Stephanie Verfuerth

Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines

Adoptive transfer of CMV-specific T cells offers the potential for reconstitution of viral immunity after allogeneic transplantation. However, the logistics of producing virus-specific T-cell clones has limited the application of cellular therapies. We treated 16 patients for CMV infection with polyclonal CMV-specific T-cell lines generated by short-term culture. Massive in-vivo expansions of CMV-specific cytotoxic T lymphocytes were observed, resulting in reconstitution of viral immunity. In eight cases antiviral drugs were not required, and subsequent episodes of reactivation occurred in only two patients. Our findings indicate that application of CMV-specific cell lines is both feasible and effective in a clinical environment.

Cytomegalovirus-Specific T Cell Immunotherapy Promotes Restoration of Durable Functional Antiviral Immunity following Allogeneic Stem Cell Transplantation

BACKGROUNDnThe profound immunodeficiency associated with allogeneic hematopoietic stem cell transplantation is permissive to uncontrolled replication of latent human herpesviridae such as cytomegalovirus. Morbidity and mortality associated with viral dissemination or its treatment are significant. Although adoptive cellular therapy with virus-specific T cells offers the potential for accelerating pathogen-specific immune reconstitution, the risk of induction of graft-versus-host disease and the logistics of production of clonal T cell populations restrict application.nnnMETHODSnWe investigated the ability of cytomegalovirus-specific mixed CD4(+) and CD8(+) T cell lines, generated by short-term ex vivo culture of donor lymphocytes with donor monocyte-derived dendritic cells pulsed with virus lysate, to restore antiviral immunity in 30 allogeneic transplant recipients at high risk of both uncontrolled viral replication and of graft-versus-host disease.nnnRESULTSnThere were no immediate toxicities and no excess of graft-versus-host disease. Massive in vivo expansions of cytomegalovirus-specific T lymphocytes occurred, temporally associating with periods of viral replication, suggesting that antigen exposure was necessary for optimal cytomegalovirus-specific immune reconstitution. The expanding populations maintained functional competence in ex vivo re-stimulation assays, promoting reconstitution of durable functional cytomegalovirus-specific immunity and effectively preventing recurrent viral infection and late cytomegalovirus disease.nnnCONCLUSIONSnThese data confirm the ability of cellular immunotherapy to hasten reconstitution of antiviral immunity following allogeneic transplantation, indicating that significant clinical benefits may be conferred in terms of reduction of secondary viral infection episodes, potentially reducing exposure to the toxicities of antiviral drugs.

Reconstitution of T-cell repertoire after autologous stem cell transplantation: Influence of CD34 selection and cytomegalovirus infection

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.

T- and B-cell immune reconstitution and clinical outcome in patients with multiple myeloma receiving T-cell-depleted, reduced-intensity allogeneic stem cell transplantation with an alemtuzumab-containing conditioning regimen followed by escalated donor lymphocyte infusions

Summary. Immune reconstitution after conventional allogeneic transplantation is a major determinant of survival. We conducted a detailed investigation of T‐ and B‐cell immune reconstitution and clinical outcome in 19 patients with multiple myeloma undergoing reduced‐intensity stem cell transplantation using in vivo T‐cell depletion with alemtuzumab. These patients experienced delayed T‐cell recovery, particularly in the naïve (CD45 RA+) CD4 compartment. T‐cell receptor spectratype analysis showed a reduced repertoire diversity, which improved rapidly after the administration of donor leucocyte infusions and subsequent conversion to full donor T‐cell chimaerism. Post‐transplant recovery of CD19+ B cells was also delayed for up to 18u2003months. Spectratype analysis of IgH CDR3 repertoire revealed a gradual normalization in IgM spectratype complexity by 6–12u2003months after transplant. There was a high incidence of viral infection, particularly cytomegalovirus reactivation, but the regimen‐related mortality was low, perhaps because of the very low incidence of acute graft‐versus‐host disease (GVHD; grade I‐II skin GVHD was seen in 5/19 patients). Over 80% of all patients have relapsed at a median of 283 (range 153–895) d after transplant, suggesting that the initially low rate of GVHD comes at a high price with regard to the desired graft‐versus‐myeloma effect.

Assessing diversity: immune reconstitution and T-cell receptor BV spectratype analysis following stem cell transplantation.

Summary. T‐cell receptor (TCR) BV spectratyping provides information concerning immune reconstitution complementary to that derived from monoclonal antibody analysis of surface antigen expression. However, the most appropriate way to analyse and represent these data, the number of subfamilies that should be analysed, and the effects of CD4/8 ratio on observed diversity, remain unclear. A diversity scoring system was developed based on analysis of 11 cord blood (CB) and 12 normal adult BV spectratypes. CB subfamily spectratypes demonstrated minor deviations from a Gaussian pattern consistent with current knowledge about germline TCR rearrangements. Diversity scores were significantly lower in myeloma patients than normal adults (Pu2003<u20030·001) and fell significantly following stem cell transplantation (Pu2003= 0·003). Subsequently increasing diversity was not detected by two previously described complexity scoring systems. The CD4/8 ratio was neither the major determinant of the absolute diversity score nor of the change in score for individual patients, suggesting that analysis of unselected mononuclear cells can provide information largely independent of CD4/8 ratios. There was a relatively low correlation between individual subfamily scores and overall diversity score. The novel and objective diversity scoring system described appears better able to document changes in spectratype patterns than previously described techniques and should aid the standardization of reporting.

One antigen mismatched related donor bone marrow transplant in a patient with acute lymphoblastic leukaemia and β-thalassaemia major : potential cure of both marrow disorders

We report a case of a 34-year-old man with T-ALL and β-thalassaemia major who underwent a one antigen mismatched related donor bone marrow transplant. Five months post transplant chimeric studies revealed full donor haemopoiesis and the patient remains leukaemia and thalassaemia free at 12 months post transplant. Cumulative risk factors contributing to the increased transplant-related mortality in patients with two different marrow disorders are discussed. Bone Marrow Transplantation (2000) 25, 677–678.

Generation of memory T cells for adoptive transfer using clinical-grade anti-CD62L magnetic beads.

Pre-clinical studies of allogeneic stem cell transplantation suggest that depletion of naive T cells from donor lymphocytes will reduce the risk of GvHD but preserve immunity to infectious pathogens. In this study, we have established a clinical-grade protocol under good manufacturing practice conditions for purging CD62L+ naive T cells from steady-state leukapheresis products using the CliniMACS system. The efficacy of immunomagnetic CD62L depletion was assessed by analysis of cell composition and functional immune responses. A median 2.9 log CD62L depletion was achieved with no evidence of CD62L shedding during the procedure and a mean T-cell yield of 47%. CD62L− cells comprised an equal mix of CD4+ and CD8+ T cells, with elimination of B cells but maintenance of regulatory T cells and natural killer cell populations. CD62L-depleted T cells were predominantly CD45RA− and CD45RA+ effector memory (>90%) and contained the bulk of pentamer-staining antivirus-specific T cells. Functional assessment of CD62L− cells revealed the maintenance of antiviral T-cell reactivity and a reduction in the alloreactive immune response compared with unmanipulated cells. Clinical-grade depletion of naive T cells using immunomagnetic CD62L beads from steady-state leukapheresis products is highly efficient and generates cells suitable for adoptive transfer in the context of clinical trials.

Early PCR-negativity after allogeneic BMT in adults with t(4;11) ALL in the absence of acute or chronic GVHD

Acute lymphoblastic leukaemia (ALL) with the t(4;11) translocation has a very poor prognosis following conventional chemotherapy. Many patients are offered an allogeneic BMT in first remission. We report on the impact of allogeneic BMT on three patients with t(4;11) ALL in first remission. Median age was 20 years. One patient received marrow from an HLA-identical sibling and the other two from unrelated donors. All three engrafted and none of the patients developed acute or chronic GVHD. Remission status was monitored using a sensitive nested RT-PCR to detect the ALL-1/AF-4 hybrid transcript. All three were PCR-negative at 3 months post-BMT. One of the unrelated recipients died of a fungal infection 4 months post-BMT. The other two are alive and in molecular remission at 21 and 24 months post-BMT. This is the first report of longitudinal follow-up of t(4;11) ALL post-allogeneic BMT by PCR. The early attainment of molecular remission in the absence of GVHD suggests that the conditioning regimen may have been more important than a graft-versus-leukaemia effect in these patients. Follow-up of larger numbers of patients will be required to confirm these preliminary observations.