Immunotherapy with CD25/CD71-allodepleted T cells to improve T-cell reconstitution after matched unrelated donor hematopoietic stem cell transplant: a randomized trial

BACKGROUND AND AIMS: Delayed immune reconstitution is a major challenge after matched unrelated donor (MUD) stem cell transplant (SCT). In this randomized phase 2 multi-center trial, Adoptive Immunotherapy with CD25/71 allodepleted donor T cells to improve immunity after unrelated donor stem cell transplant (NCT01827579), the authors tested whether allodepleted donor T cells (ADTs) can safely be used to improve immune reconstitution after alemtuzumab-based MUD SCT for hematological malignancies. METHODS: Patients received standard of care or up to three escalating doses of ADTs generated through CD25+/CD71+ immunomagnetic depletion. The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-SCT. Twenty-one patients were treated, 13 in the ADT arm and eight in the control arm. RESULTS: The authors observed a trend toward improved CD3+ T-cell count at 4 months in the ADT arm versus the control arm (230/µL versus 145/µL, P = 0.18), and three ADT patients achieved normal CD3+ T-cell count at 4 months (>700/µL). The rates of significant graft-versus-host disease (GVHD) were comparable in both cohorts, with grade ≥2 acute GVHD in seven of 13 and four of eight patients and chronic GVHD in three of 13 and three of eight patients in the ADT and control arms, respectively. CONCLUSIONS: These data suggest that adoptive transfer of ADTs is safe, but that in the MUD setting the benefit in terms of T-cell reconstitution is limited. This approach may be of more use in the context of more rigorous T-cell depletion

Anti-tumor activity without on-target off-tumor toxicity of GD2-Chimeric Antigen Receptor T cells in patients with neuroblastoma

The reprogramming of a patient’s immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity

Investigation of antigen processing using partially assembled MHC class I molecules

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

TLRs to cytokines:Mechanistic insights from the imiquimod mouse model of psoriasis

Psoriasis is an inflammatory disease of the skin affecting 2-3% of the population, characterized by a thickening of the epidermis and immune infiltrates throughout the dermis and epidermis, causing skin lesions that can seriously affect quality of life. The study of psoriasis has historically been hampered by the lack of good animal models. Various genetically induced models exist, which have provided some information about possible mechanisms of disease, but these models rely mostly on intrinsic imbalances of homeostasis. However, a mouse model of psoriasiform dermatitis caused by the repeated topical application of Aldara™ containing 5% imiquimod was described in 2009. The mechanisms of action of Aldara™ are complex. Imiquimod is an effective ligand for TLR7 (and TLR8 in humans) and also interferes with adenosine receptor signaling. In addition, isostearic acid present in the Aldara™ vehicle has been shown to be biologically active and of importance for activating the inflammasome. Interestingly, the repetitive application of Aldara™ reveals a complex aetiology involving multiple cell types, cytokines, and inflammatory pathways. In this review, we will dissect the findings of the imiquimod model to date and ask how this model can inform us about the immunological aspects of human disease.</p

What on “Irf” Is This Gene 4? Irf4 Transcription-Factor-Dependent Dendritic Cells Are Required for T Helper 2 Cell Responses in Murine Skin

Interferon regulatory factors play an important role in the transcriptional regulation of immunity. In this issue of Immunity, Kumamoto et al. (2013) and Gao et al. (2013) identify an Irf4-dependent migratory dendritic cell subset required for T helper 2 cell polarization following cutaneous challenge

Memory lapses in graft-versus-host disease

"Faster, better, more'' is the conventional benchmark used to define responses of memory T cells when compared with their naive counterparts. In this issue of the European Journal of Immunology, Mark and Warren Shlomchik and colleagues [Eur. J. Immunol. 2011. 41: 2782-2792] make the intriguing observation that murine memory CD4(+) T-cell populations enriched for alloreactive precursors are fully capable of rejecting allogeneic skin grafts but yet are incapable of inducing significant graft-versus-host disease. These observations add to the emerging concept that memory CD4(+) T-cell development is more nuanced and complex than predicted by conventional models. In particular, the data suggest that it may be just as important to consider what naive or effector cells have "lost'' in their transition to memory

Suppression of MHC class I surface expression by calreticulin's P-domain in a calreticulin deficient cell line

AbstractCalreticulin (CRT) is an important chaperone protein, comprising an N-domain, P-domain and C-domain. It is involved in the folding and assembly of multi-component protein complexes in the endoplasmic reticulum, and plays a critical role in MHC class I antigen processing and presentation. To dissect the functional role and molecular basis of individual domains of the protein, we have utilized individual domains to rescue impaired protein assembly in a CRT deficient cell line. Unexpectedly, both P-domain fragment and NP domain of CRT not only failed to rescue defective cell surface expression of MHC class I molecules but further inhibited their appearance on the surface of cells. Formation of the TAP-associated peptide-loading complex and trafficking of the few detectable MHC class I molecules were not significantly impaired. Instead, this further suppression of MHC class I molecules on the cell surface appears due to the complex missing antigenic peptides, the third member of fully assembled MHC class I molecules. Therefore the P-domain of calreticulin appears to play a significant role in antigen presentation by MHC class I molecules

Host CD11c+ Dendritic Cells Are Required for Priming the Lympho-Haematopoietic Graft-Versus-Host Response but Not Graft-Versus-Host Disease.

Abstract Abstract 2450 Poster Board II-427 Following allogeneic bone marrow transplantation (BMT), delayed donor leukocyte infusion (DLI) at a point when conditioning-induced inflammation has resolved, is employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease (GVHD). We have previously shown that host bone marrow (BM)-derived antigen presenting cells (APC) are required to prime GVL responses following delayed donor T cell transfer into MHC-mismatched allogeneic chimeras. The APC population(s) required for induction of graft-versus-host reactivity are not known, although it has been demonstrated by others that certain host-derived dendritic cell (DC) populations (in situ or transferred) are capable of inducing GVHD in models of immediate T cell transfer to freshly conditioned recipients. We hypothesised that host CD11c+ conventional DC would be required for the induction of graft-versus-host reactivity in a model of delayed T cell transfer to established mixed chimeras (MC). To test this hypothesis, we reconstituted lethally irradiated B6 (H2b) mice with a mixture of BALB/c (H2d) and B6.CD11c-DTR T cell depleted bone marrow. In the resulting MC, host CD11chigh cells (predominantly conventional DC) are uniquely sensitive to killing by diphtheria toxin (DT) due to their selective expression of a high affinity DT receptor. 8-10 week old established MC received DLI in form of congenic Thy1.1+ BALB/c splenocytes. MC were injected with DT (or PBS) every 72 hours from day -1 to day 10 following DLI. Depletion of host conventional DC (&gt;90%) abrogated accumulation of donor Thy1.1+ CD4 and CD8 T cells in recipient spleens and reduced in vivo cytotoxicity against host target B cells. These effects associated with a lack of conversion from mixed to full donor chimerism, demonstrating an absolute requirement for host CD11chigh DC in priming the lympho-haematopoietic graft-versus-host response in the steady state. We have shown previously that induction of systemic inflammation by injection of a synthetic TRL7 agonist (R-848) is sufficient to induce systemic GVHD in MC after delayed DLI (Chakraverty et al, JEM, 2006). We therefore considered the requirement for CD11chigh DC in priming graft-versus-host reactivity in the presence of inflammation. We generated BALB/c + B6.CD11c-DTR→ B6 MC as before and then used DT treatment to deplete host conventional DC in the presence of R-848 (given every 72h from day -1 to day 10 following DLI). In MC without depletion of CD11chigh DC, R-848 treatment enhanced accumulation of Thy1.1+ BALB/c CD4 and CD8 donor cells and increased in vivo cytotoxicity against host B cell targets as compared to non-R-848 treated MC. This was associated with histological evidence of GVHD in the skin, gut and liver. In MC with depletion of CD11chigh DC (&gt;90% deletion), R-848 treatment was associated with equivalent donor T cell accumulation and in vivo killing of host targets as observed in non-DC depleted controls. Furthermore, DC-depleted MC treated with R-848 also developed histological GVHD. In vitro experiments using CD11c+ stimulator cells purified from the spleens and lymph nodes of R-848 or PBS-treated mice suggested that priming of the allo-response in the presence of inflammation was not due to a residual population of CD11chigh DC remaining after DT treatment. Taken together, these data demonstrate for the first time that conventional host CD11chigh DC are required to prime lympho-haematopoietic graft-versus-host responses in the steady state following delayed DLI to MC. In contrast, their role is redundant in the context of TLR-agonist induced GVHD. This suggests that other APC populations can prime GVHD in the presence of systemic inflammation. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Clonal Expansion of Graft-Versus-Host Reactive CD8+ T Cells Is Dissociated from Full Effector Differentiation Following Delayed DLI.

Abstract Extrinsic factors within the host environment are crucial in determining recruitment of graft-versus-host (GVH)-reactive T cells to peripheral tissues and the capacity of these cells to induce graft-versus-host disease (GVHD). In this study, we have examined how the host environment influences graft-versus-leukemia (GVL) activity. Transfer of small numbers of allogeneic T cells to freshly irradiated (TBI allo) mice induces both GVL and GVHD, whereas transfer of much higher numbers of T cells to established (&amp;gt;8 weeks) mixed chimeras (MC) can induce GVL without GVHD. Using an EL4 tumor protection assay and low doses of B10.A splenocytes (3 × 10e6, a dose 1 log lower than normally transferred to MC to induce GVL), we observed that tumor-free survival of recipient mice was greater following transfer to TBI allo B6 recipients than in B6 + B10.A → B6 MC. To determine the mechanisms for this disparity, we used a BALB/c recipient/B6 donor strain combination, in which we could track the distribution of donor T cells within secondary lymphoid organs and bone marrow (BM) following transfer to TBI allo or MC recipients. Despite similar expansions within the spleen, there was a significant delay in the accumulation of polyclonal donor T cells (B6 CD45.1) and transgenic 2C GVH-reactive CD8+ T-cells (bearing TCR specific for recipient antigen) in the BM of MC compared to TBI allo mice. Moreover, in vivo cytotoxicity of host B cell targets occurred rapidly and was virtually complete in TBI allo recipients, but was absent in MC even at late time points. To evaluate the acquisition of effector functions in a clonal GVH-reactive CD8+ T cell population, we sorted 2C T cells at intervals following transfer and performed quantitative RT-PCR of molecules linked to effector differentiation. Strikingly, transcription of IFN-γ, granzyme B and TNF-α was significantly higher in cells derived from TBI allo hosts compared to MC. Similar differences in IFN-γ and granzyme B protein expression were confirmed in the polyclonal donor CD8+ T cell population. Since, full GVL activity might also depend upon the survival of GVH-reactive CTL, we also examined the viability of donor T cells during the initial response in both environments. We observed higher rates of sustained 2C CD8+ T cell apoptosis (as indicated by annexin V staining) following T cell transfer to MC. Furthermore, we also detected lower expression of common γ chain cytokine receptors that mediate responsiveness to IL-2, IL-7 and IL-15, upon donor T cells from MC. However, following secondary co-transfer to syngeneic recipients for 21 days, memory phenotype polyclonal donor CD8+ T cells derived from established MC (CD45.1+) were recovered to a greater extent than T cells initially derived from TBI allo mice (Thy1.1+), arguing against any intrinsic defect in the viability of GVH-reactive T cell populations emerging in the former setting. Indeed, when co-cultured in the presence of individual cytokines, MC-derived CD8+ T cells maintained viability to a greater (IL-2, IL-7) or equivalent (IL-15) extent as TBI allo-derived cells. Taken together, these data suggest that disparity in GVL activity following to TBI allo recipients and MC can be explained by differences in the effector function and survival of anti-host CTL in quiescent MC environment. Reduced GVL activity of donor T cells on a per-cell basis in MC can be compensated for by transferring greater numbers of cells.</jats:p