Journal of Translational Medicine
Suppression of Acute Respiratory Distress Syndrome Pulmonary Inflammation by
Transfer Factor Activated Dendritic Cells: StemVacs™
--Manuscript Draft--
Manuscript Number:
Full Title: Suppression of Acute Respiratory Distress Syndrome Pulmonary Inflammation by
Transfer Factor Activated Dendritic Cells: StemVacs™
Article Type: Research
Funding Information:
Abstract: Despite falling into disfavor amongst immunologists as a means of antigen-specific
immune modulation, the potential of Transfer Factor (TF) as to act as an inducer of
innate immunity has not been evaluated. Utilizing leukocytes from healthy donors, we
generated leukocyte lysate employing a procedure similar to that used in preparation of
TF with exception that the donors were not pre-immunized. We demonstrated superior
augmentation of costimulatory molecules, cytokine production and T cell activation by
dendritic cells treated with TF as compared to those activated using conventional
protocols. Administration of TF-activated allogeneic dendritic cells resulted in
suppression of B16 melanoma growth in an NK dependent manner. Interestingly
administration of the cells in a murine model of ARDS resulted in suppression of
pulmonary pathology. Given that StemVacs has already been used in humans and is
subject of an FDA IND application, utilization of this cellular therapy for treatment of
COVID-19 may be considered.
Corresponding Author: Thomas Ichim, MSc, Ph.D, CCRA
Therapeutic Solutions International
UNITED STATES
Corresponding Author E-Mail: thomas.ichim@gmail.com
Corresponding Author Secondary
Information:
Corresponding Author's Institution: Therapeutic Solutions International
Corresponding Author's Secondary
Institution:
First Author: James Veltmeyer, MD
First Author Secondary Information:
Order of Authors: James Veltmeyer, MD
Weiping Min, MD Ph.D
Thomas Ichim, MSc, Ph.D, CCRA
Timothy Dixon
Order of Authors Secondary Information:
Manuscript Classifications: 90: Immunovirology; 140: Patient-targeted molecular therapies
Additional Information:
Question Response
Is this study a clinical
trial?<hr>A clinical trial is defined
by the World Health Organisation as 'any
research study that prospectively assigns
human participants or groups of humans
to one or more health-related
interventions to evaluate the effects on
health outcomes'.
No
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Suppression of Acute Respiratory Distress Syndrome Pulmonary Inflammation by Transfer Factor
Activated Dendritic Cells: StemVacs™
1
James Veltmeyer, 2Wei-Ping Min, 1Thomas E Ichim, 1Timothy Dixon
1Therapeutic Solutions International, Oceanside, California; 2University of Western Ontario, London,
Canada
Manuscript Click here to access/download;Manuscript;Paper draft july 6.docx
Click here to view linked References
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Abstract:
Despite falling into disfavor amongst immunologists as a means of antigen-specific immune modulation,
the potential of Transfer Factor (TF) as to act as an inducer of innate immunity has not been evaluated.
Utilizing leukocytes from healthy donors, we generated leukocyte lysate employing a procedure similar
to that used in preparation of TF with exception that the donors were not pre-immunized. We
demonstrated superior augmentation of costimulatory molecules, cytokine production and T cell
activation by dendritic cells treated with TF as compared to those activated using conventional
protocols. Administration of TF-activated allogeneic dendritic cells resulted in suppression of B16
melanoma growth in an NK dependent manner. Interestingly administration of the cells in a murine
model of ARDS resulted in suppression of pulmonary pathology. Given that StemVacs has already been
used in humans and is subject of an FDA IND application, utilization of this cellular therapy for treatment
of COVID-19 may be considered.
Background
One of the major causes of COVID-19 associated fatality is Acute Respiratory Distress Syndrome (ARDS).
This is a condition of acute respiratory failure caused by a variety of factors which are related to
inflammation and release of various activators of the innate immune system such as cytokines and
inflammatory factors [1-9]. An ideal therapy would involve stimulation of immunity while suppressing
inflammation.
It is widely known that ARDS generally presents with progressive hypoxemia, dyspnea and increased
work of breathing. Patients often require mechanical ventilation and supplemental oxygen [10]. Over
the years, our understanding of ARDS has advanced significantly, with elucidation of several of the
molecular and cellular pathways involved in initiation, progression and resolution/fibrosis . However,
ARDS is still represents significant morbidity and mortality and therapeutic strategies to mitigate the
foregoing have resulted in limited translational success. Part of this failure stems from the very different
presentations of ARDS between people, as well as differences in their genetic composition.
ARDS is caused by many situations bacterial and viral pneumonia, sepsis, inhalation of harmful
substances, head, chest or other major injury, burns, blood transfusions, near drowning, aspiration of
gastric contents, pancreatitis, intravenous drug use, and abdominal trauma. Furthermore, those with a
history of chronic alcoholism are at a higher risk of developing ARDS [11-13]. Alcoholism affects several
parameters relevant to ARDS including: a) reduction in glutathione levels [14, 15], b) increasing levels of
adhesion molecules on lung blood vessels so as to increase recruitment of inflammatory cells [16]; c)
upregulating lung adenosine levels, resulting in impaired active Na(+) transport in the lung [17]; and d)
suppression of pulmonary immunity [18].
One of the cardinal symptoms of ARDS is fluid accumulation in the lungs. When this occurs, the elastic
air sacs (alveoli) in the lungs fill with fluid and the function of the alveoli is impaired. The result is that
less oxygen reaches the bloodstream, depriving organs of the oxygen required for normal function and
viability. In some instances, ARDS occurs in people who are already critically ill or who have significant
injuries. Severe shortness of breath, the main symptom of ARDS, usually develops within a few hours to
a few days after the precipitating injury or infection. [19]
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Unfortunately, many patients who develop ARDS do not survive. The risk of death increases with age
and severity of illness. Of the people who do survive ARDS, some recover completely while others
experience lasting damage to their lungs.
Currently there exist no effective pharmacologic therapies for treatment or prevention of ARDS. While
inhibition of fibrin formation mitigated injury in some preclinical models of ARDS, anticoagulation
therapies in humans do not attenuate ARDS and may even increase mortality. Protective lung ventilator
strategies remain the mainstay of available treatment options. Due to the significant morbidity and
mortality associated with ARDS and the lack of effective treatment options, new therapeutic agents for
the treatment of ARDS and new treatment methods for ARDS are needed.
The current paper examined whether allogeneic dendritic cells (DC) can be utilized for stimulation of NK
mediated immunity, while concurrently not exacerbating ARDS. The possibility of a dual NK stimulation
and ARDS suppression is intriguing.
Materials and Methods
Mouse Model
Female C57BL/6 mice of 8 weeks of age were inoculated with 1 million B16 cells in the flank. Mice were
administered allogeneic BALB/c bone marrow derived dendritic cells which were not antigen pulsed. For
experiments involving lung fluid content, BALB/c mice were administered 5 ug of lipopolysaccharide
intratracheally and sacrificed at the indicated timepoints. DC were administered intravenously via tail
vein injection. Lung fluid content was measured by weight the lung versus the weight of the body.
Cell Cultures
Whole blood (20–50 ml) from healthy donors (>80 participants) was mixed with Ficoll-Hypaque, and
after centrifugation the layer of mononuclear cells was collected. After lysis of RBC, the mononuclear
cells were laid on petri dishes (Costar, Cambridge, MA) for 30–60 min at 37°C to remove nonadherent
cells. After five washes with PBS, adherent cells were cultured in 3 ml of medium containing 800 U/ml
GM-CSF and 500 U/ml IL-4. The culture medium was changed every other day with 300 µl of fresh
medium containing 2400 U of GM-CSF and 1500 U of IL-4. The detached cells, the main population of
CD1a+ cells (our unpublished observations), were used for experiments after culture for 7 days.
Consistently, >95% of the cells in the gated region expressed CD1a.
Maturation of DC was performed by culture with 10ng/ml lipopolysaccharide and 10 ng/ml TNF-alpha.
Assessment of maturation was performed by flow cytometry for CD80, CD86 and IL-12 production.
Assessment of natural killer cell activity as performed using ProMega cytotoxicity kit as per
manufacturers instructions.
Cytokine Analysis
Standard ELISAs were used to measure cytokine concentrations in harvested. All determinants were
performed triplicate (IL-12) and expressed as the mean ± SD. The expression of cell surface molecules
was determined by flow cytometric analysis. Each histogram or density plot comprised at least 104
events and expressed as mean fluorescent intensity.
Transfer Factor
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Peripheral blood leukocytes were resuspended in 5 ml sterile water (Lonza). Cells were frozen and then
thawed using dry ice and 100% ethanol bath, alternating with a 37°C water bath. The freeze-thaw cycle
was performed at least 7 times. The product was then placed into dialysis bags with an 8 kDa cutoff that
had been boiled for 3 intervals of 20 min (Spectrum Labs, Rancho Dominguez, CA, USA) against 50 vol
water. Dialysis was conducted for 24 h at 4°C under constant stir. Dialysis was repeated for an additional
24 h with an identical volume of water after the first amount was removed and frozen. Subsequent
experiments using ultrafiltration were performed by filtering lysed splenocytes through a Centriprep10K cutoff ultrafiltration unit (EMD Millipore, Billerica, MA, USA). After the second dialysis or after
ultrafiltration, the contents from both dialysis sessions were combined in T225 flasks (BD Falcon; BD
Biosciences, San Jose, CA, USA), frozen to -80°C, and then lyophilized (FreeZone 2.5; Labconco, Kansas
City, MO, USA). Lyophilized remnants were resuspended in sterile water or DMEM (Thermo Fisher
Scientific) to a cell-equivalent concentration, as indicated.
Results
Induction of Dendritic Cell Maturation
Transfer Factor (TF) was generated according to previously published protocols and added to dendritic
cell progenitors. As seen in Figure 1, dendritic cells activated with TF possessed higher expression of
CD80 (Fig 1a), higher expression of CD86 (Fig 1b), lower expression of IL-12 (Fig 1c) and possessed
enhanced ability to stimulate NK cytotoxicity (Fig 1d).
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Allogeneic Non-pulsed Dendritic Cells (StemVacs™) Inhibit B16 Melanoma in an NK Dependent Manner
It is well-recognized that NK cells play a role in suppression of cancer and metastasis [20-22]. Studies
have shown that in certain situations non-pulsed DC are capable of eliciting an anticancer response
through stimulation of NK cell
therapeutic activity [23-26]. The
importance of DC to cancer can be
seen in numerous studies showing
superior prognosis in patients having
dendritic cell infiltrations in the
tumor [27, 28]. In order to assess
whether StemVacs™ allogeneic TF
activated DC are capable of
inhibiting tumor growth, we
administered cells in a wild-type B16
models (Figure 2a) and in NK
depleted mice (Figure 2b).
Strikingly, the antitumor activity was
diminished in absence of NK cells.
This indicates the possibility that
allogeneic TF pulsed DC may be
useful as a cancer therapeutic.
Suppression of Lung Inflammation by StemVacs™
One therapeutic possibility for cell therapies which activate NK cells could be in the treatment of COVID19. One of the potential drawbacks of utilizing immunotherapies in this condition is the possibility of
cytokine storm and exaggeration of pulmonary failure. Accordingly, we sought to assess whether
administration of StemVacs™ would exacerbate inflammatory reactions subsequent to endotoxin
challenge. To our surprise StemVacs™ administration was associated with enhanced protection from
lung inflammation as compared to conventionally activated DC which actually enhanced inflammation
(Figure 3).
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Discussion
We have demonstrated that transfer factor activated DC are capable of inducing potent immunity by
stimulating both T and NK cells. The current data support the translational use of StemVacs™ in
treatment of COVID-19 due to its unique ability to activate NK cells but also suppress inflammation in
the lungs.
Pulmonary inflammation such as in ARDS is associated with augmentation of interleukin-17 [29, 30]. It is
known that the cells which produce IL-17, called Th17 cells, have a reciprocal relationship with T
regulatory cells. Indee,d it may be possible that our dendritic cell therapy was stimulating NK cells which
account for its anticancer properties, while stimulating T regulatory cells, which may have been
responsible for reduction of inflammation.
In many situations it is established that mature DC activate T cells while immature ones generate T
regulatory cells. Two of the authors (WPM and TEI) more than ten years ago described the Tolerogenic
Feedback Loop in which permanent allograft tolerance is maintained through a self-amplifying loop
between regulatory T cells and tolerogenic dendritic cells [31]. We believe that further experiments are
needed to determine whether the suppression of pulmonary inflammation is occurring as a result of T
regulatory cells being elicited.
While utilization of autologous dendritic cells is well-known in the area of oncology, allogeneic dendritic
cells present the tantalizing option of an “off the shelf” therapy. Initial clinical reactions using this
approach have demonstrated safety (manuscript in preparation) and signals of efficacy. It may be that
allogeneic dendritic cells work by activating NK cells as compared to T cells. Further studies are needed
to elucidate whether it is the allogeneic nature of the stimulating cells, or whether it is the potency of
the TF in activating DC.
One unexplained observation was the finding that while TF stimulated expression of costimulatory
molecules in a manner superior to LPS and TNF-alpha, it did not stimulate IL-12 production. IL-12 is
needed for NK activation, however NK activation was higher with TF as compared to traditional means
of activating dendritic cells. We postulate that other cytokines may be involves such as IL-18, which we
are in the process of assessing.
In conclusion, StemVacs™ appears to be a novel means of stimulating innate immunity while
concurrently suppressing cytokine storm and pathology associated with adaptive immunity. These
current data will serve to provide preclinical support for the planned StemVacs™ IND application.
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