Jonathan D. Powell

HLA-Haploidentical Bone Marrow Transplantation for Hematologic Malignancies Using Nonmyeloablative Conditioning and High-Dose, Posttransplantation Cyclophosphamide

We evaluated the safety and efficacy of high-dose, posttransplantation cyclophosphamide (Cy) to prevent graft rejection and graft-versus-host disease (GVHD) after outpatient nonmyeloablative conditioning and T cell-replete bone marrow transplantation from partially HLA-mismatched (haploidentical) related donors. Patients with advanced hematologic malignancies (n = 67) or paroxysmal nocturnal hemoglobinuria (n = 1) received Cy 50 mg/kg i.v. on day 3 (n = 28) or on days 3 and 4 (n = 40) after transplantation. The median times to neutrophil (>500/microL) and platelet recovery (>20,000/microL) were 15 and 24 days, respectively. Graft failure occurred in 9 of 66 (13%) evaluable patients, and was fatal in 1. The cumulative incidences of grades II-IV and grades III-IV acute (aGVHD) by day 200 were 34% and 6%, respectively. There was a trend toward a lower risk of extensive chronic GVHD (cGVHD) among recipients of 2 versus 1 dose of posttransplantation Cy (P = .05), the only difference between these groups. The cumulative incidences of nonrelapse mortality (NRM) and relapse at 1 year were 15% and 51%, respectively. Actuarial overall survival (OS) and event-free survival (EFS) at 2 years after transplantation were 36% and 26%, respectively. Patients with lymphoid malignancies had an improved EFS compared to those with myelogenous malignancies (P = .02). Nonmyeloablative HLA-haploidentical BMT with posttransplantation Cy is associated with acceptable rates of fatal graft failure and severe aGVHD or cGVHD.

The mTOR Kinase Differentially Regulates Effector and Regulatory T Cell Lineage Commitment

Effector T cell differentiation requires the simultaneous integration of multiple, and sometimes opposing, cytokine signals. We demonstrated mTORs role in dictating the outcome of T cell fate. mTOR-deficient T cells displayed normal activation and IL-2 production upon initial stimulation. However, such cells failed to differentiate into T helper 1 (Th1), Th2, or Th17 effector cells. The inability to differentiate was associated with decreased STAT transcription factor activation and failure to upregulate lineage-specific transcription factors. Under normally activating conditions, T cells lacking mTOR differentiated into Foxp3(+) regulatory T cells. This was associated with hyperactive Smad3 activation in the absence of exogenous TGF-beta. Surprisingly, T cells selectively deficient in TORC1 do not divert to a regulatory T cell pathway, implicating both TORC1 and TORC2 in preventing the generation of regulatory T cells. Overall, our studies suggest that mTOR kinase signaling regulates decisions between effector and regulatory T cell lineage commitment.

The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2

The kinase mTOR has emerged as an important regulator of the differentiation of helper T cells. Here we demonstrate that differentiation into the TH1 and TH17 subsets of helper T cells was selectively regulated by signaling from mTOR complex 1 (mTORC1) that was dependent on the small GTPase Rheb. Rheb-deficient T cells failed to generate TH1 and TH17 responses in vitro and in vivo and did not induce classical experimental autoimmune encephalomyelitis (EAE). However, they retained their ability to become TH2 cells. Alternatively, when mTORC2 signaling was deleted from T cells, they failed to generate TH2 cells in vitro and in vivo but preserved their ability to become TH1 and TH17 cells. Our data identify mechanisms by which two distinct signaling pathways downstream of mTOR regulate helper cell fate in different ways. These findings define a previously unknown paradigm that links T cell differentiation with selective metabolic signaling pathways.

Hyaluronan Fragments Act as an Endogenous Danger Signal by Engaging TLR2

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Cζ-, and NF-κB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2null mice.

Regulation of immune responses by mTOR.

mTOR is an evolutionarily conserved serine/threonine kinase that plays a central role in integrating environmental cues in the form of growth factors, amino acids, and energy. In the study of the immune system, mTOR is emerging as a critical regulator of immune function because of its role in sensing and integrating cues from the immune microenvironment. With the greater appreciation of cellular metabolism as an important regulator of immune cell function, mTOR is proving to be a vital link between immune function and metabolism. In this review, we discuss the ability of mTOR to direct the adaptive immune response. Specifically, we focus on the role of mTOR in promoting differentiation, activation, and function in T cells, B cells, and antigen-presenting cells.

Egr-2 and Egr-3 are negative regulators of T cell activation.

T cell receptor engagement in the absence of proper accessory signals leads to T cell anergy. E3 ligases are involved in maintaining the anergic state. However, the specific molecules responsible for the induction of anergy have yet to be elucidated. Using microarray analysis we have identified here early growth response gene 2 (Egr-2) and Egr-3 as key negative regulators of T cell activation. Overexpression of Egr2 and Egr3 was associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Conversely, T cells from Egr3−/− mice had lower expression of Cbl-b and were resistant to in vivo peptide-induced tolerance. These data support the idea that Egr-2 and Egr-3 are involved in promoting a T cell receptor–induced negative regulatory genetic program.

Allogeneic Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease

BACKGROUND Myeloablative allogeneic hematopoietic stem-cell transplantation is curative in children with sickle cell disease, but in adults the procedure is unduly toxic. Graft rejection and graft-versus-host disease (GVHD) are additional barriers to its success. We performed nonmyeloablative stem-cell transplantation in adults with sickle cell disease. METHODS Ten adults (age range, 16 to 45 years) with severe sickle cell disease underwent nonmyeloablative transplantation with CD34+ peripheral-blood stem cells, mobilized by granulocyte colony-stimulating factor (G-CSF), which were obtained from HLA-matched siblings. The patients received 300 cGy of total-body irradiation plus alemtuzumab before transplantation, and sirolimus was administered afterward. RESULTS All 10 patients were alive at a median follow-up of 30 months after transplantation (range, 15 to 54). Nine patients had long-term, stable donor lymphohematopoietic engraftment at levels that sufficed to reverse the sickle cell disease phenotype. Mean (+/-SE) donor-recipient chimerism for T cells (CD3+) and myeloid cells (CD14+15+) was 53.3+/-8.6% and 83.3+/-10.3%, respectively, in the nine patients whose grafts were successful. Hemoglobin values before transplantation and at the last follow-up assessment were 9.0+/-0.3 and 12.6+/-0.5 g per deciliter, respectively. Serious adverse events included the narcotic-withdrawal syndrome and sirolimus-associated pneumonitis and arthralgia. Neither acute nor chronic GVHD developed in any patient. CONCLUSIONS A protocol for nonmyeloablative allogeneic hematopoietic stem-cell transplantation that includes total-body irradiation and treatment with alemtuzumab and sirolimus can achieve stable, mixed donor-recipient chimerism and reverse the sickle cell phenotype. (ClinicalTrials.gov number, NCT00061568.)

A Role for Mammalian Target of Rapamycin in Regulating T Cell Activation versus Anergy

Whether TCR engagement leads to activation or tolerance is determined by the concomitant delivery of multiple accessory signals, cytokines, and environmental cues. In this study, we demonstrate that the mammalian target of rapamycin (mTOR) integrates these signals and determines the outcome of TCR engagement with regard to activation or anergy. In vitro, Ag recognition in the setting of mTOR activation leads to full immune responses, whereas recognition in the setting of mTOR inhibition results in anergy. Full T cell activation is associated with an increase in the phosphorylation of the downstream mTOR target S6 kinase 1 at Thr421/Ser424 and an increase in the mTOR-dependent cell surface expression of transferrin receptor (CD71). Alternatively, the induction of anergy results in markedly less S6 kinase 1 Thr421/Ser424 phosphorylation and CD71 surface expression. Likewise, the reversal of anergy is associated not with proliferation, but rather the specific activation of mTOR. Importantly, T cells engineered to express a rapamycin-resistant mTOR construct are resistant to anergy induction caused by rapamycin. In vivo, mTOR inhibition promotes T cell anergy under conditions that would normally induce priming. Furthermore, by examining CD71 surface expression, we are able to distinguish and differentially isolate anergic and activated T cells in vivo. Overall, our data suggest that by integrating environmental cues, mTOR plays a central role in determining the outcome of Ag recognition.

High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft-versus-host disease

Because of its potent immunosuppressive yet stem cell-sparing activity, high-dose cyclophosphamide was tested as sole prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation (alloBMT). We treated 117 patients (median age, 50 years; range, 21-66 years) with advanced hematologic malignancies; 78 had human leukocyte antigen (HLA)-matched related donors and 39 had HLA-matched unrelated donors. All patients received conventional myeloablation with busulfan/cyclophosphamide (BuCy) and T cell-replete bone marrow followed by 50 mg/kg/d of cyclophosphamide on days 3 and 4 after transplantation. The incidences of acute grades II through IV and grades III through IV GVHD for all patients were 43% and 10%, respectively. The nonrelapse mortality at day 100 and 2 years after transplantation were 9% and 17%, respectively. The actuarial overall survival and event-free survivals at 2 years after transplantation were 55% and 39%, respectively, for all patients and 63% and 54%, respectively, for patients who underwent transplantation while in remission. With a median follow-up of 26.3 months among surviving patients, the cumulative incidence of chronic GVHD is 10%. These results suggest that high-dose posttransplantation cyclophosphamide is an effective single-agent prophylaxis of acute and chronic GVHD after BuCy conditioning and HLA-matched BMT (clinicaltrials.gov no. NCT00134017).

Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy

Summary: The consequences of T‐cell receptor engagement (signal I) are profoundly affected by the presence or absence of co‐stimulation (signal 2). T‐cell receptor (TCR) stimulation in the absence of CD 28‐mediated co‐stimulation not only results in little interleukin (IL)‐2 production, but induces a long lasting hyporesponsive state known as T‐cell clonal anergy The addition of CD 28 ligation to signal 1, on the other hand, results in the production of copious amounts of IL‐2. Our laboratory has utilized CD4+ Th1 clones in an effort to understand the molecular events resulting in enhanced IL‐2 production by co‐stimulation and the inhibition of IL‐ 2 production in anergy Our current studies have focused on defining the post‐transcriptional effects of CD28‐enhanced IL‐2 production The data suggest that a major component of CD28′s ability to regulate IL‐2 production occurs at the level of message stability and involves the 3′‐untranslated region of the message. In terms of anergy, our recent studies support the notion that it is not the result of TCR engagement in the absence of co‐stimulation. but rather signal 1 in the absence of IL‐2 receptor signaling and proliferation. Furthermore, T‐cell anergy appears to be an active negative state in which IL‐2 production is inhibited both at the level of signal transduction and by cis‐dominant repression at the level of the IL‐2 promoter.