Impact of Personal Protective Equipment on Pediatric Cardiopulmonary Resuscitation Performance: A Controlled Trial.

OBJECTIVES: This study aimed to determine whether personal protective equipment (PPE) results in deterioration in chest compression (CC) quality and greater fatigue for administering health care providers (HCPs). METHODS: In this multicenter study, HCPs completed 2 sessions. In session 1 (baseline), HCPs wore normal attire; in session 2, HCPs donned full PPE. During each session, they performed 5 minutes of uninterrupted CCs on a child manikin. Chest compression rate, depth, and release velocity were reported in ten 30-second epochs. Change in CC parameters and self-reported fatigue were measured between the start and 2- and 5-minute epochs. RESULTS: We enrolled 108 HCPs (prehospital and in-hospital providers). The median CC rate did not change significantly between epochs 1 and 10 during baseline sessions. Median CC depth and release velocity decreased for 5 minutes with PPE. There were no significant differences in CC parameters between baseline and PPE sessions in any provider group. Median fatigue scores during baseline sessions were 2 (at start), 4 (at 2 minutes), and 6 (at 5 minutes). There was a significantly higher median fatigue score between 0 and 5 minutes in both study sessions and in all groups. Fatigue scores were significantly higher for providers wearing PPE compared with baseline specifically among prehospital providers. CONCLUSIONS: During a clinically appropriate 2-minute period, neither CC quality nor self-reported fatigue worsened to a significant degree in providers wearing PPE. Our data suggest that Pediatric Basic Life Support recommendations for CC providers to switch every 2 minutes need not be altered with PPE use.

Reliability of the Faces, Legs, Activity, Cry, and Consolability Scale in Assessing Acute Pain in the Pediatric Emergency Department.

OBJECTIVES: The Faces, Legs, Activity, Cry, and Consolability (FLACC) scale is one of the most widely utilized observational pain assessment scales in clinical practice. Although designed and validated to assess postoperative pain, the tool is currently applied to assess acute pain in multiple settings, including the emergency department. Scarce literature exists evaluating the reliability of the FLACC scale in the nonsurgical population and none in the emergency department. We sought to investigate the reliability of the FLACC scale in assessing acute pain in the pediatric emergency department and to examine the sensitivity of FLACC scores after the administration of analgesia. METHODS: In phase 1 of this prospective study, a series of 2 independent evaluators, blinded to each other's evaluations, scored 66 patients using the FLACC tool. Degree of concordance among the 6 dyads was used to measure interrater reliability. In phase 2, FLACC scores were obtained just before the administration of analgesia in 35 patients and measured at 30 and at 60 minutes after administration. RESULTS: Among the 6 dyads of evaluators, Kendall W demonstrated a strong concordance (27 of 30 measures; range, 0.63-1.00) for individual components of the scale and for the composite scores (range, 0.85-0.96). Significant mean reductions from preanalgesia FLACC scores [5.54; 95% confidence interval (CI), 4.79-6.30] were seen at 30 minutes (2.00; 95% CI, 1.61-2.39) and 60 minutes (1.14; 95% CI, 0.79-1.50) postanalgesia (P < 0.0001 for all comparisons). CONCLUSIONS: The FLACC scale demonstrated high interrater reliability for both individual FLACC items and total scores in a convenience sample of patients aged 6 months to 5 years in a pediatric emergency department. It seems to be an appropriate observational tool to assess acute pain in this population.

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

Absence of P-selectin in recipients of allogeneic bone marrow transplantation ameliorates experimental graft-versus-host disease.

Alloreactive T cells are crucial for graft-versus-host disease (GVHD) pathophysiology, and modulating their trafficking patterns has been efficacious in ameliorating experimental disease. We report in this paper that P-selectin, a glycoprotein found on resting and inflamed endothelium, is important for donor alloreactive T cells trafficking into GVHD target organs, such as the intestines and skin. Compared with wild-type (WT) recipients of allogeneic bone marrow transplantation, P-selectin(-/-) recipients exhibit decreased GVHD mortality and decreased GVHD of the skin, liver, and small bowels. This was associated with diminished infiltration of alloactivated T cells into the Peyer's patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLOs). Surprisingly, however, donor T cells deficient for P-selectin glycoprotein ligand 1, the most well described P-selectin ligand, mediated GVHD similar to WT T cells and accumulated in SLO and target organs in similar numbers as WT T cells. This suggests that P-selectin may be required for trafficking into inflamed tissues but not SLO and that donor T cells may use multiple P-selectin ligands apart from P-selectin glycoprotein ligand 1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable strategy for GVHD prophylaxis or treatment.

Impact of Personal Protective Equipment on the Performance of Emergency Pediatric Procedures by Prehospital Providers.

BACKGROUND: Personal protective equipment (PPE) is worn by prehospital providers (PHPs) for protection from hazardous exposures. Evidence regarding the ability of PHPs to perform resuscitation procedures has been described in adult but not pediatric models. This study examined the effects of PPE on the ability of PHPs to perform resuscitation procedures on pediatric patients. METHODS: This prospective study was conducted at a US simulation center. Paramedics wore normal attire at the baseline session and donned full Level B PPE for the second session. During each session, they performed timed sets of psychomotor tasks simulating clinical care of a critically ill pediatric patient. The difference in time to completion between baseline and PPE sessions per task was examined using Wilcoxon signed-rank tests. RESULTS: A total of 50 paramedics completed both sessions. Median times for task completion at the PPE sessions increased significantly from baseline for several procedures: tracheal intubation (+4.5 s; P = 0.01), automated external defibrillator (AED) placement (+9.5 s; P = 0.01), intraosseous line insertion (+7 s; P < 0.0001), tourniquet (+8.5 s; P < 0.0001), intramuscular injection (+21-23 s, P < 0.0001), and pulse oximetry (+4 s; P < 0.0001). There was no significant increase in completion time for bag-mask ventilation or autoinjector use. CONCLUSIONS: PPE did not have a significant impact on PHPs performing critical tasks while caring for a pediatric patient with a highly infectious or chemical exposure. This information may guide PHPs faced with the situation of resuscitating children while wearing Level B PPE.

GITR activation induces an opposite effect on alloreactive CD4(+) and CD8(+) T cells in graft-versus-host disease.

Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) family that is expressed at low levels on unstimulated T cells, B cells, and macrophages. Upon activation, CD4(+) and CD8(+) T cells up-regulate GITR expression, whereas immunoregulatory T cells constitutively express high levels of GITR. Here, we show that GITR may regulate alloreactive responses during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Using a BMT model with major histocompatibility complex class I and class II disparity, we demonstrate that GITR stimulation in vitro and in vivo enhances alloreactive CD8(+)CD25(-) T cell proliferation, whereas it decreases alloreactive CD4(+)CD25(-) proliferation. Allo-stimulated CD4(+)CD25(-) cells show increased apoptosis upon GITR stimulation that is dependent on the Fas-FasL pathway. Recipients of an allograft containing CD8(+)CD25(-) donor T cells had increased GVHD morbidity and mortality in the presence of GITR-activating antibody (Ab). Conversely, recipients of an allograft with CD4(+)CD25(-) T cells showed a significant decrease in GVHD when treated with a GITR-activating Ab. Our findings indicate that GITR has opposite effects on the regulation of alloreactive CD4(+) and CD8(+) T cells.

Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation.

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-X(L) expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4(+) and CD8(+) T cells. Transplantation of RAG-2-eGFP-transgenic BM revealed that proliferating eGFP(lo)CD44(hi) donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFP(hi)CD44(lo) recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester-labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44(hi) phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.

STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.(1-7) This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD.

Enhanced responses to tumor immunization following total body irradiation are time-dependent.

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8(+) T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8(+) T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.

Organ-derived dendritic cells have differential effects on alloreactive T cells.

Dendritic cells (DCs) are considered critical for the induction of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been shown to induce organ-tropism through induction of specific homing molecules on T cells. Using adoptive transfer of CFSE-labeled cells, we first demonstrated that alloreactive T cells differentially up-regulate specific homing molecules in vivo. Host-type dendritic cells from the GVHD target organs liver and spleen or skin- and gut-draining lymph nodes effectively primed naive allogeneic T cells in vitro with the exception of liver-derived dendritic cells, which showed less stimulatory capacity. Gut-derived dendritic cells induced alloreactive donor T cells with a gut-homing phenotype that caused increased GVHD mortality and morbidity compared with T cells stimulated with dendritic cells from spleen, liver, and peripheral lymph nodes in an MHC-mismatched murine BMT model. However, in vivo analysis demonstrated that the in vitro imprinting of homing molecules on alloreactive T cells was only transient. In conclusion, organ-derived dendritic cells can efficiently induce specific homing molecules on alloreactive T cells. A gut-homing phenotype correlates with increased GVHD mortality and morbidity after murine BMT, underlining the importance of the gut in the pathophysiology of GVHD.

IFN-gamma and Fas ligand are required for graft-versus-tumor activity against renal cell carcinoma in the absence of lethal graft-versus-host disease.

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 10(6) donor CD8(+) T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8(+) T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-gamma and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-alpha, perforin, or TRAIL. Recipients of IFN-gamma(-/-) CD8(+) T cells are unable to reject RENCA compared with recipients of wild-type CD8(+) T cells and, importantly, neither group develops severe GVHD. IFN-gamma(-/-) CD8(+) T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-gamma enhances class I and FasL expression and rescues the lytic capacity of IFN-gamma(-/-) CD8(+) T cells. These results demonstrate that the addition of low numbers of selected donor CD8(+) T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-gamma and FasL.

Enhanced immune reconstitution by sex steroid ablation following allogeneic hemopoietic stem cell transplantation.

Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT) effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.

Remodeling specific immunity by use of MHC tetramers: demonstration in a graft-versus-host disease model.

Major histocompatibility complex (MHC) molecules carrying selected peptides will bind specifically to their cognate T-cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen-specific T-cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T-cell populations, while leaving the remaining T-cell repertoire and immune response intact. In this report, we successfully demonstrate that a tetramer-based depletion of T cells can be achieved in a murine model of allogeneic bone marrow transplantation. Depletion of a specific alloreactive population of donor splenocytes (< 0.5% of CD8+ T cells) prior to transplantation significantly decreased morbidity and mortality from graft-versus-host disease. There was no early regrowth of the antigen-specific T cells in the recipient and in vivo T-cell proliferation was greatly reduced as well. Survival was increased more than 3-fold over controls, yet the inherent antitumor activity of the transplant was retained. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T-cell clones, which could result in novel therapies for certain autoimmune disorders, T-cell malignancies, and solid organ graft rejection.

Absence of beta7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine.

The alpha4beta7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the beta7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive beta7-/- T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of beta7-/- donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versus-tumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of beta7-/- donor T cells. In conclusion, beta7-/- donor T cells caused less GVHD morbidity and mortality than WT donor T cells because of selectively decreased T-cell infiltration of the liver and intestines. Our data suggest that strategies to target the beta7 integrin have the clinical potential to alleviate or prevent GVHD while sparing or potentiating GVT activity.

Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions.

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity.

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-gamma and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.

Interleukin-15 enhances immune reconstitution after allogeneic bone marrow transplantation.

Interleukin-15 (IL-15) is a gamma-common cytokine that plays an important role in the development, survival, and proliferation of natural killer (NK), NK T, and CD8+ T-cells. We administered IL-15 to recipients of an allogeneic bone marrow transplantation (allo BMT) to determine its effects on immune reconstitution. Posttransplantation IL-15 administration significantly increased donor-derived CD8+ T (mostly CD122(+)CD44(+)CD8+ T-cells), NK, and NK T-cells at day +28 in young and old recipients of allo BMT. This was associated with enhanced T-cell and NK-cell function. IL-15 stimulated homeostatic proliferation of donor CD8+ T-cells in recipients of carboxyfluorescein diacetate succinimidyl ester-labeled donor T-cell infusions. Posttransplantation IL-15 administration also resulted in a decrease in apoptotic CD8+ T-cells, an increase in Bcl-2-expressing CD8+ T-cells, and an increase in the fraction of Ki67+ proliferative NK and CD8+ T-cells in recipients of allo BMT. IL-15 did not exacerbate graft-versus-host disease (GVHD) in recipients of T-cell-depleted BMT but could aggravate GVHD in some cases in recipients of a T-cell-repleted BMT. Finally, we found that IL-15 administration could enhance graft-versus-leukemia activity. In conclusion, IL-15 can be administered safely to recipients of a T-cell-depleted allo BMT to enhance CD8+ T, NK, and NK T-cell reconstitution.

CCR2 is required for CD8-induced graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8+ T cells is relevant for the control of CD8+ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2-/-) CD8+ T cells developed less damage of gut and liver than recipients of wild-type CD8+ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8+ T-cell target organ infiltration revealed that CCR2-/- CD8+ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-gamma production and cytotoxicity of CCR2-/- CD8+ T cells were intact. Interestingly, the graft-versus-tumor effect mediated by CCR2-/- CD8+ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity.

Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation.

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-gamma (IFN-gamma), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.