New Approach to Effectively Modulate and Redirect the Autoimmune ...

2012 Research Grant Program Winning Abstract
New Approach to Effectively Modulate and Redirect the Autoimmune
Response in T1D Patients: Phenotypical and Functional Analysis of the
Components
By Alessia Zoso
Type 1 diabetes (T1D) is an autoimmune disease in which the immune system
recognizes and specifically eliminates insulin producing beta cells and consequently
results in impaired blood glucose control, inducing poor blood circulation, heart disease,
kidney failure, stroke, infection, and eventually death. Currently, the main approaches
used to control disease progression consist of daily exogenous insulin administration
and pancreas transplantation. Either approach, though, does not attempt to solve the
immunological alterations that represent a crucial aspect in the development and
progression of the disease. Moreover, the complexity and polyclonal nature of the
autoimmune response in T1D patients makes any attempt to revert/modulate it
extremely challenging. Nevertheless important accomplishments have been achieved
with a new strategy, now established in our department, which is aimed at controlling
and redirecting the T1D autoimmunity by using cord blood derived stem cells as in vitro
“educators.”
Briefly, cord blood cells cultured on highly adherent surfaces for 2–3 weeks are used to
“educate” T1D patients’ peripheral blood mononuclear cells (PBMCs) by a 2-hour coincubation.
The autologous “educated PBMCs” are then reinfused in the diabetic patient.
Clinical data show that this approach significantly increases C-peptide production,
reduces glycated-hemoglobin, and promotes generation of T regulatory cells (Tregs)
able to control T1D progression. Furthermore, the approach was extremely safe, with no
graft-versus-host reaction or significant adverse events. Preclinical data in the nonobese
diabetic model indicate that this approach not only promotes the in vivo generation of
CD4 + /CD62L + Tregs, but also allows pancreatic beta cell regeneration. Thus, both
clinical and preclinical results indicate the capacity of cultured cord blood derived cells to
tolerize the patients’ PBMCs and to partially revert T1D. Nevertheless, it is still unknown
which umbilical cord blood population/s is responsible for this therapeutic effect and
what fine mechanisms are involved. Their comprehension is necessary for maximizing
the therapeutic efficacy and heading toward a cure for T1D. Toward this goal we
propose the following aims:
AIM 1: Analysis of the stem cells educator. Using several panels of specific anti-human
antibodies from BD Biosciences, we will perform an extensive flow cytometry analysis on
the cord blood units used to prepare the stem cells educator. In particular we will
evaluate the content and quality of different immune components (monocytes,
granulocytes, DCs, T cells, Tregs, B cells) at different time points: 1) before plating the
cells, 2) before adding the patient’s PBMCs to the educator cells, and 3) after the coculture
with the patient’s cells. This analysis will help us in defining possible critical and
functional changes that the initial cord blood units will undergo during the educator
preparation and co-culture with the patient’s PBMCs. Furthermore, we will functionally
analyze the educator cells with particular attention to: 1) their immune-suppressive

activity toward the proliferation of CFSE activated T cells obtained from T1D patients or
healthy donors; and 2) their capacity to induce Treg expansion. Both CD4 + and CD8 + T-
cell proliferation will be evaluated as CFSE dilution by flow cytometry.
AIM 2: Analysis of T1D patient’s educated cells. T1D and healthy donor PBMCs will be
evaluated phenotypically by flow cytometry using panels of antibodies for lymphoid (ie, T
and B cells, Tregs, etc), myeloid (macrophage, myeloid derived suppressor cells,
immature cells, etc), and DC (conventional and plasmacytoid) subsets. Functionally,
PBMCs will be tested by proliferation assay, and both phenotype and functionality will be
evaluated on fresh isolated PBMCs before and after the co-culture with the educator
cells. This set of experiments should reveal crucial changes in the patient’s PBMCs that
occur during the incubation with the educator cells.
AIM 3: Role of soluble factors in mediating the stem cells educator function. Our recent
data prove the importance of the soluble factor, released upon interaction between
activated T cells and cord blood derived suppressive cells, for the generation/expansion
of FoxP3 + CD4 + Tregs. We will compare this finding to the stem cells educator approach
by evaluating the cytokine profile of the culture media used in the apparatus at different
time points: 1) Time 0: media before starting the educator preparation; 2) Time 1: culture
media of the stem cells educator before adding the PBMCs, and 3) Time 2: culture
media at the time of PBMC collection and reinfusion in the patient. A cytokine profile also
would be run on the serum sample obtained from the patients before and 4 weeks after
cell reinfusion. Control media will be collected from the following cells cultures: a) cord
blood alone; b) PBMCs alone; c) PBMCs cultured with an irrelevant cell line (HEK-293).
All the samples will be analyzed at once for the expression of several cytokines using
the BD Cytometric Bead Array, a multiplex bead-based immunoassay.
This study will be of extreme importance, not only to better understand the stem cells
educator functionality, but also to identify new mechanisms and factors with critical roles
in the treatment of T1D.
The BD Biosciences Research Grant Program aims to reward and enable important
research by providing vital funding for scientists pursuing innovative experiments to
advance the scientific understanding of disease.
Visit bdbiosciences.com/grant to learn more and apply online.