The marginal zone B cel in concordant xenograft rejection: critical help signals in the T cel-independent type II xenoantibody response and possible targets for xeno-immunosuppression.

Allogeneic hematopoietic stem cell transplantation
(HSCT) and posttransplant adoptive cell therapy with donor leukocyte infusions
(DLI) are known to exert potent graft-versus-leukemiaeffects and convincing
evidence is emerging that this approach can also elicit responses against solid
tumors. Graft-versus-leukemia effects specifically after DLI largely rely
on lymphohematopoietic graft-versus-host T cell alloreactivity which can result
in graft-versus-host disease when it extends to epithelial tissues. Not only
for hematological but also for solid tumors the association of anti-tumor
effects with graft-versus-host disease represents the major obstacle to the
successful application of allogeneic HSCT and adoptive DLI therapyas an
immunotherapeutic strategy. Recent data in mice have shown that challenge with
recipient-type rather than donor-type leukocytes (recipient leukocyte infusion,
RLI) can equally provide antitumor effects in hematological malignancies. </>RLI provokes lymphohematopoietic host-versus-graft T
cell reactivity with rejection of the graft and associated anti-leukemic
responses and does not carry the risk of graft-versus-host disease. This
strategy is currently being explored clinically in patients with hematological
malignancies. In experimental mouse models, it was demonstrated </>that NK cells, activated in vivo in
the course of RLI-induced T cell alloreactivity, contribute to the survival
advantage produced byRLI</>.</></>

Allogeneic
hematopoietic stem cell transplantation may also hold potential for the
treatment of high-risk neuroblastoma, a common extracranial solid tumor in
childhood that carries a bleak prognosis. Despite aggressive treatment
including high-dose chemotherapy and lymphohematopoietic rescue with autologous
hematopoietic stem cells, relapse is very frequent. This spurs research into
post-transplant immunotherapeutic interventions to enhance antitumor immunity.
Neuroblastoma cells are known to be sensitive to NK cell cytotoxicity and strategies
targeting NK cells are therefore particularly of interest. Alternatively, case
reports suggest that allogeneic HSCT in high-risk neuroblastoma patients may
prolong survival and thus confers a graft-versus-neuroblastoma effect. This has
been experimentally demonstrated in mice. A phase I/II trial is currently
investigating the anti-tumoreffect of haplo-identical HSCT in children with
neuroblastoma. </></>

The overall aim of this study was to investigate the potential combined
effect of allogeneic HSCT and the novel RLI strategy on the growth of
experimentally induced neuroblastoma tumors. Given the efficacy of RLI in
enhancing graft-versus-leukemia effects in mice, the evidence that such effects
involve not only T cells but also NK cells, and the knowledge that
neuroblastoma cells are generally sensitive to NK cell cytotoxicity</></>, we hypothesized that RLI may be an
effective and safe alternative to DLI to enhance the immune-mediated graft-versus-neuroblastoma
effect. Immune interventions after standard therapy with autologous HSCT for
neuroblastoma can to some extent enhance endogenous anti-neuroblastoma immunity.
In mice it has been shown that T cells devoid of anti-host alloreactivity may
foster anti-neuroblastoma effects of allogeneic bone marrow transplantation
(BMT) in mice. This supported our hypothesis that challenge with syngeneic
mature effector cells may in addition to causing alloreactive T cell
responses foster direct anti-tumor reactions. </>

The specific aim
of this work was to study the effects of RLI adoptive therapy on the local and
metastatic growth of neuroblastoma tumors in allogeneic bone marrow chimeras.
We studied RLI in comparison with DLI since this is the standard approach used
in hematological malignancies ánd the approach currently studied in clinical
trial for high-risk neuroblastoma. We used mouse models offocal and systemic
posttransplant neuroblastoma relapse in a settingof MHC-mismatched allogeneic
BMT: we chose to inoculate mice after the transplant to study immune anti-tumor
effects independently of direct effects of recent radiation therapy on the
tumor. We used three different models. First, a focal model in which tumor
cells are injected subcutaneously and give rise to a local and clinically
measurable tumor (subcutaneous model</>).
Second, a systemic model where tumor cells are inoculated intravenously and
thus cause direct metastatic spread (intravenous
model</>). Third, a more physiological and preclinical model of metastatic
spread where tumor cells are inoculated underthe kidney capsule and where the
local tumor is removed in toto after 7 days by a nephrectomy (kidney model</>). For the systemic and
kidney models, we generated a luciferase-expressing tumor cell line and
developed a bioluminescence imaging protocol to non-invasively follow
metastasis-free survival in vivo.</>

In part I of this work we used the subcutaneous model to study the
effects of allogeneic BMT and adoptive cell therapy on local neuroblastoma
growth. </>We confirmed that allogeneic BMT in mice provides
a baseline graft-versus-neuroblastoma effect. Wenext demonstrated that both
DLI ánd RLI, when given to mixed bone marrow chimeras, further slow the local
growth of subcutaneous implanted neuroblastoma tumors. DLI provoked a rapid
conversion to full donorchimerism but mice developed severe GvHD, whereas RLI
produced complete graft-rejection but left mice healthy. The chimerism of
intratumoral leukocytes evolved in parallel with peripheral blood chimerism and
was -both after DLI and RLI- associated with increased intra-tumor CD8/CD4
ratios, CD8+ T-cell IFN-</>γ</>-expression and NK-cell Granzyme B-expression. The effect of RLI was
weaker than that of DLI but co-transfer of naïve recipient-type NK cells could
elicit a growth-limiting effect on neuroblastoma tumors in RLI chimeras. </>

Inpart II we investigated the
effects of posttransplant adoptive cell therapy on the systemic spread of
neuroblastoma. The final prognosis for patients with solid tumors, and
especially for neuroblastoma, is determined by the presence of distant therapy
non-responsive metastases. </>Novel forms of
immunotherapy therefore only hold clinical potential if they are efficacious on
both local and systemic spread of the tumor.</>
Using the intravenous tumor model we documented that allogeneic BMT provides a
significant overall survival benefit relative to syngeneic BMT. Moreover, using
luciferase-expressing tumor cells and bioluminescence imaging weshowed that
both </>DLI and RLI produce a significant delay in
metastatic spread. The bioluminescence imaging studies in the kidney model -
although preliminary -were consistent with those of the intravenous model and
support the notion that both DLI and RLI have the potential to limit metastatic
spread of neuroblastoma. </>

In part III we addressed the
question whether syngeneic BMT by itself can provide an immune
anti-neuroblastoma effect. The current standard treatment of high-risk
neuroblastoma includes high-dose chemotherapy and autologous HSCT which serves
merely as a lymphohematopoietic rescue. However, the potential intrinsic immune
effects of this procedure have so far not been investigated.</>Our data are preliminarybut show that, relative to
naïve mice, syngeneic BMT facilitates tumorigenesis of subcutaneously
inoculated tumor cells while on the other hand it delays the growth of
established tumors. CD8+ T and NK cells synergistically regulated
tumor growth. Direct evidence of CD8+ T cells could not be found.
However, the role of NK cells is supported by the evidence of increased Fas-L expression
and by the fact that adoptively transferred syngeneic NK cells could reinforce
the reduction in tumor growth.</>

In conclusion,
the data support the potential value of DLI in mediating graft-versus-solid
tumor effects. Importantly, they also deliver the first experimental evidence
that RLI is able to target a solid tumor and that it thus may represent a safe
alternative to DLI for this purpose. We document that lymphohematopoietic
alloreactivity extends to the environment of the tumor itself, indicating that
also for solid tumors it is closely related with the antitumor effect. Studies
on the leukocytes contained within the focal neuroblastoma tumors provided
evidence strongly suggesting that the antitumor effect of both DLI and RLI
involves not only CD8+ T cells but also cytotoxic NK cells. Whereas
in the DLI setting,the antitumor effect may rely predominantly on alloreactive
CD8+ T and NK cells, we provided evidence that the T cell
alloreactivity provoked by an RLI challenge suffices to provide syngeneic naïve
NK cellswith the potential to attack syngeneic neuroblastoma cells in vivo. We
conclude therefore that RLI and DLI in allogeneic chimeras can provoke an in vivo</> lymphokine-activated killer
phenomenon leading to cytotoxically active NK cells, even in the syngeneic
setting. The alloreactive T cell response produced by RLI and DLI may also
facilitate the activation and expansion of tumor-specific donor or host CD8+
T cells. Consistent with prior studies in leukemia models the RLI effect was
weaker than the DLI effect but this could be compensated by the additional
infusion of syngeneic NK cells, a procedure that does not compromise the safety
profile of RLI. Lastly, although to some extent preliminary, the data indicate
that the effects of both DLI and RLI are not limited to the local growth of a
tissue-localized tumor but can also limit the metastatic spread of a solid
tumor. This is a critical requirement for a novel form of immunotherapy to be
clinically relevant. </>

The data may be
particularly helpful to understand the value of allogeneic HSCT and adoptive
cell therapy for neuroblastoma. We demonstrate that this approach may provide a
synergistic T cell and NK cell interaction resulting in an effective antitumor
response against neuroblastoma, even in the setting where RLI is used. This is
particularly interesting since neuroblastoma is wellknown to be sensitive to
NK cell cytotoxicity. With this respect, the data also suggest that NK cells equally
contribute to the baseline antitumor effect of allogeneic HSCT in
neuroblastoma. Last, our preliminary observations in the syngeneic BMT model
show that the effects of conditioning and autologous lymphohematopoietic
reconstitution on neuroblastoma growth are complex and may include paradoxical
effects on tumorigenesis and tumor progression.</>



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