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Page 8 of 15 Gururangan et al. Neuroimmunol Neuroinflammation 2018;5:45 I http://dx.doi.org/10.20517/2347-8659.2018.44

Ex-vivo T cell expansion
The discovery in 1976 of IL-2, a T-cell growth factor, in the supernatant of phytohemagglutinin-activated
[62]
human lymphocytes , facilitated ex-vivo expansion of autologous T-cells without loss of effector function
[3]
as well as the parenteral use of this cytokine to induce T-cell proliferation in the host . Using this approach,
impressive responses, albeit transient, were seen with bulky tumors in patients with lymphomas and
metastatic melanomas. In a search of reactive T-cells and cognate tumor antigens, it was found that certain
tumors, especially melanomas, harbored reactive lymphocytes in the tumor stroma that were of both CD4+
[63]
and CD8+ . These cells provided a rich source of possible TILs that if harvested, expanded ex-vivo, and
[63]
reinfused could provide better therapeutic benefit . Hence, TILs obtained from a resected melanoma
[63]
and injected into the same host resulted in objective tumor regressions . However, T-cells administered
following such ex-vivo TILs expansion did not survive more than a few days due to immunosuppressive
[63]
factors related to the tumor . This obstacle was circumvented by using host NMA lymphodepletive
conditioning using cyclophosphamide and fludarabine immediately prior to TIL transfer that led to increased
longevity of the infused T cells and more oligoclonal expansion directed at specific antigenic epitopes (see
[64]
later under “lymphodepletion with chemotherapy”) . While TIL expansion and transfer can theoretically be
possible in many human tumors with T-cell infiltration, it appears to be particularly suitable in melanomas
due to high mutation burden and immunogenicity, characteristics of a carcinogen - induced tumor.

While it seems attractive to employ utilize this strategy in children with brain tumors, the lack of a
prominent lymphocyte infiltrate in such tumors makes this an unsuitable approach in this patient
population. However, the approach in our lab involves using ttRNA DCs to “educate” and expand
naïve autologous lymphocytes ex vivo and reinfuse these lymphocytes (ttRNA-xALT) into the host after
lymphodepletive conditioning using either MA or NMA chemotherapy followed by hematopoietic stem
cell (HSC) rescue and sustained the expansion of these “educated” lymphocytes by regular administration
of ttRNA DCs. We have demonstrated the capacity to induce anti-tumor lymphocytes in vitro and in vivo
using tumor RNA-pulsed DCs from a variety of human and murine tumors, including malignant brain
tumors [50,52,55,65-73] . We have explored the capacity to enhance the yield of antigen-specific T cells ex-vivo
through a rapid expansion protocol (REP) developed at the National Cancer Institute for expansion of
melanoma-reactive tumor-infiltrating lymphocytes . The REP employs irradiated allogeneic PBMCs as
[74]
feeder cells, low-dose anti-CD3 monoclonal antibodies (OKT3), and IL-2 for the rapid expansion of activated
[74]
lymphocytes in culture . This process has the capacity to expand lymphocytes stimulated by RNA-pulsed
DCs 200-500 fold and is possible to achieve greater than 1 × 10 T-cells with an input of 1 × 10 activated
8
10
lymphocytes. The addition of IL-21 and IL-7 during co-culture of T cells with RNA-pulsed DCs prior to
REP leads to a greater generation of central memory antigen-reactive T cells which have been shown to be
superior in anti-tumor efficacy.

Lymphodepletion with chemotherapy augments immune responses
[75]
After episodes of lymphopenia, host recovery ensues before regaining normal lymphocyte counts .
After profound lymphopenia [75,76] , there may be an increase in cytokines (i.e., IL-7, IL-15) which can
induce lymphocyte differentiation into effector memory T cells imbued with memory recall against target
[77]
antigens . However, under these conditions, competition for homeostatic cytokines remains a potent
[75]
barrier for lymphocyte proliferation , which may allow antigen-specific B- or T-cells (generated through
vaccination) to predominate during recovery from lymphodepletive therapy; data to support this has been
[80]
shown in murine model [78,79] and in humans . The predominance of these cell types may augment anti-
tumor response [78,79,81] , but may theoretically also precipitate autoimmunity [82,83] .

[84]
Rosenberg et al. used NMA lymphodepletion to sustain and augment these antigen specific memory
T cell subsets against refractory malignancies, and achieved impressive responses in patients [84-88] despite
autoimmunity [64,86] . Under these NMA contexts, adoptively transferred T cells proliferate intensely following
lymphopenia and comprise the bulk of a T cell repertoire in a treated host, which persists for months after
cell transfer [64,89] . Moreover, disease regression strongly associates with the amount and persistence of these