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Gururangan et al. Neuroimmunol Neuroinflammation 2018;5:45 I http://dx.doi.org/10.20517/2347-8659.2018.44 Page 9 of 15

antigen-specific T cells [84-88,90] . This anti-tumor effect can also be significantly enhanced by MA conditioning
regimens coupled with autologous HSC support. Interestingly, HSCs were found to confer an enhancing
effect on the in vivo expansion and persistence of tumor-specific lymphocytes transferred simultaneously
[91]
with HSCs independent of the effects of lymphopenia . Pursuant to these findings, Rosenberg and
colleagues proceeded to evaluate MA conditioning regimens coupled with peripheral blood stem cell (PBSC)
rescue in patients with malignant melanoma receiving ACT. ALT was administered in the peri-transplant
[92]
period within 24 h of PBSC infusion and was feasible and safe in humans . As demonstrated in murine
studies, this conditioning regimen enhanced anti-tumor responses in patients with refractory metastatic
disease, resulting in increased objective clinical responses from 30%-50% of patients with NMA regimens to
over 70% in patients receiving MA conditioning coupled with PBSC infusion.

The mechanisms by which lymphodepletion leads to an enhancement of immune responses in humans
are not well elucidated but elegant murine studies have implicated the following important processes:
(1) increased in production of homeostatic cytokines such as IL-7 and IL-15 that drive lymphocyte
[93]
proliferation ; (2) decreased competition with adoptively transferred tumor-specific lymphocytes through
removal of “cytokine sinks” consisting of host lymphocytes and NK cells that decrease the bioavailability
[94]
[95]
of growth factors ; (3) removal of CD4+CD25+Fox P3+ tregs that attenuate anti-tumor immunity ; (4)
increased toll-like receptor agonistic signals and inflammatory cytokines through release of gut microbial
[96]
antigens such as endotoxin during damage to gut endothelium by MA therapy ; and (5) direct enhancing
[97]
effects of HSC transplant on the in vivo expansion and function of adoptively transferred lymphocytes .
HSCs augment immune responses during ttRNA-DC + ttRNA-xALT therapy
MA chemotherapy with HSC rescue is frequently used in children with primary brain tumors including
medulloblastoma, other central primitive neuro-ectodermal tumors (PNETs), and in malignant glioma both
at diagnosis as well as at recurrence . The HSC rescue serves to repopulate the bone marrow and recovery
[98]
following myeloablation and additionally helps in the reconstitution of the host immune system. Since MA
conditioning with HSC rescue is used in our adoptive T-cell therapy protocols, we wanted to explore potential
immune-modulatory effects of HSC in addition to its role in recovery from lethal bone marrow damage. It
has been previously shown that HSCs can promote the expansion and function of CD8+ T-cells by secreting
[91]
homeostatic cytokines IL-7 and IL-15 . In a pre-clinical highly invasive chemotherapy and radiotherapy
resistant orthotopic glioma mouse model (KR158B glioma), administration of MA conditioning + HSC +
x-ALT + ttRNA DC (× 3 vaccines) produced significantly improved survival and cures in 30% of animals
as compared to tumor bearing controls (no treatment, MA conditioning alone, MA + HSC + x-ALT only,
[99]
or ttRNA DC only) . The x-ALT cells were syngeneic splenocytes harvested from tumor antigen-primed
mice and expanded ex-vivo with ttRNA DCs in the presence of IL-2. The HSCs were found to migrate into
the tumor and attract activated T-cells into the tumor. Correlative studies found that tumor elaborating
C-X-C motif chemokine 12 (CXCL12) attracted the HSCs into the tumor by expressing the cognate receptor
C-X-C motif chemokine receptor 4 (CXCR4). T-cells (both CD4+ and CD8+) were attracted into the tumor
[99]
milieu following secretion of CCL3 by the HSCs . In addition, maintenance DC vaccines were crucial in
[99]
maintaining this immune response . In probing the role of HSCs further in inducing immune responses,
it was found that in addition to attracting effector T-cells into the tumor microenvironment, HSC infusion
precipitated production of activated CD86+CD11c+MHC class II+ cells consistent with a DC phenotype in
[100]
this tumor milieu and replacement of host MDSCs . This was attributed to the differentiation of the HSCs
[100]
into DC under the influence of T-cell secreted IFN-γ .
Pre-clinical and clinical studies of ttRNA DC vaccine -/+ x-ALT in primary CNS tumors
In our laboratories and those of others, systemic immunization using DCs co-cultured with uncharacterized
[55]
tumor homogenate [44,101] , whole tumor RNA , unidentified peptides eluted from tumor cells by gentle acid
[103]
[102]
washing , or a distinct peptide encompassing the tumor-specific EGFRvIII mutation have been shown
to induce humoral and cell-mediated systemic immune responses and to prolong the survival of mice
with intracranial brain tumors. We have used a strain of mice (VMDk) that is susceptible to experimental