Late diversification in the clonal composition of human cytomegalovirus-specific CD8+ T cells following allogeneic hemopoietic stem cell transplantation

To investigate the mechanisms of human T-cell reconstitution following allogeneic hemopoietic stem cell transplantation (alloSCT), we analyzed the clonal composition of human cytomegalovirus (HCMV)-specific or Epstein-Barr virus (EBV)-specific CD8+ T cells in 10 alloSC transplant recipients and their donors. All virus-specific CD8+ T-cell clones isolated from recipients after alloSCT contained DNA of donor origin. In all 6 D+/R+ sibling alloSCTs from seropositive donors into seropositive recipients, donor virus-specific clones transferred in the allograft underwent early expansion and were maintained long term in the recipient. In contrast, in 2 of 3 HCMV D+/R- alloSC transplant recipients in whom there was no detectable HCMV infection, donor HCMV-specific clones were undetectable, whereas donor EBV-specific clones were maintained in the same EBV-seropositive recipients, suggesting that transferred clones require antigen for their maintenance. Following D-/R+ transplantation from 3 seronegative donors into seropositive recipients, a delayed primary virus-specific CD8+ T-cell response was observed, in which the T cells contained donor DNA, suggesting that new antigen-specific T cells arose in the recipient from donor-derived progenitors. In 2 of 4 HCMV D+/R+ sibling allograft recipients the clonal composition underwent diversification as compared with their donors, with delayed persistent expansion of HCMV-specific clones that were undetectable in the donor or in the recipient during the early months after transplantation; this diversification may represent expansion of new clones generated from donor-derived progenitors. We conclude that, following alloSCT, late diversification of the HCMV-specific CD8+ T-cell clonal repertoire can occur in response to persistent viral antigen.     

Immune tolerance to hepatitis C virus acquired during engraftment of bone marrow transplant

The CD4+ T-cell response appears to be important for clearance of hepatitis C virus (HCV) in the majority of individuals. We have recently described a series of human leucocyte antigen (HLA)-DR11-restricted T-cell epitopes derived from HCV proteins which enables distinct populations of memory CD4+ T cells to be detected and counted in all nonviraemic HCV subjects. We examined the case of an HLA-DR11+ recipient of a haematopoietic stem-cell transplant who was concurrently infected with HCV from an HLA-DR11+ donor sibling. An acute HCV hepatitis developed and was treated with type I interferon. After successful viral clearance, the recipient demonstrated a selective lack of HCV epitope-specific CD4+ T cells and absence of serological responses compared with the treated donor. The recipient had no evidence of any nonspecific immunosuppression. The subsequent effects of concurrent infection during immune reconstitution are not known in adult humans, but data from murine models suggest this can lead to a skewing of the T-cell repertoire because of thymic selection. From the above observations, it is plausible that the introduction of foreign viral antigen into the thymus may lead to subsequent acquired central tolerance.     

Evolution of the donor T-cell repertoire in recipients in the second decade after allogeneic stem cell transplantation

After allogeneic stem cell transplantation (SCT), T lymphocyte function is reestablished from the donor's postthymic T cells and through thymic T-cell neogenesis. The immune repertoire and its relation to that of the donor have not been characterized in detail in long-term adult SCT survivors. We studied 21 healthy patients in their second decade after a myeloablative SCT for hematologic malignancy (median follow-up, 12 years). Immune profiles were compared with donor samples cryopreserved at transplant and beyond 10 years from SCT. Only one recipient was on continuing immunosuppression. Compared with the donor at transplant, there was no significant difference in CD4, CD8, natural killer, and B-cell blood counts. However, compared with donors, recipients had significantly fewer naive T cells, lower T-cell receptor excision circle levels, fewer CD4 central memory cells, more effector CD8(+) cells, and more regulatory T cells. TCR repertoire analysis showed no significant difference in complexity of TCRVβ spectratype between recipients and donors, although spectratype profiles had diverged with both gain and loss of donor repertoire peaks in the recipient. In conclusion, long-term allogeneic SCT survivors have subtle defects in their immune profile consistent with defective thymic function but compatible with normal health. This study is registered at http://www.clinicaltrials.gov as NCT00106925.     

T lymphocytes of recipient origin may contribute to the recovery of specific immune response toward viruses and fungi in children undergoing cord blood transplantation

Patients undergoing allogeneic cord blood transplantation (CBT) benefit from a low risk of graft-versus-host disease (GVHD), but there are still concerns that they be able to recover an effective immune capacity early after transplantation. We investigated the ability to develop in vitro T-lymphocyte-mediated immune response toward human cytomegalovirus and Candida albicans antigens, early and late after transplantation, in children given cord blood transplants from either an HLA-identical sibling or an unrelated donor. Proliferative capacity and frequency of antigen-specific T cells were evaluated; antigen-specific CD4(+) T-cell clones were also generated and characterized for T-cell receptor repertoire diversity, cytokine phenotype, and their origin (either from donor or patient). We found that the majority of recipients can develop a specific response to viral or fungal antigens already early after transplantation. Antigen-specific T-cell clones of both donor and recipient origin contributed to the reconstitution of immune response. Antigen-specific T lymphocytes of recipient origin were detected in patients receiving a transplant from a relative, after a chemotherapy-based conditioning regimen, and who did not have GVHD. Our results document, at a clonal level, that after CBT recovery of either polyclonal or pauciclonal T-cell response toward widespread pathogens is prompt, with some patients benefiting from a contribution of recipient-derived cells.     

Inhibitory effect of a CD4-CDR3 peptide analog on graft-versus-host disease across a major histocompatibility complex-haploidentical barrier

A structure-based designed peptide has been engineered to exhibit the same molecular surface as a portion of the CDR3-like region in domain 1 of the murine CD4 molecule. Earlier in vitro experiments indicated that this analog, known as rD-mPGPtide, inhibited T-cell proliferation in mixed lymphocyte reactions and blocked activation of both normal CD4+ T cells and T-cell lines after T-cell receptor triggering. In addition, rD-mPGPtide proved to be a potent inhibitor in vivo of CD4+ T-cell- mediated experimental allergic encephalomyelitis disease in the SJL mouse model. In this current report, we have evaluated the potential of rD-mPGPtide for suppressing the development of graft-versus-host disease (GVHD) in an irradiated major histocompatibility complex (MHC)- haploidentical murine bone marrow transplantation (BMT) model [(B6 x DBA/2)F1-->(B6 x CBA)F1 (950 cGy)]. Our results indicated that early administration of rD-mPGPtide was effective in the inhibition of alloreactive responses of the donor T cells against the host and thus delayed or prevented the onset of GVHD. The median survival time of animals treated with rD-mPGPtide was enhanced as much as four-fold with as little as a single dose of peptide at the time of transplant. Decreased alloreactivity was indicated by phenotypic and functional analysis of positively selected thoracic duct lymphocytes 4 days after transplant and by histopathological examination of skin and gastrointestinal tissue samples 4 weeks later. Therefore, the administration of a CD4-CDR3 peptide is an efficacious approach against the development of GVHD during allogeneic BMT.     

T-cell immune constitution after peripheral blood mononuclear cell transplantation in complete DiGeorge syndrome

Complete DiGeorge syndrome (cDGS) is a congenital disorder characterized by typical facies, thymic aplasia, susceptibility to infections, hypoparathyroidism and conotruncal cardiac defect. Fetal thymus or post-natal thymus tissue transplantations and human leucocyte antigen (HLA)-genoidentical bone marrow transplantations were followed in a few cases by immune reconstitution. More recently, a peripheral blood mononuclear cell transplantation (PBMCT) was performed with an HLA-genoidentical donor and followed by a partial T-cell engraftment and immune reconstitution. We report a boy with cDGS, without cardiac defect, who suffered recurrent severe infections. At the age of 4 years, he underwent PBMCT from his HLA-genoidentical sister. He received no conditioning regimen, but graft-versus-host disease (GVHD) prophylaxis was with oral cyclosporin A and mycophenolate mofetil. Toxicity was mild, with grade I acute GVHD. The patient is currently 2.5 years post-PBMCT with excellent clinical performances. Mixed chimaerism can only be observed on the T-cell population (50% donor T cells). T-lymphocyte count fluctuated (CD3 more than 400 x 10(6)/l at d 84 and CD4 more than 200 x 10(6)/l at d 46). Exclusive memory phenotype T cells and absence of new thymic emigrants suggest expansion of infused T cells. T-cell mitogen and tetanus antigen responses normalized a few months after transplantation. After immunizations, specific antibodies were produced. PBMCT from an HLA identical sibling could be an efficient treatment of immune deficiency in cDGS.     

Similar pattern of thymic-dependent T-cell reconstitution in infants with severe combined immunodeficiency after human leukocyte antigen (HLA)-identical and HLA-nonidentical stem cell transplantation

Donor T cells after stem cell transplantation reconstitute by 2 different pathways: by expansion from grafted, mature T cells and by intrathymic maturation from progenitor cells. This study characterized thymic-dependent reconstitution of CD4(+) T cells following different transplant modalities in patients with severe combined immunodeficiency (SCID). Three groups of patients were studied: one group after transplantation from human leukocyte antigen (HLA)-identical siblings with unmanipulated grafts without conditioning, a second group after transplantation from HLA-nonidentical parents with T-cell-depleted grafts without preconditioning, and a third group with prior conditioning. Reconstitution of the T-cell compartment was monitored by determining the expression of CD45 isoforms by developing CD4(+) cells in the peripheral blood and in discriminating expanded (CD45RO(+)) and newly generated (CD45RA(+)) T cells. Concomitantly, changes in the size of the thymus were evaluated sequentially by ultrasonography. Reconstitution of CD4(+)CD45RA(+) cells was delayed in all patients for several months, including patients after HLA-identical transplantation, and was always paralleled by normalization of the size of the thymus. No engraftment of donor progenitor cells was observed, as studied in one patient transplanted without conditioning. CD4(+)CD45RO(+) cells were detected early after transplantation only in patients given unmanipulated grafts. The study showed that thymic-dependent T-cell maturation in these patients with SCID runs an autonomous course, independent of graft manipulation, of major HLA disparities, and of whether conditioning is used or not. In addition, thymic maturation may not require engraftment of donor-derived CD34(+) cells in the marrow. (Blood. 2000;96:4344-4349)     

Successful generation of primary virus-specific and anti-tumor T-cell responses from the naive donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regulatory T cells

Background

One of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire.

Design and Methods

Antigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers.

Results

Using this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO⁺ responder cells resulted in improved priming and expansion of antigen-specific precursor T cells.

Conclusions

In conclusion, we present a robust method for the in-vitro induction and isolation of antigen-specific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.     

Effective graft depletion of MiHAg T-cell specificities and consequences for graft-versus-host disease

Minor histocompatibility antigen (MiHAg) differences between donor and recipient in MHC-matched allogeneic hematopoietic stem cell transplantation (allo-HSCT) often result in graft-versus-host disease (GVHD). While MiHAg-specific T-cell responses can in theory be directed against a large number of polymorphic differences between donor and recipient, in practice, T-cell responses against only a small set of MiHAgs appear to dominate the immune response, and it has been suggested that immunodominance may predict an important contribution to the development of GVHD. Here, we addressed the feasibility of graft engineering by ex vivo removal of T cells with 1 or more defined antigen specificities in a well-characterized experimental HSCT model (B6 --> BALB.B). We demonstrate that immunodominant H60- and H4-specific CD8(+) T-cell responses can be effectively suppressed through MHC class I tetramer-mediated purging of the naive CD8(+) T cell repertoire. Importantly, the development of GVHD occurs unimpeded upon suppression of the immunodominant MiHAg-specific T-cell response. These data indicate that antigen-specific graft engineering is feasible, but that parameters other than immunodominance may be required to select T-cell specificities that are targeted for removal.     

Co-transplantation of pure blood stem cells with antigen-specific but not bulk T cells augments functional immunity

Impaired immunity is a fundamental obstacle to successful allogeneic hematopoietic cell transplantation. Mature graft T cells are thought to provide protection from infections early after transplantation, but can cause life-threatening graft-vs.-host disease. Human CMV is a major pathogen after transplantation. We studied reactivity against the mouse homologue, murine CMV (MCMV), in lethally irradiated mice given allogeneic purified hematopoietic stem cells (HSCs) or HSCs supplemented with T cells or T-cell subsets. Unexpectedly, recipients of purified HSCs mounted superior antiviral responses compared with recipients of HSC plus unselected bulk T cells. Furthermore, supplementation of purified HSC grafts with CD8(+) memory or MCMV-specific T cells resulted in enhanced antiviral reactivity. Posttransplantation lymphopenia promoted massive expansion of MCMV-specific T cells when no competing donor T cells were present. In recipients of pure HSCs, naive and memory T cells and innate lymphoid cell populations developed. In contrast, the lymphoid pool in recipients of bulk T cells was dominated by effector memory cells. These studies show that pure HSC transplantations allow superior protective immunity against a viral pathogen compared with unselected mature T cells. This reductionist transplant model reveals the impact of graft composition on regeneration of host, newly generated, and mature transferred T cells, and underscores the deleterious effects of bulk donor T cells. Our findings lead us to conclude that grafts composed of purified HSCs provide an optimal platform for in vivo expansion of selected antigen-specific cells while allowing the reconstitution of a naive T-cell pool.     

Expansion and activation of minor histocompatibility antigen HY-specific T cells associated with graft-versus-leukemia response

The immune system of females is capable of recognizing and reacting against the male-specific minor histocompatibility antigen (mHA), HY. Thus, cytotoxic T-lymphocytes (CTLs) recognizing this antigen may be useful in eradicating leukemic cells of a male patient if they can be generated in vivo or in vitro from a human leukocyte antigen (HLA)-identical female donor. The HLA-A*0201-restricted HY antigen, FIDSYICQV, is a male-specific mHA. Using HLA-A2/HY peptide tetrameric complexes, we reveal a close association between the emergence of HY peptide-specific CD8(+) T cells in peripheral blood and molecular remission of relapsed BCR/ABL(+) chronic myelogenous leukemia in lymphoid blast crisis in a patient who underwent female-to-male transplantation. Assessment of intracellular cytokine levels identified T cells that produce interferon-gamma in response to the HY peptide during the presence of HY tetramer-positive T cells. These results indicate that transplant with allogeneic HY-specific CTLs has therapeutic potential for relapsed leukemia, and that expansion of such T cells may be involved in the development of a graft-versus-leukemia response against lymphoblastic leukemia cells.     

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.     

Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo

Although CD4+CD25+ regulatory T cells are pivotal in the prevention of autoimmunity and appear to mediate transplantation tolerance, little is known concerning their antigen specificity. Here we describe the induction of a human CD4+CD25+ regulatory T-cell line specific for a defined peptide alloantigen (human leukocyte antigen A2 [HLA-A2] 138-170) by priming purified CD4+CD25+ cells ex vivo. The regulatory cells were anergic and retained their ability to suppress antigen-driven responses of CD4+CD25- cells. They inhibited not only interleukin 2 (IL-2) secretion by CD4+CD25- T cells specific for the same peptide but also direct alloresponse of naive CD4+CD25- T cells stimulated by semiallogeneic dendritic cells (DCs) in the presence of the peptide ("linked suppression"). They also suppressed the response of CD4+ T cells specific for viral and bacterial antigens. The suppressive T-cell line showed sustained high CD25 expression. These findings suggest that peripheral CD4+CD25+ regulatory cells are a precommitted cell lineage from which cells with specificity for non-self-peptides can be selected. This may pave the way for inducing and expanding peptide antigen-specific regulatory T cells ex vivo for cell therapy in transplantation, allergy, and autoimmune disease.     

In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.     

Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo

T-cell anergy is a tolerance mechanism defined as a hyporesponsive status of antigen-specific T cells upon prior antigen encounter and is believed to play a critical role in the evasion of tumor immunity and the amelioration of allogeneic transplant rejection. Molecular mechanisms in controlling T-cell anergy are less known. We show here that administration of an agonistic monoclonal antibody (mAb) to CD137, a member of the tumor necrosis factor receptor superfamily, prevents the induction of CD8+ cytolytic T-lymphocyte (CTL) anergy by soluble antigens. More importantly, CD137 mAb restores the functions of established anergic CTLs upon reencountering their cognate antigen. As a result, infusion of CD137 mAb inhibits progressive tumor growth that is caused by soluble tumor antigen-induced tolerance in a P815R model. CD137 mAb also restores proliferation and effector functions of anergic alloreactive 2C T cells in a bone marrow transplantation model. Our results indicate that ligation of CD137 receptor delivers a regulatory signal for T-cell anergy and implicate manipulation of the CD137 pathway as a new approach to break T-cell tolerance.     

High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT

Regulatory T cells (T(reg)s) that constitutively express FOXP3 are instrumental to the maintenance of tolerance and may suppress graft-versus-host disease (GVHD) in humans. To determine whether regulatory T cells in allogeneic stem cell transplants (SCTs) ameliorate GVHD after transplantation, we quantitated the coexpression of FOXP3 on CD4(+) T cells in 32 donor SCTs infused into HLA-matched siblings and examined GVHD incidence in respective recipients. High CD4(+)FOXP3(+) T-cell count in the donor was associated with a reduced risk of GVHD. We monitored T(reg)s during immune reconstitution in 21 patients with leukemia undergoing a T-cell-depleted allogeneic SCT. Early after SCT, there was a significant expansion in the CD4(+)FOXP3(+) T-cell compartment. A low CD4(+)FOXP3(+) T-cell count early after SCT (day 30) was associated with an increased risk of GVHD, and the ratio of CD4(+)FOXP3(+) T cells to CD4(+)CD25(+)FOXP3(-) T cells was significantly reduced in patients with GVHD, suggesting diminished control of effector T cells. Our findings suggest that graft T(reg) content may predict for risk of GVHD after SCT. Determining the T(reg) levels in the donor and manipulating T(reg)s early after transplantation may provide a new approach to controlling GVHD.     

Rapid helper T-cell recovery above 200 x 10 6/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation

The current study evaluates the role of quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes including overall survival (OS) and non-relapse mortality (NRM) after allogeneic stem cell transplantation. A total of 69 allogeneic recipients were included with following diagnoses: acute myeloid leukemia 42, acute lymphoblastic leukemia 5, chronic myeloid leukemia 15, non-Hodgkin's lymphoma 5 and high-risk myelodysplastic syndrome 2. The peripheral lymphocyte subset counts (CD3+ T cells, CD3+4+ helper T cells, CD3+8+ cytotoxic T cells, CD19+ B cells, and CD56+ natural killer cells) were measured at 3, 6 and 12 months. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (P<0.0001), NRM (P=0.0007), and opportunistic infections (P=0.0108) at the cutoff value of 200 x 10(6)/l CD4(+) helper T cells. Rapid CD4+ helper T-cell recovery was also associated with a higher CD4+ helper T-cell transplant dose (P=0.006) and donor type (P<0.001). An early CD4+ helper T-cell recovery at 3 months correlated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate analysis, rapid recovery of CD4+ helper T cells at 3 months was a favorable prognostic factor together with higher CD34+ cell transplant dose in terms of OS (P=0.001) and NRM (P=0.005).     

CD62L- memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L(-)) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L(-) T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L(-) T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L(-) T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L(-) T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L(-) T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L(-) T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L(-) T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L(-) T cells and their potential applications in clinical hematopoietic cell transplantation.     

Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation

We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence.     

Low-intensity transplant regimens facilitate recruitment of donor-specific regulatory T cells that promote hematopoietic engraftment

Low- or reduced-intensity conditioning regimens for allogeneic hemopoietic stem cell transplantation are effective at establishing donor hematopoietic engraftment and host-vs.-graft (HvG) tolerance. We investigated the mechanisms of HvG tolerance induction and maintenance in an animal model in which transplantation of sublethally irradiated female recipients with bone marrow (BM) from syngeneic male donors produces mixed chimerism. Splenocytes from chimeric mice inhibited HY-specific CD8(+) T cell responses both in vitro and in vivo, and their adoptive transfer facilitated donor hematopoietic engraftment. These properties were contained within the CD4(+)CD25(+) population. The conditioning protocol alone led to a proportional expansion of regulatory T cells (T(regs)), but the inhibitory activity was induced only if male BM was infused. The administration of anti-CD25-depleting antibodies to conditioned recipients at time of BM transplantation prevented donor-recipient chimerism but did not affect engraftment if performed after the establishment of chimerism, thus indicating that recipient T(regs) are required for the generation but not the maintenance of HvG tolerance. We conclude that donor-specific T(regs) of recipient origin are recruited when the donor antigens are present during reduced-intensity conditioning-induced T(reg) expansion.