Cytomegalovirus-specific regulatory and effector T cells share TCR clonality--possible relation to repetitive CMV infections.

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T-cell response, CMV-IE-1 antigen-specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE-1/pp65-specific T-cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV-induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV response. Analysis of in vitro expanded CD4+ epitope-specific cells revealed that CMV-specific CD4+CD25(high) Treg cells functionally suppress CD25(low) effector T cells (Teff) upon epitope-specific reactivation. Their phenotype is similar to iTreg - CD39(high) /Helios-/IL-2(low) /IFNγ(high) /IL-10±/TGFß-LAP±/FOXP3+ and methylated foxp3 locus. Remarkably, in vitro expanded CD4+CD25(high) iTreg share the same dominant TCR-Vß-CDR3 clones with functionally distinct CD4+CD25(low) Teff. Moreover, the same clones were present in freshly isolated CD4+CD25(high) and CD4+CD25(low) T cells suggesting their in vivo generation. These findings directly demonstrate that Teff and iTreg can differentiate from one "mother" clone with specificity to the same viral epitope and indicate that peripheral iTreg generation is related to frequent antigen appearance.

Adoptive T-cell therapy of a lung transplanted patient with severe CMV disease and resistance to antiviral therapy.

Infections with cytomegalovirus (CMV) can induce severe complications after transplantation, particularly in patients resistant to virostatic therapy. Adoptive transfer of CMV-specific T-cell lines has demonstrated promising results in patients after hematopoietic stem cell transplantation. However, the generation of specific T-cell lines ex vivo and their function in vivo is complicated in solid organ transplant (SOT) recipients. Here, we present the successful adoptive transfer of autologous CMV-specific T cells to a lung transplant recipient with ganciclovir-resistant CMV-pneumonia requiring mechanical ventilation. Infused T cells rapidly expanded in vivo and efficiently inhibited viral replication as confirmed by extensive longitudinal immunological monitoring. After full recovery, the patient was released from the clinic. After 4 weeks, the infection reappeared and persisted at a low level even after a second T-cell infusion. Our experimental data indicate that this could be the consequence of the late differentiated phenotype of the infused T cells and therefore their insufficient longevity in vivo. In summary, our report signifies the high therapeutic potential of adoptive immunotherapy in the treatment of SOT recipients when all other measures show no effect. Further studies have to elucidate the most potent strategies to generate antigen-specific T cells with high functional capacity and robust long-term persistence.

Development of Kaposi's sarcoma under sirolimus-based immunosuppression and successful treatment with imiquimod.

Kaposi's sarcoma (KS) is a vascular neoplasm typically observed in the immunocompromised patient populations, such as acquired immunodeficiency syndrome or transplant patients. KS can appear simultaneously at multiple sites as red to purple, maculo-papular or nodular cutaneous lesions sometimes showing a visceral extension. Sirolimus, an immunosuppressive agent with potent antitumor activity, has been effective in combating post-transplant KS. However, an aggressive regimen of immunosuppression for therapy of severe acute rejection episodes may abolish the antitumor effects of sirolimus. The following is a description of KS development under immunosuppressive therapy with sirolimus, and the successful treatment of KS lesions utilizing the topical application of imiquimod 5% cream, an immune response modifier.

HLA type-independent method to monitor polyoma BK virus-specific CD4 and CD8 T-cell immunity.

(Re)activation of quiescent viral diseases is a major problem in immunosuppressed transplant patients. Polyoma BK virus-associated nephropathy (PVAN) caused by active polyoma BK virus (BKV) infection became a main reason for graft loss in kidney transplantation. After diagnosis, most transplant centers react by reducing immunosuppression (IS) to allow the immune system to control the infection. However, the impact of reduced IS on BKV immunity is not well researched. Here we present an HLA type-independent method to monitor BKV-specific T-cell immunity. Applying our method, viral protein 1-specific CD4+ and CD8+ T-cell responses were detected in patients with serum BKV-DNA levels >250 000 copies/mL. In addition, specific T-cell responses were also found in allograft-infiltrating cells. The method can be used to assess the impact of decreased immunosuppression on BKV immunity and to clarify the role of specific T cells in the pathogenesis of PVAN. We strongly recommend its implementation in future clinical studies.

[Adoptive transfer of Epstein-Barr virus-specific T-lymphocytes in chronic active Epstein-Barr infection]. / Adoptiver Transfer EBV-spezifischer T-Lymphozyten bei chronisch aktiver EBV-Infektion.

HISTORY: A 27-year-old man was admitted because of intermittent fever, fatigue, lymphadenopathy and splenomegaly for 20 years. Chronic administration of 6 - 8 g aspirin per day (self-prescribed) resulted in limited control of symptoms and in the development of analgesic nephropathy. INVESTIGATIONS: The patient had prominent splenomegaly and lymphadenopathy without histological signs of malignancy. Monocytosis and T-lymphopenia were also present. Infectious disease testing revealed IgG+/IgM- EBV serology and EA-EBV-mRNA nested PCR clearly demonstrated the presence of lytic EBV-proteins in PBMCs. TREATMENT AND COURSE: As chronic active Epstein-Barr virus infection (CAEBV) was highly probable, treatment with aciclovir, gancilovir and steroids was started. Because treatment failed adoptive T-cell transfer with autologous EBV-specific T-cells was performed. After three consecutive infusions the patient responded with a complete remission of fever, fatigue, lymphadenopathy and splenomegaly without adverse effects. Retrospective real-time PCR analysis showed a decrease in viral load from 62847 copies/ microg DNA to 45 - 250 copies after treatment. The patient remains in stable remission without signs of CAEBV (> 4 years). CONCLUSION: Adoptive transfer of autologous, EBV-specific T-lymphocytes is a promising treatment in CAEBV.

Homing of in vitro-generated donor antigen-reactive CD4+ T lymphocytes to renal allografts is alpha 4 beta 1 but not alpha L beta 2 integrin dependent.

The extravasation and sequestration of Ag-reactive T lymphocytes into vascularized organ allografts depend on a cascade of complex interactions among circulating lymphocytes, endothelial cells, and extracellular matrix proteins. Ag-activated donor-specific CD4 T cells are major initiators and effectors in the allograft rejection response. Interfering with the intragraft homing of activated CD4 T cells may represent a novel therapeutic approach in transplant recipients. We have developed a FACS-based short-term homing assay that allows tracing in vitro-generated Ag-reactive CD4 T cells after adoptive transfer in test rat recipients. Allospecific cell lines were preincubated with anti-alpha(4)beta(1) or anti-alpha(L)beta(2) mAb, because of enhanced expression of both integrin receptors after alloactivation. The pretreated Lewis(BN) lymphocytes were carboxyfluorescein diacetate succinimidyl ester labeled and adoptively transferred into Lewis rat recipients of Brown Norway kidney allografts. The injection of equal numbers of PKH-26-labeled untreated cells allowed quantitative comparison of both populations in the same animal. Ex vivo treatment with anti-alpha(4)beta(1) mAb diminished intragraft infiltration of adoptively transferred T cells by 85% in a donor-specific fashion. In contrast, treatment with anti-alpha(L)beta(2) mAb did not affect intragraft cell sequestration. Hence, blocking alpha(4)beta(1) integrin interactions represents a novel strategy in preventing local intragraft recruitment of Ag-reactive CD4 T cells in transplant recipients.

Potential of allospecific gene-engineered T cells in transplantation gene therapy: specific T cell activation determines transgene expression in vitro and in vivo.

T lymphocytes, regardless of their specificity, are considered key targets for genetic modification in the treatment of inherited or acquired human diseases. In this study, we generated Lewis T cell lines specific for Dark Agouti rat alloantigens and tested the potential of allospecific T lymphocytes as carriers of genes encoding therapeutic proteins in transplantation gene therapy. These allospecific T lymphocytes were successfully, stably transduced with enhanced green fluorescent protein (EGFP) by an Mo-MuLV-based retrovirus vector. A novel gene delivery protocol was utilized, resulting in nearly 100% EGFP-expressing T cells. This approach enabled tracking of allospecific transduced T cells in vivo and illustrates their transgene production by fluorometric determination after ex vivo isolation. Quantitation of EGFP transgene expression was used to determine the influence of T cell receptor-specific activation on transgene regulation. A strict positive correlation between activation state and expression level was detected in vitro and in vivo. The activation-induced increase in transgene expression could be blocked by interference with T cell activation signaling pathways by cyclosporin A, anti-CD4 MAb, or CTLA4-Ig. These data provide strong evidence that direct or indirect effects caused by activation-induced transcription factors are crucial in transgene upregulation. Allospecific activation in spleens, lymph nodes, and transplanted grafts can be considered as antigen-specific targeting strategy. This activation might be useful in expressing therapeutic proteins such as TGF-beta or IL-10 specific to these sites. T lymphocyte priming and activation might be prevented or altered by modification of the local microenvironments, thereby exerting a therapeutic influence on acute and chronic graft rejection processes.