Jeff K. Davies

T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production

T-cell exhaustion, originally described in chronic viral infections, was recently reported in solid and hematologic cancers. It is not defined whether exhaustion contributes to T-cell dysfunction observed in chronic lymphocytic leukemia (CLL). We investigated the phenotype and function of T cells from CLL patients and age-matched controls. CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD244, CD160, and PD1, with expansion of a PD1+BLIMP1HI subset. These molecules were most highly expressed in the expanded population of effector T cells in CLL. CLL CD8+ T cells showed functional defects in proliferation and cytotoxicity, with the cytolytic defect caused by impaired granzyme packaging into vesicles and nonpolarized degranulation. In contrast to virally induced exhaustion, CLL T cells showed increased production of interferon-γ and TNFα and increased expression of TBET, and normal IL2 production. These defects were not restricted to expanded populations of cytomegalovirus (CMV)–specific cells, although CMV seropositivity modulated the distribution of lymphocyte subsets, the functional defects were present irrespective of CMV serostatus. Therefore, although CLL CD8+ T cells exhibit features of T-cell exhaustion, they retain the ability to produce cytokines. These findings also exclude CMV as the sole cause of T-cell defects in CLL.

Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

We report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 x10(6)/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

An update on the management of severe idiopathic aplastic anaemia in children.

The current outlook for a child with severe idiopathic aplastic anaemia (AA) is very much better than in previous decades. In part, this may reflect better differentiation of idiopathic and inherited marrow failure. For children with idiopathic AA and a human leucocyte antigen (HLA)‐matched sibling donor (MSD), allogeneic haematopoietic stem‐cell transplantation (AHSCT) is the primary therapy of choice, offering long‐term disease‐free survival of 90%, although graft‐versus‐host disease remains a cause of long‐term morbidity. A greater treatment challenge remains for those children without a MSD. Combination immunosuppressive therapy (IST) is associated with response rates of 70% or more. However, relapse and clonal evolution with transformation to myelodysplasia or acute myeloid leukaemia remain significant problems after IST and long‐term event‐free survival rates are less impressive. For children who do not have a sustained response to IST, alternate donor AHSCT should be considered. New HLA typing technologies, novel stem cell sources, reduced‐intensity conditioning and graft engineering have reduced toxicity and improved the outcome after alternate donor AHSCT. Emerging therapies that capitalise on recent advances in our understanding of the pathophysiology of idiopathic AA and the immunobiology of AHSCT and IST may further improve the long‐term outcome of this disease.

Ligation of CD8α on human natural killer cells prevents activation‐induced apoptosis and enhances cytolytic activity

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross‐linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK‐cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK‐cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti‐MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells.

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34+CD38− stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34+CD38+ progenitors were reduced. Residual normal CD34+ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.

CpG Oligodeoxynucleotides Alter Lymphocyte and Dendritic Cell Trafficking in Humans

Purpose: CpG oligodeoxynucleotides (CpG-ODN) are being investigated as cancer vaccine adjuvants because they mature plasmacytoid dendritic cells (PDC) into potent antigen-presenting cells. CpG-ODN also induce PDC to secrete chemokines that alter lymphocyte migration. Whether CpG-ODN TLR signals enhance antigen-specific immunity and/or trafficking in humans is unknown. Experimental Design: We conducted a phase I study of CpG-ODN (1018 ISS) given as a vaccine adjuvant with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce T-cell immunity to a peptide vaccine from the tumor-associated antigen hTERT. Results: The adjuvant effect was limited; only 1 of 16 patients showed a high-frequency hTERT-specific tetramer CD8+ T-cell response. However, CpG-ODN induced marked, transient peripheral blood lymphopenia. Biopsies showed dense lymphocytic infiltration at the vaccine site clustered around activated PDC. In vitro, CpG-ODN-treated PDC induced T-cell migration, showing that CpG-ODN stimulation of human PDC was sufficient to chemoattract T cells. Conclusions: Our results show that (a) CpG-ODN with GM-CSF may not be an effective adjuvant strategy for hTERT peptide vaccines but (b) GM-CSF/CpG-ODN causes a PDC-mediated chemokine response that recruits T-cell migration to the peripheral tissues. These findings suggest a novel therapeutic role for targeted injections of CpG-ODN to direct lymphocyte migration to specific sites such as the tumor bed.

Combining CD19 Redirection and Alloanergization to Generate Tumor-Specific Human T Cells for Allogeneic Cell Therapy of B-Cell Malignancies

Allogeneic hematopoietic stem-cell transplantation can cure some patients with high-risk B-cell malignancies, but disease relapse following transplantation remains a significant problem. One approach that could be used to augment the donor T-cell-mediated antitumor effect is the infusion of allogeneic donor-derived T cells expressing a chimeric antibody receptor (CAR) specific to the B-cell antigen CD19. However, the use of such cells might result in toxicity in the form of graft-versus-host disease mediated by CD19-specific (CD19-CAR) T cells possessing alloreactive endogenous T-cell receptors. We therefore investigated whether nonalloreactive tumor-specific human T cells could be generated from peripheral blood mononuclear cells of healthy donors by the combination of CD19 redirection via CAR expression and subsequent alloanergization by allostimulation and concomitant blockade of CD28-mediated costimulation. Alloanergization of CD19-CAR T cells resulted in efficient and selective reduction of alloresponses in both CD4(+) and CD8(+) T cells, including allospecific proliferation and cytokine secretion. Importantly, T-cell effector functions including CAR-dependent proliferation and specific target cytolysis and cytokine production were retained after alloanergization. Our data support the application of CD19 redirection and subsequent alloanergization to generate allogeneic donor T cells for clinical use possessing increased antitumor activity but limited capacity to mediate graft-versus-host disease. Immunotherapy with such cells could potentially reduce disease relapse after allogeneic transplantation without increasing toxicity, thereby improving the outcome of patients undergoing allogeneic transplantation for high-risk B-cell malignancies.

A juicy problem

thrombocytopenia. In a serum screen for platelet antibodies using an indirect platelet immunofluorescence test, no platelet antibodies were detected in the patient’s serum when screened against a standard panel of human platelet antigen-typed and HLA-typed platelets, even when quinine was added to the test system. This did not support a diagnosis of quinine-induced immune thrombocytopenia. However, when dialysed cranberry juice of the same brand and batch as the patient had drunk was added to the test system, both IgM and IgG anti-platelet antibodies were detected in the patients serum. Subsequent sandwich ELISA with purified platelet glycoproteins (monoclonal antibody immobilisation of platelet antigens) could not identify the particular platelet glycoprotein antigen specificity of the cranberry juice-dependent IgM and IgG antibodies detected. Nevertheless, the detection of anti-platelet antibodies in the patient’s serum only in the presence of cranberry juice dialysate is good evidence for an immune mechanism for the thrombocytopenia with a constituent of cranberry juice acting as the hapten.

Induction of alloanergy in human donor T cells without loss of pathogen or tumor immunity

Background. Human leukocyte antigen (HLA)-mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is limited by acute graft-versus-host disease (aGvHD). Nonselective T-cell depletion effectively prevents severe aGvHD but profoundly impairs donor-derived immune reconstitution, increasing infection and disease relapse. The strategy of induction of alloantigen-specific hyporesponsiveness (“alloanergization”) in donor bone marrow by allostimulation with costimulatory blockade before haploidentical transplantation has demonstrated early promise in reducing severe aGvHD. However, the differential effect of alloanergization on CD4+ and CD8+ donor T-cell subsets and the degree to which beneficial pathogen- and tumor-immune responses are retained have not been extensively examined. Methods. We used an in vitro model of alloanergization by allostimulation of human donor T cells with irradiated unrelated recipient peripheral blood mononuclear cells and costimulatory blockade with humanized monoclonal anti-B7.1 and B7.2 antibodies. Residual alloresponses were assessed by proliferation (thymidine uptake, carboxyfluorescein diacetate succinimidyl ester dye dilution) and cytotoxicity assays. Retention of human herpes virus and tumor-associated antigen (TAA)-specific immunity was measured with HLA-class I-restricted pentamers, intracellular cytokine secretion, and CD107a assay using 5-color flow cytometry. Results. Alloanergization of HLA-mismatched donor T cells efficiently and selectively abrogated recipient-specific alloproliferation in both CD4+ and CD8+ cells while preserving functional CD4+ and CD8+ immune responses to clinically important human herpes viruses and to the TAA WT1. Conclusions. Retention of pathogen- and TAA-specific immunity after alloanergization demonstrates that this methodology, which is simple to apply, has potential to improve immune reconstitution while limiting alloreactivity after HLA-mismatched hematopoietic stem cell transplantation, and deserves additional evaluation in further human clinical trials.

Expansion of Allospecific Regulatory T Cells After Anergized, Mismatched Bone Marrow Transplantation

Transplantation of donor bone marrow cells treated to be tolerant to patient antigens results in expansion of regulatory T cells that suppress immune responses that mediate graft-versus-host disease. One of the most successful applications of regenerative medicine to date, hematopoietic stem cell transplants have healed numerous patients with blood-based disorders such as thalassemia, anemias, and certain cancers. Each of these maladies emanates from defective blood cells, which normally arise from stem cells in the bone marrow. Davies et al. have deduced the mechanism by which a particular treatment of bone marrow before infusion into a patient results in favorable outcome even when the donor and the patient are not matched for tissue antigens. Hematopoietic stem cell transplantation requires elimination of the patient’s faulty bone marrow with radiation or chemotherapy, after which the bone marrow niche is repopulated by the infusion of donor cells into the patient. Although it has been in use for decades, hematopoietic stem cell transplantation carries a number of risks. Among the most serious of these is graft-versus-host disease (GVHD), a complication that occurs when donor immune cells recognize the patient’s tissues—often liver, skin, or the gastrointestinal tract—as foreign, thus promoting inflammation and organ damage. This condition is a reversal of typical transplant rejection in which the patient’s immune cells reject the foreign donor tissue. GVHD often can be avoided if the donor and patient have completely matched major histocompatibility (human leukocyte) antigens. Such precise matching is not common, however, and many more patients could benefit from hematopoietic stem cell transplantation if partially matched patients and donors could be paired without fear of GVHD. To this end, scientists have developed several procedures that deplete, before infusion, donor immune cells including those that react against the patient’s organs (alloreactive cells). However, this process interferes with the reconstitution of crucial components of the patient’s immune system and thus suppresses their ability to fight infection or cancer. In a more incisive approach, scientists can remove or destroy only donor T cells specific for patient antigens before transplantation. Alternatively, donor T cells specific for patient antigens can be inactivated prior to transplantation by the process of alloanergization, by stimulation with patient antigens and simultaneous blocking of a cell surface receptor called CD28. Transplanting donor bone marrow containing immune cells that have been so treated (alloanergized) into patients results in less GVHD than expected and yet preserves the ability of the patient to fight infection. Davies et al. have closely examined the T cell populations in patients who have received alloanergized bone marrow transplants and find that the decreased incidence of GVHD is not just a result of the inactivation of alloreactive cells through the in vitro treatment. Transplantation of alloanergized bone marrow also results in an increase in a subset of T cells that suppress responses of other immune cells. Unexpectedly, the responses that are suppressed are specifically those directed at the patient tissues. Thus, any transplanted immune cells directed against patient tissues that might have survived in vitro inactivation are inhibited by these suppressive T cells. This extra benefit of anergized donor bone marrow cells is especially important because the ability to generate T cells that suppress responses to defined and controllable antigens could be harnessed in the treatment of other diseases in which unwanted immune responses cause pathology. Transplantation of hematopoietic stem cells from healthy donors can cure patients with many diseases. Donor T cells can protect against recurrence of infection and disease, but some of these (alloreactive) T cells recognize patient tissues as foreign, causing graft-versus-host disease. Removing T cells from donor grafts before transplantation reduces graft-versus-host disease but increases infection and disease recurrence. Inactivation of alloreactive T cells by inducing tolerance to patient cells (anergization) before transplantation preserves beneficial donor T cell effects while reducing graft-versus-host disease. We show that this approach also results in expansion of regulatory T cells that specifically suppress alloreactive donor T cell responses in the recipient. In addition to reducing graft-versus-host disease, antigen-specific regulatory T cells generated with this strategy could suppress unwanted T cell responses that cause rejection of solid organ transplants and tissue damage in autoimmune disorders.