NIH response criteria measures are associated with important parameters of disease severity in patients with chronic GVHD.

Lack of standardized criteria measuring therapeutic response remains an obstacle to the development of better treatments for chronic GVHD (cGVHD). This cross-sectional prospective study examined the concurrent and predictive validity of 18 clinician-reported ('Form A') and 8 patient-reported ('Form B') response measures proposed by NIH criteria. Concurrent parameters of interest were NIH global score, cGVHD activity, Lee symptom score and SF36 PCS. Patient cohort included 193 adults with moderate-to-severe cGVHD. Measures associated with the highest number of outcomes were lung function score (LFS), 2-min walk, grip strength, 4-point health-care provider (HCP) and patient global scores, 11-point clinician- and patient-reported global symptom severity scores, and Karnofsky performance score (KPS). Measures associated with survival in univariate analyses led to a Cox model containing skin erythema, LFS, KPS, eosinophil count and interval from cGVHD diagnosis to enrollment as jointly associated with survival. In conclusion, 4-point HCP and patient global scores and 11-point clinician- and patient-reported global symptom severity scores are associated with the majority of concurrent outcomes. Skin erythema is a potentially reversible sign of cGVHD that is associated with survival. These results define a subset of measures that should be prioritized for evaluation in future studies.

Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes.

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

Neoantigen and tumor antigen-specific immunity transferred from immunized donors is detectable early after allogeneic transplantation in myeloma patients.

To enhance the therapeutic index of allogeneic hematopoietic SCT (HSCT), we immunized 10 HLA-matched sibling donors before stem cell collection with recipient-derived clonal myeloma Ig, idiotype (Id), as a tumor antigen, conjugated with keyhole limpet hemocyanin (KLH). Vaccinations were safe in donors and recipients. Donor-derived KLH- and Id-specific humoral and central and effector memory T-cell responses were detectable by day 30 after HSCT and were boosted by post-transplant vaccinations at 3 months in most recipients. One patient died before booster vaccinations. Specifically, after completing treatment, 8/9 myeloma recipients had persistent Id-specific immune responses and 5/9 had improvement in disease status. Although regulatory T cells increased after vaccination, they did not impact immune responses. At a median potential follow-up period of 74 months, 6 patients are alive, the 10 patients have a median PFS of 28.5 months and median OS has not been reached. Our results provide proof of principle that neoantigen and tumor antigen-specific humoral and cellular immunity could be safely induced in HSCT donors and passively transferred to recipients. This general strategy may be used to reduce relapse of malignancies and augment protection against infections after allogeneic HSCT.

Ultra-short course sirolimus contributes to effective GVHD prophylaxis after reduced-intensity allogeneic hematopoietic cell transplantation.

Reduced-intensity conditioning (RIC) allo-SCT is a potentially curative treatment approach for patients with relapsed Hodgkin's or non-Hodgkin's lymphoma. In the present study, 37 patients underwent RIC allo-SCT after induction treatment with EPOCH-F(R) using a novel form of dual-agent immunosuppression for GVHD prophylaxis with CsA and sirolimus. With a median follow-up of 28 months among survivors, the probability for OS at 3 and 5 years was 56%. Treatment-related mortality was 16% at day +100 and 30% after 1 year of transplant. Acute GVHD grades II-IV developed in 38% of patients, suggesting that the regimen consisting of CsA and an ultra-short course of sirolimus is effective in the prevention of acute GVHD.

Clinical laboratory markers of inflammation as determinants of chronic graft-versus-host disease activity and NIH global severity.

Chronic graft-versus-host disease (cGVHD) remains a major cause of non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Currently there are no accepted measures of cGVHD activity to aid in clinical management and disease staging. We analyzed clinical markers of inflammation in the sera of patients with established cGVHD and correlated those with definitions of disease activity. In all, 189 adults with cGVHD (33% moderate and 66% severe according to National Institutes of Health (NIH) global scoring) were consecutively enrolled onto a cross-sectional prospective cGVHD natural history study. At the time of evaluation, 80% were receiving systemic immunosuppression and failed a median of four prior systemic therapies (PST) for their cGVHD. Lower albumin (P<0.0001), higher C-reactive protein (P = 0.043), higher platelets (P = 0.030) and higher number of PST (P<0.0001) were associated with active disease defined as clinician's intention to intensify or alter systemic therapy due to the lack of response. Higher platelet count (P = 0.021) and higher number of PST (P<0.0001) were associated with more severe diseased defined by NIH global score. This study identified common laboratory indicators of inflammation that can serve as markers of cGVHD activity and severity.

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model.

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.

EPOCH-F: a novel salvage regimen for multiple myeloma before reduced-intensity allogeneic hematopoietic SCT.

There exists a need for effective salvage regimens for multiple myeloma patients being considered for reduced-intensity allogeneic hematopoietic SCT (RI-alloHSCT). We developed EPOCH-F, a regimen consisting of infusional etoposide, VCR and adriamycin with prednisone, CY and fludarabine to achieve both tumor control and host lymphocyte depletion to facilitate engraftment before RI-alloHSCT. In all, 22 multiple myeloma patients were treated with EPOCH-F before RI-alloHSCT. The median age was 53 years (range 36-65), and the median number of previous therapies was 2 (range 1-8). Patients received a median of three cycles (range 1-5) of EPOCH-F. Toxicities were primarily hematologic and manageable. Median lymphocyte counts decreased from 1423/µL (range 335-2788) to 519/µL (range 102-1420; P=0.0002). The overall response (≥PR) to EPOCH-F was 22 with 13% achieving a CR/near-complete response (nCR); only 1 patient progressed while on therapy. A total of 20 patients underwent RI-alloHSCT. Median day +100 donor chimerism was 100% (range 60-100). In all, 70% of patients achieved very good partial response or better response after transplant; 40% of patients achieved CR/nCR. TRM at 100 days and 5 years was 5 and 30%, respectively. Median OS after RI-alloHSCT was 46.1 months. EPOCH-F provides disease control and host lymphocyte depletion with consistent full donor engraftment in multiple myeloma patients undergoing RI-alloHSCT.

Determinants of functional performance in long-term survivors of allogeneic hematopoietic stem cell transplantation with chronic graft-versus-host disease (cGVHD).

This study examined factors accounting for functional performance limitations in 100 long-term survivors of allogeneic hematopoietic stem cell transplantation with chronic graft-versus-host disease (cGVHD). Functional performance, measured by the SF-36 physical component summary score, was substantially lower (mean=36.8+/-10.7) than the US population norm of 50 (P<0.001). The most severe decrements were in physical function (mean=38.8+/-10.9) and physical role function (mean=37.88+/-11.88); 68% of respondents exceeded the five-point threshold of minimum clinically important difference below the norm on these subscales. Controlling for age and gender, six variables explained 56% of the variance in functional performance: time since cGVHD diagnosis, cGVHD severity, intensity of immunosuppression, comorbidity, functional capacity (distance walked in 2 min, grip strength, and range of motion), and cGVHD symptom bother (F=11.26; P<0.001). Significant independent predictors of impaired performance were intensive systemic immunosuppression, reduced capacity for ambulation, and greater cGVHD symptom bother (P<0.05). Symptom bother had a direct effect on functional performance, as well as an indirect effect partially mediated by functional capacity (Sobel test, P=0.004). Results suggest two possible mechanisms underlying impaired functional performance in survivors with cGVHD and underscore the importance of testing interventions to enhance functional capacity and reduce symptom bother.

Novel application of lentiviral vectors towards treatment of graft-versus-host disease.

Allogeneic transplantation of hematopoietic stem cells and lymphocytes is a curative treatment for malignant and non-malignant disease. However, the primary complication limiting the safety of transplantation is graft-versus-host disease (GvHD), which is mediated by donor T cells. Strategies for pre- and post-transplant manipulation of graft cells are not yet optimal for balancing GvHD severity with beneficial Graft-versus-Leukemia (GvL) effects. Emerging cell fate control gene therapy (CFCGT)-based strategies, such as 'suicide' gene therapy for donor T cell regulation, can supplement existing transplantation approaches by providing a safety element to reduce GvHD. Past uses of CFCGT in the clinic have provided proof-of-principle that GvHD can be controlled by such a strategy. However, there exists a need for improved transgene delivery and suicide control systems. Recently, lentiviral vectors (LVs) have emerged as effective gene delivery vehicles for the clinic. Combining lentiviral gene delivery with newer generations of 'suicide' systems that possess improved enzyme/prodrug specificities, activities, and reduced immunogenicity, could provide the necessary degree of control required to more successfully manage GvHD. Improving the safety of transplantation through successful CFCGT will serve to expand the potential donor pool and the spectrum of disorders that can be treated by this therapeutic schema.

Retrovirally transduced murine T lymphocytes expressing FasL mediate effective killing of prostate cancer cells.

Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2(b) expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy.

Clinical evidence of a graft-versus-lymphoma effect against relapsed diffuse large B-cell lymphoma after allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: A graft-versus-lymphoma effect against diffuse large B-cell lymphoma (DLBCL) is inferred by sustained relapse-free survival after allogeneic stem-cell transplantation; however, there are limited data on a direct graft-versus-lymphoma effect against DLBCL following immunotherapeutic intervention by either withdrawal of immunosuppression or donor lymphocyte infusion (DLI). MATERIALS AND METHODS: An analysis was carried out to determine whether a direct graft-versus-lymphoma effect exists against DLBCL. The analysis was restricted to patients with DLBCL, who were either not in complete remission at day +100 after allogeneic stem-cell transplantation or subsequently relapsed beyond this time point. RESULTS: Fifteen patients were identified as either not in complete remission (n = 13) at their day +100 evaluation or subsequently relapsed (n = 2) and were assessed for subsequent responses after withdrawal of immunosuppression or DLI. Eleven patients were treated with either withdrawal of immunosuppression (n = 10) or a DLI (n = 1) alone; four patients received chemotherapy with DLI to reduce tumor bulk. Nine (60%) patients subsequently responded (complete = 8, partial = 1). Six responses occurred after withdrawal of immunosuppression alone. Six patients are alive (range 42-83+ months) in complete remission without further treatment. CONCLUSION: The demonstration of sustained complete remission following immunotherapeutic intervention provides direct evidence of a graft-versus-lymphoma effect against DLBCL.

Chronic graft-versus-host disease in the era of reduced-intensity conditioning.

In the past decade the field of hematopoietic stem cell transplantation has entered a new era with the introduction of reduced intensity conditioning (RIC) regimens. The impact of RIC on the incidence of chronic graft-versus-host disease (GVHD) has not been evaluated systematically. Factors confounding such analyses include short follow-up in studies, absence of prospective comparison trials, use of a variety of RIC regimens, lack of uniform GVHD prophylaxis and lack of rigorous criteria for the diagnosis and staging of chronic GVHD. This review discusses factors that appear to influence the incidence and clinical presentation of chronic GVHD in the RIC transplantation era. Overall, RIC seems to decrease the incidence and severity of acute GVHD through day 100 post-transplant when compared to conventional conditioning; however, there is little evidence to suggest that chronic GVHD is reduced after RIC. For the more definitive assessments of chronic GVHD after RIC it will be important to study this question in prospective comparison trials with long duration of follow-up. The recent National Institutes of Health chronic GVHD consensus project recommendations provide now the critically needed standardized guidelines for the diagnosis, classification and staging of chronic GVHD.

Animal models of acute and chronic graft-versus-host disease.

Graft-versus-host disease (GVHD) represents a special situation in transplantation immunology in which immunocompetent donor cells are engrafted into recipients that are incapable of rejecting them due to tolerance, immaturity, or radiation- or chemotherapy-induced immune deficiency. Donor T cells encountering allogeneic stimulators become activated, secrete cytokines, proliferate, and differentiate into effectors; this in vivo immune response is known as the graft-versus-host reaction (GVHR). The systemic effects of this initial donor anti-host reaction comprise a multiorgan syndrome, graft-versus-host disease (GVHD). Murine GVHD experiments have been utilized to model the clinical disorders of acute and chronic GVHD (AGVHD and CGVHD) that occur after allogeneic bone marrow transplantation, and also to study T cell regulation, induction of tolerance, and autoimmune diseases. Presented in this unit are methods for generating and assessing both AGVHD and CGVHD in mice. While the two syndromes differ markedly in immunopathogenesis, both can be induced by the two main methods presented: transfer of allogenic donor lymphocytes and stem cells into irradiated hosts, and transfer of parental strain lymphocytes and stem cells into unirradiated, immune-competent F1 strain hosts. Several endpoints of AGVHD and CGVHD should be evaluated in experimental mice, with comparisons made to the syngeneic transplant control or the T cell-depleted allogeneic control. To this end, protocols are provided for the assessment of survival rates, weight loss, chimerism, donor-host cytotoxicity, and cytokine and proliferative responses to mitogenic or allogeneic stimuli. Histopathology and assays of B cell immune function are also described for evaluation of the pathogenesis of GVHD.

Th2 and Tc2 cells in the regulation of GVHD, GVL, and graft rejection: considerations for the allogeneic transplantation therapy of leukemia and lymphoma.

Allogeneic stem cell transplantation (SCT) represents a curative treatment option for patients with leukemia and lymphoma. T lymphocytes contained in the allograft mediate a graft-versus-leukemia (GVL) effect and prevent graft rejection; however, T cells also initiate graft-versus-host disease (GVHD). Identification of T cell populations which mediate a GVL effect and prevent rejection with reduced GVHD will likely improve transplantation outcome. T cells exist in four functionally-defined populations, the CD4+, Th1/Th2 and CD8+, Tc1/Tc2 subsets. Th1-type CD4 cells primarily secrete type I cytokines (IL-2 and IFN-gamma), whereas Th2 cells secrete type II cytokines (IL-4, IL-5, and IL-10). Similarly, the CD8+ Tc1 and Tc2 cells differentially secrete the type I and type II cytokines, respectively. In addition to cytokine secretion, Tc1 and Tc2 populations mediate cytolytic effects, with Tc1 cells utilizing both perforin- and fas-based killing pathways, whereas Tc2 cells primarily utilize perforin-mediated cytolysis. In murine transplantation models of graft rejection, GVHD, and GVL effects, we have evaluated such functional T cell subsets for their ability to differentially mediate and regulate transplantation responses. These studies demonstrate that donor Th2 cells do not initiate acute GVHD, and can regulate the GVHD mediated by unmanipulated donor T cells without impairing alloengraftment. Additional experiments have shown that allospecific donor Tc2 cells result in reduced GVHD, and mediate a significant GVL effect. Thirdly, we have demonstrated that non-host reactive Tc2 cells with veto-like activity can potently abrogate marrow rejection independent of GVHD. Together, these results demonstrate that functionally-defined donor Th2 and Tc2 populations play an important role in the regulation of GVHD, the prevention of graft rejection, and the mediation of GVL effects, and suggest that utilization of Th2 and Tc2 cells in clinical allogeneic SCT may have potential for improving treatment outcome.

An immunoablative regimen of fludarabine and cyclophosphamide prevents fully MHC-mismatched murine marrow graft rejection independent of GVHD.

The prevention of graft rejection in the setting of nonmyeloablative transplant approaches might be mediated by chemotherapy-induced host immunoablation and by the graft-promoting effects of graft-versus-host disease (GVHD). To evaluate whether host immunoablation alone might allow for alloengraftment, we developed an F1-into-parent murine marrow rejection model using host preparative regimens of lethal total body irradiation (TBI; 950 cGy), sublethal irradiation (600 cGy), or combinations of fludarabine (Flu) and cyclophosphamide (Cy). A preparative regimen selectivity index (SI) was calculated to determine whether host lymphocytes were preferentially depleted relative to myeloid cells (SI = number of host myeloid/number host T lymphoid cells remaining after preparative regimen administration). Saline-treated recipients were assigned an SI value of 1.0. Recipients of lethal TBI had reduced myeloid cells relative to T cells (SI = 0.6). In contrast, all Flu/Cy regimens preferentially depleted host T cells: recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 10 days (SI = 28.1); recipients of Flu (100 mg/kg per day)/Cy (100 mg/kg per day) for 10 days (SI = 64.1); and recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 19 or 27 days (SI = 74.6). The 10-day regimen of Flu/Cy (50 mg/kg per day) did not severely reduce host T cell numbers, nor did it prevent F1 marrow rejection (<1% chimerism, n = 14). In contrast, the 10-day regimen of Flu/Cy (100 mg/kg per day) reduced T-cell numbers below that of lethal TBI recipients and prevented F1 marrow rejection (11.4% chimerism, n = 15); donor chimerism was predominant in lymphoid cells and was stable through day 240 post-BMT. Additionally, the 19- or 27-day regimen of Flu/Cy, which most selectively depleted host T cells, also prevented F1 marrow rejection (6.3% chimerism, n = 15). These results therefore demonstrate that optimized Flu-containing, immunoablative preparative regimens can prevent fully MHC-disparate marrow rejection independent of GVHD.

Selection and expansion of T cell from untreated patients with CLL: source of cells for immune reconstitution?

BACKGROUND: Lymphocyte-derived malignancies can be treated with combinations of drugs that efficiently eradicate normal and malignant lymphocytes. Lack of T lymphocytes after treatment of B lymphocyte CLL (B-CLL) makes the patients susceptible to serious infections and may limit the benefit of the therapy. The aim of the study was to purify and culture-expand normal T lymphocytes from B-CLL patients prior to therapy. These cells could be frozen and given to the patients in the lymphopenic period post-chemotherapy. METHODS: T lymphocytes were isolated from the mononuclear cell apheresis products from five patients with previously untreated B-CLL. The apheresis products were red-cell depleted by density gradient centrifugation. B-lymphocyte purging was performed by incubating with MAbs to four different B-cell epitopes, followed by magnetic-bead depletion. One round of negative selection removed >90% of the B lymphocytes. The T-lymphocyte enriched cell suspension was cultured for 10/11 days in the presence of IL-2 and the anti-T cell receptor Ab OKT3. In addition, in some cultures anti-CD22 ricin immunotoxin was added. RESULTS: T cells from CLL patients expanded 4.7-21-fold over a 10/11 days culture interval. After culture, CLL cells could no longer be identified by flow cytometric evaluation. The cultured T lymphocytes were predominantly CD8(+), and were capable of lysing autologous CLL cells through a fas-dependent mechanism. DISCUSSION: Selection and expansion of T lymphocytes by this method may represent a strategy for enhancing immunity in the lymphopenic period following CLL treatment.

fas-mediated lysis of chronic lymphocytic leukaemia cells: role of type I versus type II cytokines and autologous fasL-expressing T cells.

Given the known role of the fas cytolytic pathway in B-cell regulation, we evaluated whether fas-fasL interactions might induce chronic lymphocytic leukaemia (CLL) cell death. De novo CLL cells expressed a low level of surface fas, and were not lysed by fasL-bearing cells. CLL cells cultured in media containing the type I cytokines interleukin (IL)-12 or interferon (IFN)-alpha had increased fas expression, and were readily lysed by fasL-bearing cells. In contrast, the type II cytokine IL-4 did not increase CLL cell fas, and abrogated type I cytokine-induced fas up-regulation. With prolonged culture, IL-4 exposed CLL cells expressed an intermediate level of fas; however, such CLL cells were resistant to fas-mediated lysis. These results indicate that IL-4 inhibits fas-mediated killing of CLL cells at the level of both fas receptor expression and post-receptor events. Additionally, we have defined in vitro culture conditions which generate fasL-bearing T cells from CLL patients; such T cells efficiently mediated fas-based lysis of autologous fas-positive CLL cells. We therefore conclude that type I and type II cytokines differentially regulate the fas pathway in CLL cells, and that a combination of type I cytokines and fasL-expressing T cells may represent a new approach to the immunotherapy of CLL.

CD8+ T cells of Tc2 phenotype mediate a GVL effect and prevent marrow rejection.

Donor T cells mediate both beneficial and detrimental immune reactions in the setting of allogeneic BMT (alloBMT). T cells mediate the GVL effect and prevent marrow rejection, but also induce GVHD. In an attempt to favorably influence the balance of these allogeneic responses, we have evaluated the effect of donor CD4+, Th1/Th2 and CD8+, Tc1/Tc2 functional T cell subsets in murine marrow transplantation models. Our studies have identified the CD8+ Tc2 population (which is a cytolytic effector secreting the type II cytokines IL-4, IL-5, and IL-10) as a subset capable of mediating the GVL effect and preventing marrow rejection with reduced GVHD. We have also shown that the Tc2 subset can be generated in humans. These studies indicate that administration of donor CD8+ T cells of Tc2 phenotype represents a strategy for improving allo BMT outcome.

Non-host-reactive donor CD8+ T cells of Tc2 phenotype potently inhibit marrow graft rejection.

Donor CD8+ T cells capable of host reactivity inhibit marrow graft rejection, but also generate graft-versus-host disease (GVHD). To evaluate whether the Tc1- and Tc2-type subsets of CD8 cells might inhibit rejection without host reactivity, we established an F1 into-parent murine bone marrow transplant model. Donor Tc1 and Tc2 cells were generated that preferentially secreted type I or type II cytokines; both subsets possessed potent cytolytic function, and clonally deleted host-type allospecific precursor CTL in vitro. B6 hosts receiving 950 cGy irradiation did not reject the donor marrow (F1 chimerism of 78.6%; n = 10), whereas hosts receiving 650 cGy rejected the donor marrow (3.8% chimerism; n = 8). At 650 cGy irradiation, the addition of Tc2 cells to the F1 marrow resulted in extensive F1 chimerism (70.8%) in 8 of 8 recipients; in contrast, alloengraftment was not consistently observed in mice receiving Tc1 cells or unmanipulated CD8 cells. Furthermore, when the preparative regimen was further reduced to 600 cGy, only hosts receiving the Tc2-type cells did not reject the F1 marrow. We conclude that Tc2 cells potently inhibit marrow graft rejection without inducing an alloaggressive response and that non-host-reactive Tc2 cells therefore facilitate engraftment across genetic barriers with reduced GVHD.

In vitro generation of allospecific human CD8+ T cells of Tc1 and Tc2 phenotype.

We have previously shown that allospecific murine CD8+ T cells of the Tc1 and Tc2 phenotype could be generated in vitro, and that such functionally defined T-cell subsets mediated a graft-versus-leukemia (GVL) effect with reduced graft-versus-host disease (GVHD). To evaluate whether analogous Tc1 and Tc2 subsets might be generated in humans, CD8+ T cells were allostimulated in the presence of either interleukin-12 (IL-12) and transforming growth factor-beta (TGF-beta) (Tc1 culture) or IL-4 (Tc2 culture). Tc1-type CD8 cells secreted the type I cytokines IL-2 and interferon gamma (IFN-gamma), whereas Tc2-type cells primarily secreted the type II cytokines IL-4, IL-5, and IL-10. Both cytokine-secreting populations effectively lysed tumor targets when stimulated with anti-T-cell receptor (TCR) antibody; allospecificity of Tc1- and Tc2-mediated cytolytic function was demonstrated using bone marrow-derived stimulator cells as targets. In addition, both Tc1 and Tc2 subsets were capable of mediating cytolysis through the fas pathway. We therefore conclude that allospecific human CD8+ T cells of Tc1 and Tc2 phenotype can be generated in vitro, and that these T-cell populations may be important for the mediation and regulation of allogeneic transplantation responses.