Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor-T cell manufacture.

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)-T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection.

HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

NKp44 expression, phylogenesis and function in non-human primate NK cells.

Molecular and functional characterization of the natural cytotoxicity receptor (NCR) NKp44 in species other than Homo sapiens has been elusive, so far. Here, we provide complete phenotypic, molecular and functional characterization for NKp44 triggering receptor on Pan troglodytes NK cells, the closest human relative, and the analysis of NKp44-genomic locus and transcription in Macaca fascicularis. Similar to H. sapiens, NKp44 expression is detectable on chimpanzee NK cells only upon activation. However, basal NKp44 transcription is 5-fold higher in chimpanzees with lower differential increases upon cell activation compared with humans. Upon activation, an overall 12-fold lower NKp44 gene expression is observed in P. troglodytes compared with H. sapiens NK cells with only a slight reduction in NKp44 surface expression. Functional analysis of 'in vitro' activated purified NK cells confirms the NKp44 triggering potential compared with other major NCRs. These findings suggest the presence of a post-transcriptional regulation that evolved differently in H. sapiens. Analysis of cynomolgus NKp44-genomic sequence and transcription pattern showed very low levels of transcription with occurrence of out-of-frame transcripts and no surface expression. The present comparative analysis suggests that NKp44-genomic organization appears during macaque speciation, with considerable evolution of its transcriptional and post-transcriptional tuning. Thus, NKp44 may represent an NCR being only recently emerged during speciation, acquiring functional relevance only in non-human primates closest to H. sapiens.

Chimpanzee CD4+ T cells are relatively insensitive to HIV-1 envelope-mediated inhibition of CD154 up-regulation.

CD40-CD154 interaction forms a key event in regulation of crosstalk between dendritic cells and CD4 T cells. In human immunodeficiency virus (HIV)-1 infected patients CD154 expression is impaired, and the resulting loss of immune responsiveness by CD4+ T cells contributes to a progressive state of immunodeficiency in humans. Although chimpanzees are susceptible to chronic HIV-1/SIVcpz infection, they are relatively resistant to the onset of AIDS. This relative resistance is characterized by maintenance of CD4+ T cell populations and function, which is highly compromised in human patients. In our cohort of chronically HIV-1- and SIVcpz-infected chimpanzees, we demonstrated the capacity to produce IL-2, following CD3/CD28 stimulation, as well as preserved CD154 up-regulation. Cross-linking of CD4 with mAb was found to inhibit CD3/CD28-induced up-regulation of CD154 equally in chimpanzees and humans. However, specific cross-linking with trimeric recombinant HIV-1 gp140 revealed reduced sensitivity for inhibition of CD154 up-regulation in chimpanzees, requiring fourfold higher concentrations of viral protein. Chimpanzee CD4+ T cells are thus less sensitive to the immune-suppressive effect of low-dose HIV-1 envelope protein than human CD4+ T cells.

Differential NKp30 inducibility in chimpanzee NK cells and conserved NK cell phenotype and function in long-term HIV-1-infected animals.

HIV-1 infection in chimpanzees, the closest human relative, rarely leads to disease progression. NK cells contribute to the shaping of adaptive immune responses in humans and show perturbed phenotype and function during HIV-1 infection. In this study, we provide full phenotypic, molecular, and functional characterization for triggering molecules (NKp46, NKp30 NKp80, and NKG2D) on Pan troglodytes NK cells. We demonstrate that, in this AIDS-resistant species, relevant differences to human NK cells involve NKp80 and particularly NKp30, which is primarily involved in NK-dendritic cell interactions. Resting peripheral chimpanzee NK cells have low or absent NKp30 molecule expression due to posttranscriptional regulation and increase its levels upon in vitro activation. Following long-standing HIV-1 infection, peripheral NK cells in chimpanzees have conserved triggering receptor expression and display moderate phenotypic and functional decreases only once activated and cultured in vitro. These data suggest that one of the keys to successful lentivirus control may reside in part in a different regulation of NK cell-triggering receptor expression.

Transmission of simian immunodeficiency virus SIVcpz and the evolution of infection in the presence and absence of concurrent human immunodeficiency virus type 1 infection in chimpanzees.

Current data suggest that the human immunodeficiency virus type 1 (HIV-1) epidemic arose by transmission of simian immunodeficiency virus (SIV) SIVcpz from a subspecies of common chimpanzees (Pan troglodytes troglodytes) to humans. SIVcpz of chimpanzees is itself a molecular chimera of SIVs from two or more different monkey species, suggesting that recombination was made possible by coinfection of one individual animal with different lentiviruses. However, very little is known about SIVcpz transmission and the susceptibility to lentivirus coinfection of its natural host, the chimpanzee. Here, it is revealed that either infected plasma or peripheral blood mononuclear cells readily confer infection when exposure occurs by the intravenous or mucosal route. Importantly, the presence of preexisting HIV-1 infection did not modify the kinetics of SIVcpz infection once it was established by different routes. Although humoral responses appeared as early as 4 weeks postinfection, neutralization to SIVcpz-ANT varied markedly between animals. Analysis of the SIVcpz env sequence over time revealed the emergence of genetic viral variants and persistent SIVcpz RNA levels of between 10(4) and 10(5) copies/ml plasma regardless of the presence or absence of concurrent HIV-1 infection. These unique data provide important insight into possible routes of transmission, the kinetics of acute SIVcpz infection, and how readily coinfection with SIVcpz and other lentiviruses may be established as necessary preconditions for potential recombination.

Lentivirus infections and mechanisms of disease resistance in chimpanzees.

One year after the human immunodeficiency virus (HIV) was pinpointed as the etiological agent of the acquired immunodeficiency syndrome (AIDS) in humans, chimpanzees were identified as one of the few living species also capable of sustaining persistent HIV-1 infection. During the mid to late 1980s, as the AIDS epidemic spread globally in humans, the chimpanzee was eagerly looked to for answers concerning effective AIDS therapies and a possible HIV vaccine. Although from the complicated inter-relationship of the AIDS virus with the human immune system, neither an effective vaccine nor a therapy has emerged, one remarkable observation has been that, unlike humans, chimpanzees are relatively resistant to the development of AIDS. In the meantime, HIV-1 vaccine and therapy research has moved on to the SHIV/SIVmac rhesus model as an alternative AIDS model for which disease intervention studies can be better performed, and chimpanzees are rarely studied anymore. However, pertinent questions about the mechanisms of resistance to AIDS in this species beg to be answered. After more than twenty years, the spotlight has recently been turned once again on to the chimpanzee, in the intense search for the origin of the AIDS epidemic. Here we review the history of HIV-1 infection in this species as well as the observations that have led to some of the current leading hypotheses regarding the resistance to AIDS in naturally infected African primates.