV curative treatment (40 vs. 60 days, p < 0.0001). As a corollary, we found that Vδ2^neg γδ T cell expansion was faster in LOD than in EOD (31 vs. 168 days after the beginning of CMV disease, p < 0.0001). In D+R? KTRs, universal prophylaxis is associated with more LOD, which had better infection management and a faster immune response. These results support the use of universal prophylaxis over a preemptive strategy and reappraise outcomes of LOD.     

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.

     

High‐Quality CMV‐Specific CD4+ Memory Is Enriched in the Lung Allograft and Is Associated With Mucosal Viral Control

The maintenance of CMV‐specific T cell memory in lung transplant recipients (LTRs) is critical for host defense and allograft durability, particularly in donor⁺/recipient^? (D⁺R^?) individuals who demonstrate increased mortality. We studied CD4⁺ and CD8⁺ CMV‐specific memory responses to phosphoprotein 65 (pp65) in a prospective cohort of 18 D⁺R^? LTRs, from bronchoalveolar lavage (BAL)‐obtained lung mononuclear cells (LMNC) and PBMC. Unexpectedly, pp65‐specific CD4⁺ and CD8⁺ IFN‐γ memory responses from LMNC were similar, in contrast to persistent CD8⁺ predominance in PBMC. Unlike the pulmonary CD8⁺ predominance during acute primary infection, compartmental equalization occurred in the CMV‐specific CD8⁺ memory pool during chronic infection, whereas CMV‐specific CD4⁺ memory was enriched in the bronchoalveolar space. Moreover, CMV‐specific CD4⁺ memory T cells with multifunctional production of IFN‐γ, TNF‐α, IL‐2 and MIP‐1β were significantly increased in LMNCs, in contrast to similar intercompartmental CD8⁺ memory function. Moreover, the absolute number of CMV‐specific CD4⁺IFN‐γ⁺ memory cells in BAL was significantly increased in LTRs exhibiting viral control compared to those with CMV early antigen positivity. Collectively, these data demonstrate both preferential distribution and functional quality of CMV‐specific CD4⁺ memory in the lung allograft during chronic infection, and show an important association with CMV mucosal immunity and viral control.     

UVB‐induced depletion of donor‐derived dendritic cells prevents allograft rejection of immune‐privileged hair follicles in humanized mice

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor‐ and recipient‐derived pathways. Donor‐derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor‐derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti‐CD154 antibody (Ab) into recipients in an MHC‐mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long‐term survival with newly growing pigmented hair shafts in both Ab‐treated groups (Ab‐only and UV plus Ab) and in the UV‐only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3⁺ T cell and MHC class II⁺ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV‐only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen‐specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.     

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.     

Donor‐specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients

Cell therapy with autologous donor‐specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor‐specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR‐Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor‐specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti‐HLA‐A2‐specific CAR were administered to Bl/6 mice at the time of transplanting an HLA‐A2⁺ Bl/6 skin graft. Donor‐specific CAR‐Tregs, but not irrelevant‐CAR Tregs, significantly delayed skin rejection and diminished donor‐specific antibodies (DSAs) and frequencies of DSA‐secreting B cells. Donor‐specific CAR‐Treg–treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen‐specific suppression. When donor‐specific CAR Tregs were tested in HLA‐A2‐sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor‐specific CAR‐Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.     

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen‐Pulsed Autologous Regulatory Dendritic Cells

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte‐derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg‐pulsed autologous DCreg (1.4–3.6 × 10⁶/kg) were administered intravenously, 1 day before MHC‐mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag‐pulsed DCreg (n = 5) was associated with evidence of modulated host CD4⁺ and CD8⁺ T cell responses to donor Ag and attenuation of systemic IL‐17 production. Circulating anti‐donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag‐pulsed DCreg rejected its graft in association with progressively elevated anti‐donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag‐pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.     

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.     

Cytomegalovirus‐Associated CD4+CD28null Cells in NKG2D‐Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction

Emerging data suggest that expansion of a circulating population of atypical, cytotoxic CD4⁺ T cells lacking costimulatory CD28 (CD4⁺CD28^null cells) is associated with latent cytomegalovirus (CMV) infection. The purpose of the current study was to increase the understanding of the relevance of these cells in 100 unselected kidney transplant recipients followed prospectively for a median of 54 months. Multicolor flow cytometry of peripheral blood mononuclear cells before transplantation and serially posttransplantation was undertaken. CD4⁺CD28^null cells were found predominantly in CMV‐seropositive patients and expanded in the posttransplantation period. These cells were predominantly effector‐memory phenotype and expressed markers of endothelial homing (CX3CR1) and cytotoxicity (NKG2D and perforin). Isolated CD4⁺CD27^?CD28^null cells proliferated in response to peripheral blood mononuclear cells previously exposed to CMV‐derived (but not HLA‐derived) antigens and following such priming incubation with glomerular endothelium resulted in signs of endothelial damage and apoptosis (release of fractalkine and von Willebrand factor; increased caspase 3 expression). This effect was mitigated by NKG2D‐blocking antibody. Increased CD4⁺CD28^null cell frequencies were associated with delayed graft function and lower estimated glomerular filtration rate at end follow‐up. This study suggests an important role for this atypical cytotoxic CD4⁺CD28^null cell subset in kidney transplantation and points to strategies that may minimize the impact on clinical outcomes.     

Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC‐Specific Chimeric Antigen Receptor

CD4⁺CD25^highFOXP3⁺ regulatory T cells (Tregs) are involved in graft‐specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft‐specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2‐CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2‐CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2‐CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2‐CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg‐supported weaning after allogeneic transplantation.     

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.