T-cell Depleted Bone Marrow Transplantation Research Articles

Long-Term Immune Reconstitution and Outcome After HLA-Nonidentical T-Cell–Depleted Bone Marrow Transplantation for Severe Combined Immunodeficiency: A European Retrospective Study of 116 Patients

AbstractWe have performed a retrospective analysis of the development of T- and B-cell functions after HLA-nonidentical T-cell–depleted bone marrow transplantation (BMT) performed in 193 patients with severe combined immunodeficiency (SCID) at 18 European centers between December 1982 and December 31, 1993. One hundred sixteen of 193 patients were alive with evidence of engraftment 6 months after BMT. Development of T-cell function occurred earlier than B-cell function and was achieved more frequently up to the time of last follow-up. The median time to achieve normal T-cell function was 8.7 months, whereas the median time to achieve normal B-cell function was 14.9 months. Twenty-four patients died later than 6 months post-BMT, mainly due to chronic graft-versus-host disease (cGVHD) and/or viral infection. Absence of T-cell reconstitution 6 months after BMT, unlike absence of B-cell reconstitution, was associated with a poor outcome. Two additional factors were associated with a poor outcome: presence of cGVHD 6 months after BMT and B− SCID versus B+ SCID. However, two of these three factors remained as significant prognostic factors in a multivariate analysis: the absence of T-cell function and the presence of cGVHD 6 months after BMT. Analysis of the factors influencing the development of immune reconstitution showed that T- and B-cell functions occurred earlier and more frequently in B+ SCID versus B− SCID patients. Acute GVHD was associated with a slower development of T-cell function at 6 months, and cGVHD had a negative influence on the development of T-cell function afterwards, but neither acute nor chronic GVHD was found to influence the development of B-cell function. Once engraftment occurred, whether patients had or had not received Busulfan in the conditioning regimen did not influence the kinetics and quality of T-cell function development. In a multivariate study, two factors were found to influence the T-cell function 6 months after BMT: type of SCID and acute GVHD. The results of this retrospective analysis should lead to new protocols adapted to SCID disease, considering that disease-related as well as BMT-related parameters influence the development of immune function and thereby long-term outcome after HLA-nonidentical T-cell–depleted BMT.

CD19 CAR T Cells Maintain Efficacy in the Allogeneic Environment but Mediate Acute Graft-Versus-Host-Disease Only in the Presence CD19+ Acute Lymphoblastic Leukemia

A significant portion of patients with relapsed acute lymphoblastic leukemia (ALL) who are eligible for treatment with chimeric-antigen-receptor (CAR) T cells have undergone a previous hematopoietic stem cell transplantation. We and others have previously demonstrated that the allogeneic environment, even in the presence mild graft-versus-host disease (GVHD), severely impairs tumor-directed T-cell immunity. To study the behavior of CAR cells in the allogeneic setting we chose a murine model of minor mismatched allogeneic T-cell depleted bone marrow transplantation (BMT) followed by CAR T (CD19-28-z) cell infusion in recipients bearing a murine B-precursor ALL model (positive for CD19). Importantly, the leukemia persists following myeloablative radiation thus mimicking post-transplant minimal residual disease. Donor-derived, post-transplant injection of CD19 CAR T cells eliminated residual leukemia in the syngeneic as well as the allogeneic settings. CD19 CAR T cells harvested from allogeneic recipients maintained in-vitro ability to produce IFN-gamma and degranulate in the presence of ALL ex vivo, at levels comparable to syngeneic CD19 CAR T cells. However, CAR T cells administered in the allogeneic environment had the potential to mediate severe acute GVHD with early mortality of recipients not typically seen in this minor mismatch model. This occurred across multiple T cell doses capable of clearing leukemia (0.3e6-5e6), in transduced CD19 CAR T cells generated from donors tolerized to allogeneic antigens, and when CD19 CAR T cell infusion was delayed following BMT. In-vivo tracking of transferred cells showed comparable expansion and persistence of the CD19 CAR T cells in the allogeneic and syngeneic environments. However, syngeneic CAR T cells tended to develop later into central memory T cells (CD62L+CD44+), whereas the profile of allogeneic cells was significantly skewed toward effector T cells (CD62L-CD44+). Remarkably, the process of CAR-driven acute GVHD in this minor mismatch model was only seen in the presence of leukemia, indicating a CAR-target interaction influences the induction of GVHD. Indeed, pro-inflammatory cytokines IFN-gamma and IL-6 were elevated only in the presence of both ALL and CAR T cells, whereas TNF alpha levels were undetectable in all instances. We are currently testing whether neutralization of cytokines can prevent GVHD in these models. Altogether, these data demonstrate efficacy of CD19 CAR to clear residual leukemia in an immunocompetent mouse model, and maintain initial cytotoxicity despite the potentially suppressive allogeneic environment. However, we demonstrated potential risks of allogeneic CAR T cells aggravating GVHD in the presence of residual leukemia, likely via cytokine-mediated manner. Clinically, as IL-6 was shown to be significant in cytokine release syndrome, this may represent a murine model to study potential interventions. DisclosuresNo relevant conflicts of interest to declare.

MyD88 in Donor Bone Marrow Cells Is Critical for Protection from Acute Intestinal Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation is an important therapeutic modality used to treat malignancies of hematopoietic origin, such as leukemia and lymphoma. However, development of graft-versus-host disease (GVHD) causes non-relapse mortality and substantial morbidity of recipients. Myeloid differentiation factor 88 (MyD88), a major adaptor mediating TLR signaling, is also known to deliver pro-inflammatory signals. Activation of inflammatory signaling through MyD88 plays a key role in the expansion of myeloid-derived suppressor cells (MDSC) which are a heterogeneous population of immature myeloid ells with anti-inflammatory activity. To explore the contribution of MyD88 expressed by donor bone marrow (BM) cells to development of GVHD, we induced GVHD using T-cell-depleted BM (TCD-BM) isolated from MyD88-deficient (MyD88KO) mice and T cells isolated from wild-type (WT) mice. We employed C57BL/6 (H-2b) → B6D2F1 (H-2b/d) mouse model of GVHD, which differ at major and minor histocompatibility loci. Lethally irradiated B6D2F1 recipient mice were transplanted with either T cell-depleted bone marrow (TCD BM, 5 x 106) from either WT or MyD88KO mice together with WT spleen T cells (1 x 106). Transplantation with MyD88KO TCD BM aggravated GVHD; serious gut damage was evident, with infiltration of T cells specifically into the intestines of recipients. GVHD hosts transplanted with MyD88KO TCD BM exhibited markedly reduced expansion of MDSC. GVHD aggravation after transplantation with MyD88KO TCD-BM, associated with high-level T cell infiltration into the intestine and insignificant expansion of MDSC, was reproduced in another minor histocompatibility mismatch model (C57BL/6 → BALB.B). We next examined allogeneic T cells in the spleens of GVHD hosts in terms of the expression levels of CCR9, which are known to be associated with T cell migration to the intestinal mucosa and the proportion of CCR9 positive cells in CFSE low CD8+ T cells was higher in recipients of MyD88KO TCD BM than WT controls. In parallel, the levels of CCL25 were more highly expressed in the gut of MyD88KO recipients than WT controls. Mixed leukocyte cultures of CFSE- labeled C57BL/6 T cells and irradiated B6DF1 feeder cells were prepared in the presence of MDSC isolated from MyD88KO or WT mice. T cells, co-incubated with MDSC isolated from MyD88KO BM, exhibited a greater extent of CFSE dilution and less Annexin V staining, compared to T cells co-incubated with cells from WT BM. Moreover, MDSC from recipients of MyD88KO TCD BM exhibited a reduced suppressive function, compared to their WT counterparts. Next, we determined whether insufficient expansion of and ineffective suppression by MDSC caused severe GVHD in recipients of MyD88KO TCD BM. Supplementation of transplanted mice with MDSC from WT mice, not from MyD88KO mice, attenuated the severity of GVHD and reduced intestinal T cell infiltration in recipients of MyD88KO TCD BM. To verify the importance of MyD88-mediated signaling by MDSC in protection against severe GVHD, we determined if transplantation with TCD-BM cells containing high levels of MDSC attenuated the severity of GVHD. Pre-treatment of BM donors with lipopolysaccharide increased the frequencies of MDSC and the amounts of MyD88 transcripts in TCD-BM transplant, and alleviated the severity of GVHD and intestinal T-cell infiltration. To explore whether MDSC expansion levels could be used to predict the severity of intestinal GVHD, the T/MDSC ratios were calculated in blood of patients at the time of engraftment and were significantly higher in patients with intestinal GVHD ≥ grade 2. In conclusion, we have shown that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal acute intestinal GVHD. DisclosuresNo relevant conflicts of interest to declare.

Effects of easy-to-perform procedures to reduce bacterial colonization withStreptococcus mutansandStaphylococcus aureuson toothbrushes

It is well known that dental caries and periodontitis are the consequence of bacterial colonization and biofilm formation on the enamel surface. The continuous presence of bacterial biofilms on the tooth surface results in demineralization of the tooth enamel and induces an inflammatory reaction of the surrounding gums (gingivitis). The retention and survival of microorganisms on toothbrushes pose a threat of recontamination especially for certain patients at risk for systemic infections originating from the oral cavity, e.g., after T-cell depleted bone marrow transplantation. Thus, the effects of different decolonization schemes on bacterial colonization of toothbrushes were analyzed, in order to demonstrate their applicability to reduce the likelihood of (auto-)reinfections. Toothbrushes were intentionally contaminated with standardized suspensions of Streptococcus mutans or Staphylococcus aureus. Afterwards, the toothbrushes were exposed to rinsing under distilled water, rinsing and drying for 24 h, 0.2% chlorhexidine-based decolonization, or ultraviolet (UV) radiation. The remaining colony forming units were compared with freshly contaminated positive controls. Each experiment was nine-fold repeated. Bi-factorial variance analysis was performed; significance was accepted at P < 0.05. All tested procedures led to a significant reduction of bacteral colonization irrespective of the toothbrush model, the brush head type, or the acitivity state. Chlorhexidine-based decolonization was shown to be superior to rinsing and slightly superior to rinsing and drying for 24 h, while UV radiation was similarly effective as chlorhexidine. UV radiation was slightly less prone to species-dependent limitations of its decolonizing effects by bristle thickness of toothbrushes than chlorhexidin. Reduction of bacterial colonization of toothbrushes might reduce the risk of maintaining bacterial infections of the upper respiratory tract. Accordingly, respective procedures are advisable, particularly as they are cheap and easy to perform.

Unmanipulated Bone Marrow Transplantation From Haploidentical Related Donor for Patients with High-Risk Hematological Malignancies

AbstractAbstract 2350In this study we investigated the feasibility and clinical value of non T-cell depleted bone marrow transplantation (BMT) from HLA haploidentical related donor in patients with high risk hematological malignancies. Materials and Methods.Between August 2005 and May 2010, 71 patients were transplanted for acute myeloid leukemia (AML) (n=42), acute lymphoblastic leukemia (n=13), chronic myeloid leukemia (n=5), Hodgkin lymphoma (n=5), plasmacell leukemia (n=3), myelofibrosis (n=2) and myelodisplastic syndrome (n=1). Their median age was 35 years (5-71). At time of BMT, all patients were at very high risk: 39 were in early (complete remission 1 or 2) and 31 in more advanced stage of disease. Seventeen of them were given a previous transplant either autologous (n=14) or allogenic (n=3). All donors were HLA identical at 1 haplotype and mismatched for 2 (n=24) or 3 (n=47) loci on the unshared haplotype. As pretransplant regimen, 10 patients received a reduced intensity conditioning consisting of Fludarabine (Flu) alone (n=1), Flu + Thiotepa (Thio) + Melphalan (n=2) or Flu + Thio + i.v.Busulphan (Bu) (n=7), and 61 patients received a myeloablative therapy consisting of Aracytin + Cyclophosphamide combined with TBI (n=7) or Treosulphan (n=11) or Bu (n=11), whereas the last 32 consecutive patients underwent transplant after conditioning with the association of Thio + Flu + i.v. Bu. All patients received an identical graft-versus-host disease (GvHD) prophylaxis consisting of Fresenius Antithymocyte Globulin (5 mg/Kg/d from day -4 to -1) combined with Cyclosporine (1,5-3 mg/Kg/day i.v. from day -7 to +28 and orally 5 mg/Kg until day +365), Methotrexate (15 mg/sqm on day +1 and 10 mg/sqm on day +3, +6 and +11), Mycophenolate Mofetil (1 g/d from day +7 to +100) and the anti-CD 25 monoclonal antibody Basiliximab (20 mg i.v. on day 0 and +4). Bone marrow cells were harvested from all donors after priming with Filgrastim at 3–4 microg/Kg/d from day -7 to -1. Bone marrow cells were infused fresh and unmanipulated on day 0. Results.The median dose of total nucleated, CD34+ and CD3+ cells infused was 7.8 (1-28) ×108/kg, 2.1 (0.8-11) ×106/Kg and 28 (10-98) ×106/Kg, respectively. One patient had a primary graft failure and 5 patients died too early to be evaluated for engraftment. Results in terms of cumulative incidence (CI) of PMN engraftment, acute and chronic GvHD, relapse, transplant related mortality (TRM) and overall survival (OS) estimated with the Kaplan Meyer method are given in the Table. After a median follow-up of 16 (3-56) months, the 3 years probability of OS and disease-free survival (DFS) was 43% and 37% respectively for all patients (see Figure). The 3 years OS for the 42 patients with AML was 48% (61% for 30 patients transplanted in early stage and 18% for 12 patients transplanted in advanced stage, P=0.01). The 1 year OS for the 32 patients transplanted with the conditioning therapy employed in the last 2 years (Thio + Flu + i.v. Bu) was 72% in 18 patients transplanted in early stage and 52% in 14 patients transplanted in advanced stage (P=0.47).Early stageAdvanced stageAll patientsP valueN.393271CI of PMN engraftment at day 3087±0.381±0.588+0.7NSCI of acute GvHD grade II - IV34±0.716±0.626±0.3NSCI of acute GvHD grade III - IV9±0.43±0.16±0.1NSCI of chronic GvHD8±0.214±0.811±0.1NSCI of relapse at 2 years30±0.737±133±0.4NSCI of TRM at 1 year21±0.441±0.530±0.3NSCI of TRM at 2 years24±0.548±1.336±0.6NSOS at 3 years54±9.226±9.943±70.03 [Display omitted] Conclusions.BMT from haploidentical donor using G-CSF primed, unmanipulated bone marrow cells and an intensive regimen for GVHD prophylaxis is correlated with high engraftment rate, low incidence of acute and chronic GVHD, acceptable TRM and favourable patient outcome. The results seem particularly encouraging for AML patients grafted at an early disease stage. In alternative to transplant from matched unrelated donor or cord blood, this approach can be offered to high risk patients with hematological malignancies particularly for those who are on urgency for a transplant.Results are given as % ± standard error Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Host Tolerance in Dogs Following T-Cell Depleted Bone Marrow Transplantation with Absorbed ATG or CD6-Antibody.

Graft-versus-host disease (GvHD) is the major obstacle of allogeneic stem cell transplantation. Depletion of T-cells from the graft reduces the risk of GvHD, but results in a higher risk of leukemia relapse. Adoptive immunotherapy with donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after T-cell depleted allogeneic stem cell transplantation. However, GvHD may occur, if DLT is given too early after transplantation. In canine models of DLA-identical and DLA-haploidentical bone marrow transplantation, we compared different methods of T-cell depletion (TCD) and investigated the potential of DLT at different times after transplantation to induce GvHD. T-cell depletion was performed either with absorbed anti-thymocyte globuline (aATG) or with a combination of CD6-antibody and baby rabbit complement. ATG was absorbed with erythrocytes, liver, kidney and spleen for eliminating antibodies against stem cells. CD6-antibody (M-T606) and rabbit complement depleted T-cells effectively without affecting hematopoietic progenitor cells. Unlike aATG, monoclonal CD6-antibody spares natural killer (NK) cells and some CD8-positive cells. Treatment of bone marrow with aATG prevented GvHD in 9 dogs following DLA-identical transplantation. DLT on days 1 and 2 or 21 and 22 induced fatal GvHD in two dogs each. However, it did not induce GvHD when given on days 61 and 62 and later. In DLA-haploidentical bone marrow recipients, non-manipulated marrow produced fatal GvHD in all dogs (n=7), whereas marrow treated with aATG (vol:vol 1:100 and 1:200) produced fatal GvHD in 5 out of 16 dogs only. CD6-depletion prevented GvHD in 3 of 3 DLA-haploidentically transplanted dogs. DLT produced fatal GvHD in one dog each, when given on day 3, 7 or 14 after CD6-depleted haploidentical bone marrow transplantation. However, it produced fatal GvHD in only 2 of 4 dogs transfused on day 20 post grafting. Thus, DLT could be given earlier in DLA-haploidentical animals transplanted with CD6-depleted marrow than in DLA-identical animals transplanted with aATG treated marrow without producing GvHD. These findings support the hypothesis that graft-versus-host tolerance can be induced earlier with grafts not depleted of NK cells. NK cells in the graft may inactivate host dendritic cells necessary for the induction of GvHD. In grafts depleted with aATG, NK cells are depleted as well, because aATG still retains broad specificity despite extensive absorptions. This leaves host DCs unaffected. Transfused donor T-cells encountering this environment will thus be activated which results in severe GvHD. In contrast, monoclonal CD6-antibody spares NK cells, so that donor lymphocytes cannot be activated by host DCs at the time of DLT and thus won't trigger GvHD. CD6-depletion is the preferred method if adoptive immunotherapy with DLT is planned.

Early Immune Response Against Retrovirally Transduced Herpes Simplex Virus Thymidine Kinase-Expressing Gene-Modified T Cells Coinfused with a T Cell-Depleted Marrow Graft: An Altered Immune Response?

Administration of herpes simplex thymidine kinase (HSV-tk)-expressing, gene-modified T cells (GMCs) with T cell-depleted bone marrow transplantation (TCD-BMT) can allow modulation of posttransplantation alloreactivity. Twelve patients received 2 x 10(5) to 2 x 10(6) CD3+ donor GMCs per kilogram with HLA-identical sibling TCD-BMT. Despite extensive T cell depletion of bone marrow, an intensive conditioning regimen, and immunosuppressive graft-versus-host disease (GvHD) prophylaxis, infusion at the time of TCD-BMT of this low number of GMCs sufficed to induce a rapid GMC-specific immune response, as detected by interferon- enzyme- linked immunospot assay in six of eight patients, preferentially targeting HSV-tk. Maximal responses were reached early (median time, 49 [35-68] days post-BMT), with a subsequent rapid and significant decrease in five of six evaluable patients. Immune responses were negatively correlated with the maximal circulating GMC counts. However, such immune response did not result in the elimination of circulating GMCs and was not associated with measurable ex vivo cytotoxic activity against GMCs. Furthermore, alloreactive GMCs still could induce GCV-sensitive GvHD in one patient despite an ongoing immune response. Overall, infusion of HSV-tk-expressing GMCs at the time of BMT results in an early immune response. Such immune response may be altered and may not prevent persistent GCV-sensitive alloreactivity.

Vaccination of Children following Allogeneic Stem Cell Transplantation

Vaccination of Children following Allogeneic Stem Cell Transplantation

Deletions within the HSV-tk transgene in long-lasting circulating gene-modified T cells infused with a hematopoietic graft

AbstractIn our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk+)/neomycin phosphotransferase (NeoR+)–expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell–depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk–polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel “transgene walking” PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti–HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell–depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.

Interleukin 7 receptor α-chain-mutation severe combined immunodeficiency without lymphopenia: correction with haploidentical T-cell–depleted bone marrow transplantation

Severe combined immunodeficiency (SCID) is a rare primary immunodeficiency characterized by abnormal lymphocyte development and lymphopenia. It often presents during the first year of life with recurrent, opportunistic infections, failure to thrive, and malabsorption. To advocate newborn screening for SCID. We reviewed the case histories of dizygotic twins with Pneumocystis carinii pneumonia (PCP) at the age of 6 months. Full-term fraternal twin girls were born to nonconsanguineous parents. Twin A developed recurrent oral candidiasis at 2 months, followed by pneumococcal bacteremia and PCP. At 5 months she had failure to thrive, but her absolute lymphocyte count was normal (5,887 cells/mm3). Subsequently, twin B presented with acute respiratory distress and was also diagnosed as having PCP; her absolute lymphocyte count was 5,852 cells/mm3. Flow cytometric analysis of peripheral blood lymphocytes from both girls demonstrated a T-B+NK+ phenotype consistent with interleukin 7 receptor alpha-chain-mutation SCID. Both girls received a haploidentical T-cell-depleted bone marrow transplant from their mother. In the interim, a homozygous point mutation in the interleukin 7 receptor alpha-chain gene was identified in twin B. Both parents were found to be carriers. Twin A died of chronic lung disease 8 months after transplantation; twin B is currently thriving. Early diagnosis and treatment of SCID are associated with an increased rate of survival and improved long-term outcome. Some patients with SCID can present without lymphopenia. Thus, we advocate that more sensitive screening tests be considered for inclusion in the newborn screening program currently used in most states.

Anti-CD40L Monoclonal Antibodies Can Replace Anti-CD4 Monoclonal Antibodies for the Nonmyeloablative Induction of Mixed Xenogeneic Chimerism

We have previously demonstrated that xenogeneic bone marrow engraftment and donor-specific tolerance can be induced in mice receiving anti-CD4, -CD8, -Thy-1.2, and -NK1.1 monoclonal antibodies (mAbs) on Days -6 and -1, 3 Gy total body irradiation (TBI), and 7 Gy thymic irradiation on Day 0, followed by injection of T-cell depleted (TCD) rat bone marrow cells. We have recently demonstrated that anti-CD40L mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance in an allogeneic combination. We investigated the ability of anti-CD40L mAb to promote mixed xenogeneic chimerism and donor-specific tolerance in B6 mice receiving anti-CD8, -Thy1.2 and -NK1.1 mAbs and 3 Gy TBI followed by TCD bone marrow transplantation (BMT) from F344 rats. Administration of anti-CD4 mAb in this model could be completely replaced by one injection of anti-CD40L mAb. Evidence for deletional tolerance was obtained in mixed chimeras prepared with this anti-CD40L-based regimen. However, anti-NK1.1 and anti-Thy1.2 mAb could not be replaced by anti-CD40L mAb. These results demonstrate that anti-CD40L in combination with xenogeneic BMT can tolerize preexisting peripheral and intrathymic CD4 cells to xenoantigens. However, anti-CD40L does not prevent NK cell and/or gammaDelta cell-mediated rejection of xenogeneic bone marrow.

T-cell signaling in allogeneic bone marrow transplantation

We examined the signaling profiles of T cells in murine allogeneic bone marrow transplantation with a novel high-throughput technique to analyze activation of signaling pathways by flow cytometry. We first studied normal splenic, liver, and peripheral lymph node (PLN) T cells. Relative to naive CD4 and CD8 cells, effector and memory cells displayed nearly global increases in activation of signaling pathways including MEK1/2 and PKC (both up ∼10-fold), and S6 ribosomal protein (∼6 fold) (Table 1). In the thymus, there was a general lack of signaling activity in CD4+CD8+ cells, and these did not respond to cytokines. By contrast, CD4−CD8− (DN) cells of the DN1 and DN2 stage had the greatest signaling activity of all subsets. CD4+, CD8+, and DN cells responded to the cytokines IL-7 and IL-15 by phosphorylating STAT-5A (∼10 fold). We then studied alloreactive and homeostatically proliferating T cells by infusion of CFSE labeled cells into irradiated hosts. We noted a gradual and global increase in signaling activity with each cell division in the spleen, liver, and PLN. Molecules that were phosphorylated included c-Raf, MEK1/2, ERK1/2, Akt, STAT-1, STAT-3, STAT-4, and S6 ribosomal protein. Tacrolimus and cyclosporin inhibited both cell cycling and increases in signaling. We defined slow-cycling cells as homeostatically expanding and fast-cycling cells as alloreactive, and found a distinct signaling pattern in homeostatically expanding T cells, with lower levels of MAP kinase pathway activity and STAT pathway activity. We hypothesize that this difference can be attributed to strong IL-2 and TCR signaling in alloreactive cells through the MAP kinases, Akt and STAT-3, by IFNγ for STAT-1, and by IL-12 for STAT-4. In the setting of T-cell depleted bone marrow transplantation, we found in several models that bone marrow-derived splenic T cells had signaling levels generally comparable with that of the naive populations in a normal spleen, but with a deficiency in STAT-3 phosphorylation. In the thymus, we observed a defect in Akt signaling in all subsets relative to signaling levels in the normal thymus, with up to 100-fold decreases. The defect persisted up to day 28 post-transplant. We conclude that the high-throughput analysis of signaling activity in T cells during allogeneic BMT allows for the definition of distinct signaling profiles. These signaling signatures can be used for the development of targeted therapies to enhance or inhibit specific T cell subsets.Table 1Signaling in the C57BL/6 SpleenCD4CD8Naive(CD44lo CD62Lhi)Effector(CD44hi CD62Llo)Memory(CD44hi CD62Lhi)Naive(CD44lo)Effector/Memory (CD44hi)c-Raf (pS259)12.21.811.2MEK 1/2 (pS217/pS221)12.79.712.2ERK 1/2 (pT202/pY204)11.61.811.2JNK (pT180/pY182)11.16.512.3pan-p38 (pT180/pY182)12.13.011.8pan-Akt (pT308)12.13.811.4pan-Akt (pS473)12.63.911.7Pan-PKC (βII pS660)12.710.111.4STAT-1 (pY701)10.96.312.4STAT-3 (pY705)11.41.811.3STAT-3 (pS727)11.21.311.3STAT-4 (pY693)12.02.311.2STAT-5A (pY694)11.31.711.3S6 ribosomal protein (pS235/pS236)13.06.311.8Histone H2B (pS14)12.64.211.8PIP211.30.410.6Ratio of activation (phosphorylation) of signaling molecules in effector or memory CD4 or CD8 cells compared with corresponding naive CD4 or CD8 cells. Open table in a new tab Ratio of activation (phosphorylation) of signaling molecules in effector or memory CD4 or CD8 cells compared with corresponding naive CD4 or CD8 cells.

Signaling Profiles of T Cells during Allogeneic Hematopoietic Stem Cell Transplantation.

In this study we characterized the signaling profiles of T cells in murine allogeneic bone marrow transplantation (BMT) models. We used a novel high-throughput technique to analyze activation of signaling pathways by flow cytometry.We first studied normal splenic T cells (Table 1) and found that relative to naïve CD4 and CD8 cells, effector and memory cells displayed nearly global increases in activation of signaling pathways including the MAP kinases and STATs. We observed similar increases in signaling activity in effector/memory vs. naive T cells in the mesenteric and peripheral lymph nodes (PLN) and the liver.Signaling in the C57BL/6 spleenCD4CD8Naïve (CD44lo CD62Lhi)Effector (CD44hi CD62Llo)Memory (CD44hi CD62Lhi)Naïve (CD44lo)Effector/Memory (CD44hi)c-Raf (pS259)12.21.811.2MEK 1/2 (pS217/pS221)12.79.712.2ERK 1/2 (pT202/pY204)11.61.811.2JNK (pT183/pY185)11.16.512.3Pan-p38 (pT180/pY182)12.13.011.8Pan-Akt (pT308)12.13.811.4Pan-Akt (pS473)12.63.911.7Pan-PKC ( β II pS660)12.710.111.4STAT-1 (pY701)10.96.312.4STAT-3 (pY705)11.41.811.3STAT-3 (pS727)11.21.311.3STAT-4 (pY693)12.02.311.2STAT-5A (pY694)11.31.711.3S6 Ribosomal Protein13.06.311.8(pS235/pS236)Histone H2B (pS14)12.64.211.8Cleaved Caspase 3 (D175)12.03.811.3PIP211.30.410.6Ratios of activation of signaling molecules in effector or memory CD4 or CD8 cells compared with naïve cells.In the thymus, there was a general lack of signaling activity in CD4+CD8+ (DP) cells, and these responded poorly to cytokine stimulation. By contrast, CD4−CD8− (DN) cells of the DN1 and DN2 stage had the greatest signaling activity of all thymocytes. We then studied alloreactive and homeostatically proliferating T cells by infusion of carboxyfluorescein (CFSE) labeled T cells into irradiated hosts. We noted a gradual and global increase in signaling activity with each cell division in the spleen, liver, and PLN. Peak increases relative to the undivided CFSEhi population of up to 2.5-fold (ERK 1/2) in the CD4 cells by the 4th cell division, with similar increases in CD8 cells, were found. Even the undivided CD4 CFSEhi population itself displayed large increases in signal (~4 fold for p38 and ERK 1/2) relative to naïve splenic CD4 cells. We defined slow-cycling cells as homeostatically expanding and fast-cycling cells as alloreactive, and found a distinct signaling pattern in homeostatically expanding T cells, with lower levels of MAP kinase pathway activity, JAK/STAT pathway activity, and pro-apoptotic proteins such as cleaved caspase 3 and phosphorylated histone H2B. However, PKC phosphorylation appeared to be higher in homeostatically proliferating cells than alloreactive T cells.In the setting of T-cell depleted bone marrow transplantation, we found in several models that newly generated, donor-derived splenic T cells had signaling levels generally comparable with that of the naïve populations in a normal spleen, but with a marked deficiency in STAT-3 (pS727) phosphorylation. In the thymus, we observed defects in Akt and STAT-3 (pS727 and pY705) signaling in the DN, DP, CD4, and CD8 subsets relative to signaling levels in the normal thymus. We conclude that the high-throughput analysis of signaling activity in T cells during allogeneic BMT allows for the definition of distinct signaling profiles. These signaling signatures can be used for the development of targeted therapies to enhance or inhibit specific T cell subsets.

Cytokines transduced bone marrow stromal cell lines promote immunohematopoietic reconstitution in mice after allogeneic bone marrow transplantation

Impaired immune reconstitution following allogeneic T-cell depleted bone marrow transplantation (allo-TCD-BMT) is a major obstacle to its clinical application. Stromal cell line QXMSC1, established from bone marrow cells of BALB/c(H-2 d), was transfected with murine IL-3 and/ or IL-2 gene, and injected into lethally irradiated C57BL/6(H2 b) mice. We evaluated its effects on immunologic and hematopoietic reconstitution after allo-TCD-BMT. The results showed that QXMSC1-IL-3 + IL-2 could significantly increase the numbers of hematopoietic primitive progenitors (CFU-S), committed progenitors (CFU-GM, and BFU-E), and lymphocytes (CD8 + cells, CD4 + cells, and B cells). Similarly, immune functions of recipient mice were significantly enhanced in the QXMSC1-IL-3 + IL-2 group. In addition, QXMSC1-IL-3 or QXMSC1-IL-2 also exerted apparent effects on accelerating immune reconstitution, but these effects were far less than that of QXMSC1-IL-3 + IL-2. Our results demonstrated that stromal cell-mediated IL-3 and IL-2 gene therapy may be a potent approach in promoting immunologic and hematopoietic reconstitution after allo-TCD-BMT.

Anti-NK Cell Treatment Induces Stable Mixed Chimerism in MHC-Mismatched, T-Cell Depleted, Nonmyeloablative Bone Marrow Transplantation.

Objective: To clarify Natural killer (NK) cell-mediated resistance under cytoreductive conditioning and T-cell depleted bone marrow transplantation (TCD-BMT), we investigated the effects of host NK cell depletion on engraftment and induction of stable mixed chimerism.Methods: BALB/c mice (H-2kd) were injected intraperitoneally with anti-asialoGM1 antibody (anti-NK Ab) on day -1. On day 0, they received TBI at a dose of 500 cGy, followed by intravenous infusion of 2 x 107 T-cell-depleted (TCD) bone marrow (BM) cells from C57BL/6 mice (H-2kb). Early engraftment and chimerism were determined by the relative ratio of peripheral blood (PB) lymphocytes expressing either H-2kd or H-2kb on day +21. Long-term engraftment and chimerism were evaluated on PB and spleen by multi-color flow cytometry.Results: Although no recipients treated with TBI alone showed engraftment, all the recipients conditioned with anti-NK Ab and TBI showed successful engraftment as well as a donor-dominant pattern of mixed chimerism in both PB and spleen. Spleen cells from recipients with mixed chimerism showed specific tolerance to both host and donor strains, but not to a third-party (C3H/He). None of the reconstituted mice showed signs of graft versus host disease, and all survived up to day +330.Conclusion: These observations indicate that host NK cell depletion may be used to reduce the intensity of conditioning regimens for engraftment of TCD grafts, and can contribute to establishment of stable mixed chimerism in MHC-mismatched NMT.

Growth hormone accelerates immune recovery following allogeneic T-cell–depleted bone marrow transplantation in mice

Objective To test in a murine model whether recombinant human growth hormone can promote immune recovery after allogeneic T-cell–depleted bone marrow transplantation. Materials and Methods Lethally irradiated (8.5 Gy) BALB/c mice (H2 d) were transplanted with 5×10 6 T cell–depleted bone marrow cells from C57BL/6 mice (H2 b). Recipient mice were injected intraperitoneally with recombinant human growth hormone (20 μg/dose/day) or saline for the first 4 weeks after transplantation. These animals were followed for phenotypic and functional immune recovery. Results Administration of human recombinant growth hormone improved the CD4 + T-cell counts in peripheral blood on day +14 (44±14 vs 33±7/μL blood, p<0.05) and day +21 (281±109 vs 187±76/μL blood, p<0.01) compared with the saline control. These differences were no longer significant by day +28 despite continued growth hormone administration. Similar effects were also observed on CD8 + T cells and B220 + B cells. The improvements in peripheral T-cell counts were at least partially as a result of enhanced thymopoiesis because there was an increase in total thymocytes after treatment with growth hormone. T-cell–depleted bone marrow recipients treated with growth hormone rejected the third-party grafts faster than those treated with saline control (median survival time: 20 days vs 26 days, p<0.05). Conclusions These data demonstrated that recombinant human growth hormone can accelerate phenotypic and functional immune reconstitution following allogeneic T-cell–depleted bone marrow transplantation in mice.

Interleukin-7 improves T-cell recovery after experimental T-cell–depleted bone marrow transplantation in T-cell–deficient mice by strong expansion of recent thymic emigrants

AbstractInterleukin-7 (IL-7) has been shown to enhance thymic output of newly developed T cells following bone marrow transplantation (BMT) in mice. In addition, IL-7 may affect peripheral expansion of T cells. In order to study the relative contribution of thymopoiesis versus peripheral T-cell expansion in the setting of compromised thymopoiesis, we have applied IL-7 in an experimental stem cell transplantation model using T cell–deficient RAG-1–/–mice. C57BL/6 RAG-1–/–mice received transplants of syngeneic T-cell–depleted (TCD) bone marrow (Ly5.1) with or without supplemented T cells (Ly5.2). IL-7 was administered until day 63 after BMT. Peripheral blood T- and B-cell recovery was quantified by flow cytometry and thymopoiesis was studied by quantification of T-cell receptor rearrangement excision circles (TRECs). In mice receiving a T-cell–replete BMT, IL-7 selectively expanded mature CD45.2+T cells without affecting the recovery of new bone marrow–derived CD45.1+T cells. In contrast, IL-7 significantly enhanced the recovery of bone marrow–derived T cells after TCD BMT. Quantification of TRECs in mice receiving a TCD BMT revealed that enhanced T-cell recovery following IL-7 treatment resulted from a strong expansion of newly developed naive T cells. These results suggest that peripheral expansion of recent thymic emigrants or mature T cells may be a preferential mechanism by which IL-7 enhances T-cell recovery after BMT.

Blockade of the CD40/CD154 pathway enhances T-cell-depleted allogeneic bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy.

T-cell-depleted bone marrow transplantation (TDBMT) can prevent graft-versus-host disease (GvHD). However, depleting T cells from allogeneic bone marrow often results in failure of bone marrow engraftment under irradiation conditioning. It is not know whether donor T cells are essential for bone marrow engraftment and whether blocking the CD40/CD154 pathway promotes allogeneic TDBM engraftment under nonmyeloablative and irradiation-free fludarabine phosphate and cyclophosphamide conditioning therapy. Using fully major histocompatibility complex (MHC)-matched mouse models, we investigated whether donor T cells are essential for bone marrow engraftment under fludarabine phosphate and cyclophosphamide conditioning therapy. We also determined whether the barrier of allogeneic TDBM could be overcome by blocking the CD40/CD154 pathway. Donor chimerism was detected by flow cytometric analysis. Donor-specific tolerance through establishing mixed chimerism was tested in vivo by skin transplantation and in vitro by mixed leukocyte reaction and enzyme-linked immunospot (ELISPOT) assay. Compared with unmodified bone marrow, TDBM resulted in poor engraftment when fully MHC-mismatched donors were used. However, anti-CD154 monoclonal antibody (mAb) treatment significantly enhanced donor TDBM engraftment. TDBM engraftment was also seen in CD154 knockout mice. A stable and high level of multilinage donor chimerism was achieved. Recovery of host CD3 T cells was suppressed, and recovery of donor CD3 T cells was promoted, after TDBMT and anti-CD154 mAb treatment. Donor chimerism was established by TDBMT induced donor-specific tolerance in vivo and in vitro. Donor T cells facilitate bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy, and the blocking the CD40/CD154 pathway can replace donor T cells to promote TDBM engraftment.

Interleukin 7 worsens graft-versus-host disease

Impaired immune reconstitution has moved to the forefront of clinical problems limiting progress in allogeneic bone marrow transplantation (BMT). The identification of therapies that can enhance immune reconstitution by increasing thymopoiesis is critical to solving this problem. Interleukin 7 (IL-7) is the most potent thymopoietic cytokine identified thus far. To study the effects of IL-7 on immune reconstitution and graft-versus-host disease (GVHD) following allogeneic BMT, we administered recombinant human IL-7 (rhIL-7) in a murine parent into an F1 model. Results showed that rhIL-7 therapy lowered the "threshold" T-cell dose required to induce both clinical signs of GVHD as well as lethal GVHD. Histologic analysis of GVHD target tissues revealed that rhIL-7 increased the degree of inflammation and tissue damage observed at all T-cell doses studied, but did not change the pattern of organs affected or the histologic appearance of the GVHD within target organs. In addition, we evaluated the capacity for rhIL-7 to enhance thymopoiesis in the setting of allogeneic T cell-depleted (TCD) and T-cell-replete BMT. We observed that rhIL-7 therapy enhanced thymic function in TCD allogeneic BM transplant recipients, but not in animals that received even modest doses of T cells presumably due to thymic toxicity of the graft-versus-host reaction. Thus, caution must be exercised as IL-7 is developed clinically as an immunorestorative agent for use in the setting of allogeneic BMT. These results suggest that use of IL-7 should be limited to the setting of TCD BMT to obtain the greatest benefit on immune competence with the least toxicity.

Addition of a second, different allogeneic graft accelerates white cell and platelet engraftment after T-cell–depleted bone marrow transplantation

AbstractSignificant delays in engraftment and lymphoid recovery are the 2 major challenges in cord blood transplantation. The cause for this delay is presumed to be the low numbers of hematopoietic precursors found in one unit of cord blood. One approach to increase the stem cell doses could be to combine cord blood units from different donors differing at the major histocompatibility complex (MHC). As a first step toward this goal, the kinetics of hematologic engraftment and immune reconstitution were compared between 1 unit (2.5 × 106 cells) of T-cell–depleted bone marrow cells from a single donor (C57BL/6 [H2b] or SJL/J [H2s]) and 2 units from different donors (C57BL/6 + SJL/J) after transplantation into lethally irradiated (8.5 Gy) BALB/c recipients (H2d). Addition of T-cell–depleted bone marrow from an MHC-mismatched allogeneic donor doubled the white blood counts compared with recipients of one single unit on days +10 and +14. Similar effects were also observed on platelets. The beneficial effect of additional cells on peripheral T-cell counts were first observed on day +14. Cells both from donors (C57BL/6 and/or SJL/J) and recipients (BALB/c) contributed to myeloid and lymphoid reconstitution. The chimeras containing cells from 3 strains of mice were able to mount a recall immune response. Our data suggest that combining stem cells from MHC-mismatched allogeneic donors is feasible, that it has beneficial effects on myeloid engraftment and T-cell phenotypic recovery, and that the long-term stable mixed chimeras are immunologically normal following T-cell–depleted bone marrow transplantation.