CD4+ and CD8+ T Cells in Guillain Barre Syndrome: Correlation with Disease

Severity and Outcome

A dissertation submitted to the Department of Biochemistry & Microbiology,

North South University in the partial accomplishment of Degree of Masters of
Science in Biotechnology

Syed Akib Hossain

NSU ID 1935401070
Department of Biochemistry and Microbiology
School of Health and Life Science
North South University
Dhaka, Bangladesh
DECLARATION

We the members of this thesis defense committee certify that we have carefully read and
recommended to the Department of Biochemistry and Microbiology, North South University for
approval of this thesis entitled:

“CD4+ and CD8+ T Cells in Guillain Barre Syndrome: Correlation with

Disease Severity and Outcome”

Submitted by Syed Akib Hossain, NSU ID No. 1935401070, in partial fulfillment of the
requirements for the Degree of Master of Science in Biotechnology.

___________________________ ____________________________
Abdul Khaleque, PhD Dr. Zhahirul Islam
Professor and Supervisor Scientist & Head
Department of Biochemistry & Microbiology Laboratory of Gut-Brain Signaling
School of Health and Life Sciences Laboratory Sciences and Services Division
North South University icddr,b

___________________________ ____________________________
Dr. Nayeema Bulbul Dr. Hasan Mahmud Reza
Associate Professor & Chair Dean
Department of Biochemistry & Microbiology School of Health and Life Sciences
School of Health and Life Sciences North South University
North South University
 Acknowledgement
It is my immense pleasure and honor to write this acknowledgement to show gratitude to people
associated and who helped me throughout my thesis project and dissertation. First and foremost, I
would emphasize in thanking Almighty Allah for providing me the opportunity and blessing me
the strength, ability and patience to accomplish my work successfully. I am and will forever be
grateful to Dr. Zhahirul Islam, Scientist and Head, Gut-Brain Signaling Laboratory, Laboratory
Sciences and Services Division, icddr,b for giving me the exceptional opportunity to work and
learn simultaneously with a dynamic group of intellectual people along with an international
laboratory facility to conduct my thesis research project. My sincere gratitude goes to my
supervisor Abdul Khaleque, PhD, Professor, Department of Biochemistry and Microbiology,
School of Life Sciences, North South University for always supporting, guiding and motivating
me to reach my research goal and successfully complete the project.

I would like to express highest gratitude and honor to Dr. Nayeema Bulbul, Associate Professor
and Chair, Department of Biochemistry and Microbiology, School of Life Sciences, North South
University, Dr. Sohidul Islam, Professor and former chair, Department of Biochemistry and
Microbiology, School of Life Sciences, North South University for serving me with the
opportunity to conduct my master’s research project at icddr,b and always encouraging me to
successfully complete it with flying colors.

I am grateful and would like to share my sincere gratitude towards Ms. Israt Jahan, Research
Investigator, Gut-Brain Signaling Laboratory, LSSD, icddr,b for letting me learn and grow under
her guidance and mentorship with an exceptional role in designing my research project and its
execution. My respected mentor’s relentless immense support and trust let me overcome all the
challenges and hurdles which allowed me to complete the project in due time.

I am also thankful to Dr. Shoma Hayat, Dr. Nowshin Papri, Dr. Farzana Shaon, Mr. Asaduzzaman,
Ms. Moriam Akter Munni, Mr. Rasel Ahmed, Mr. Jigishu Ahmed, Ms. Sarah Khurshid, Ms. Ruma
Begum, Ms. Moriam Akter Mukta, Ms. Fahmida Habib Nabila, Mr. Kazi Saif for their valuable
suggestions and extending their hands when needed. I am also thankful for receiving constant
support from Ms. Smriti Akter, Mr. Md. Hossain and Mr. Khalid Hassan.
Successful completion of my project and dissertation submission required more than academic
support, and I have many, many people to thank for listening to and, at times, having to tolerate
me over the past one and half years. I cannot begin to express my gratitude and appreciation for
their friendship. Especially Nayeem Faruque, Naushin Tabassum, Md. Abu Jeher Nayeem, Jannat
Ara Riya and Uditi Paul have been unwavering in their personal and professional support during
the time I spent at the project.

Finally, I would like to wholeheartedly thank my family and members for the unceasing
encouragement, support, and attention and for believing in me more than I ever have myself. I also
place on record, my sense of gratitude to one and all, who directly or indirectly, have lent their
hand in this project.

Syed Akib Hossain

July, 2021
 ABSTRACT
Background: Guillain Barre Syndrome is an immune mediated peripheral nerve disorder where
aberrant T cell activities, autoantibody generation, and gene expression dysregulation have
previously been identified. Although it is often preceded by infections, nowadays the cellular
immune responses are shown to extensively account for the pathophysiology of it. T cells, in
addition to humoral immunity, are crucial in the pathological process of immune-mediated nerve
injury. A better knowledge about the role of the immune system in GBS pathogenesis could lead
to more effective targeted therapies and treatments.

Objective: Our study intended to determine the percentages of CD4+, and CD8+ T cell within
peripheral blood of patients and healthy individuals and find the CD4+, and CD8+ T cells
percentages in patients with GBS and healthy controls and association between CD4+ and CD8+ T
cell percentages and disease pathogenesis, severity, and clinical outcome of GBS.

Method: The association among CD4+ and CD8+ T cell percentages and disease progress were
investigated in 26 patients, and 10 healthy individuals. For analysis, blood samples were collected
along with other demographical data such as age, sex, antecedent infection, GBS disability score,
MRC sum score with standard questionnaire. After separating peripheral blood mononuclear cell
from blood sample; surface staining was done and the samples were run to count CD4+ and CD8+
T cells percentages by using Flow cytometer. Statistical analysis included unpaired t-test, paired
t-test, simple linear regression of correlation, ordinary one-way ANOVA, ratio paired t-test, one
sample t and Wilcoxon test.

Results: Patients with GBS had a median age of 33.5 years [IQR; 19-40 years], with male
predominance of 69.2%. Axonal variant was found in two in third of the GBS patients. The CD8+
T cells percentage was found higher (mean ± SD; 22.50 ± 10.47 vs. 19.71 ± 8.702) and the CD4+
T cells percentage was lower (mean ± SD; 27.72 ± 4.283 vs. 30.34± 4.116) compared to healthy
controls at the onset of disease. The mean number of CD8+ T cell percentage showed gradual
increased from onset of the disease to thirteen weeks follow-up (mean ± SD; 22.50 ± 10.47 to
27.49 ± 7.781). Increasing number of CD8+ T cells (mean ± SD; 27.49 ± 7.781) and decreasing
number of CD4+ T cells (mean ± SD; 25.23 ± 2.892) were found after 13 weeks since the onset of
the disease. There has been a correlation among CD8+ T cell and GBS-DS (r = 0.05853); but no
significant correlation has been observed between CD4+ T cell and GBS-DS. The mean number
of CD8+ T cell percentage was higher in axonal subtype than in demyelinating subtype (mean ±
SD; 25.87 ± 10.32 vs. 21.77 ± 5.715). Similar finding was observed with CD4+ T cell percentage
varied among axonal subtype and demyelinating subtype (mean ± SD; 26.51 ± 3.748 vs. 28.64 ±
4.756). The geometric mean of ratios (CD4+/CD8+) was found 1.290 with the standard deviation
of log (ratios) 0.1692.

Conclusion: This study is conducted to assess the percentage of CD4+, and CD8+ T cells within
GBS patients in a Bangladeshi population. The variance in the percentages of CD4+ and CD8+ T
cell among the patients with GBS and healthy controls indicate their probable role in pathogenesis
and clinical outcome in GBS. Further transparent and extensive investigation of respective subsets
with larger sample size may lead us to reveal a biomarker for therapeutic intervention of GBS.
 Table of contents

Serial No. Title Page/s

INTRODUCTION 1-19
1.1 Background 2-3
1.2 Epidemiology 3-4
1.3 Preceding infections in GBS 4-8
1.4 Clinical Features of GBS 9
1.5 Variants of Guillain-Barre Syndrome 9-10
1.6 Immune-pathogenesis of GBS 11-17
1.7 Diagnosis and Detection of GBS 18
1.8 Treatment of GBS 18
1.9 Hypothesis 18
1.10 Study Objectives 19
Materials and Methods 20-26
2.1 Study subjects 21
2.2 Work outline 22
2.3 Sample collection 23
2.4 Separation of PBMC 23
2.5 Surface staining 23-24
2.6 Flowcytometry panel designing and gating strategy 24-25
2.8 Statistical Analysis 25-26
Results 27-37
3.1 Demography of the study participants 28-29
3.2 Prognosis of GBS patients 30
3.3 Illustrative Analysis 31-37
Discussion 38-42
References 43-52
Appendices 53-57
Introduction
 Page |2

1.1 Background

Guillain Barre Syndrome (GBS) has been characterized as peripheral nervous system
disorder which is being presented as rapidly progressive, ascending, and flaccid paralysis of body
parts often including whole body with reduced or missing reflexes [1]. Though J.B. Landry in 1859
reported acute ascending paralysis as ascending post-infectious polyneuropathy but it was named
in 1916 after a combination of clinical and experimental symptoms given via Guillain, Barre, and
Strohl, while Strohl's name was removed during the early twentieth century [2]. It is an exceptional
condition as the immune system of the body destructs a component of the outer nerve. And outside
the brain, signals are carried from the brain towards the muscles by the peripheral nervous system.
Because the immune response targets the nervous system in GBS, nerve signal transmission is
impeded and muscle activity is diminished, signals out from brain to the body muscles cannot be
transmitted as quickly as they should be. GBS is also characterized as a polyradiculoneuropathy
with a sudden or sub-acute onset that typically comes after a respiratory infection or
gastrointestinal infection [3].
It has been found that approximately two in third GBS patients had an infection three weeks
earlier to the commencement of the condition [4]. Fever, cold, pharyngitis, runny nose, and
diarrhea are the most common signs [4]. In majority of the studies concerning GBS, the
predominant symptoms of a previous infection have been reported in the gastrointestinal tract or
upper respiratory tract, between other types of infections. The most frequent causative agent
reported are Campylobacter jejuni., Mycoplasma pneumoniae, Cytomegalovirus, Haemophilus
influenza, and Epstein–Barr virus are some of the other causative microorganisms [5, 6].
Numbness, muscle weakness, and tingling sensations are characteristic signs of GBS, which
gradually worsen until muscle action is completely lost, resulting in paralysis [7]. It is been
observed that maximal weakness in patients is generally reported in 4 weeks of the beginning of
the disease, but within two weeks maximum patients reach their highest weakness [8].
Though the mechanism of GBS is poorly understood, it’s most likely associated with an
immune response; for example, an infection that affects peripheral nerves a result of molecular
mimicry. Within a month of clinical symptoms, upper respiratory tract infection or gastrointestinal
infection occurs. GBS has a wide range of probable diagnoses, including flaccid paralysis, effects
on brain function, or a mix of the two. Diagnosis of the brain, spinal cord, nervous system, and
 Page |3

muscles may be present. Cerebrospinal fluid (CSF) investigations, electroencephalography, and,

less particularly, magnetic resonance imaging (MRI) are the most useful diagnostic procedures for
GBS [9].
In the early stages of GBS treatment, patients' motor, autonomic nervous system, and lung
functions are closely monitored in the hospital. An effective treatment option for GBS patients
include Plasmapheresis, a procedure that removes autoantibodies, cytokines, and complement
from the blood stream. Another treatment option involves infusion of intravenous human
immunoglobulin to offset activated antibodies. Oral or intravenous corticosteroids weren’t
effective in patients [10].

1.2 Epidemiology
1.2.1 Global incidences:
The global incidents of GBS were very low and accounts between 1.1 and 1.8 per 100 000
per year in the time interval of 1980 and 2008 [11]. It rises with age, with estimates ranging from
1.7 to 3.3 every 100,000 per year for those over 50. The axonal subtypes affect about 5 percent of
GBS patients in Northern American continent and European continent but axonal subtypes account
for 30% to 47% of GBS cases in Central and South America, Japan, and China [12]. Around 5%
of GBS cases is associated with MFS in Western countries whereas cases are higher in Eastern
Asian countries [12-15]. Study suggests that Men are more often affected than women which is
quite uncommon in other autoimmune disorders. This incidence increases with age and accounts
for around 4.7 per 100 000 above 75 years of age [16-18]. Around the globe, GBS is more common
in the winter than in the summer, which could be related to the preparatory phases of specific
pathogenic organisms [19].

1.2.2 Comparative GBS incidences in Bangladesh and developed countries:

In comparison to countries with lower income, countries with higher income have less case
fatalness from GBS patients. In the Netherlands, Singapore and Japan, the case-fatality in patients
with GBS are 2.8%, 2%, and 3% respectively [20]. The true incidence or frequency of patients
with GBS in Bangladesh remains unknown as patients who have mild disease or show mild
symptoms does not seek medical support in hospitals. The associated GBS in Bangladesh consists
of higher mortality rate and poor outcome of disease treatment which might be owing to
 Page |4

preponderance of axonal type, deficiency in treatment measures and short of appropriate medical
facilities to effectively treat this disease [21]. Prospective earlier studies have noted high fatality
of GBS cases with around 14% in percentage in Bangladesh, which is not only higher in compared
to developed nations, and also greater in relation to neighboring nations like India with 3.4% and
Pakistan with 2.4% [21]. The study also noted around 12% of death cases in the GBS population
in Bangladesh within 6 months from the disease onset but most of the deaths occurred within one
month of disease onset. The maximum overall deaths in Bangladesh occurred during the severe
disease development phase, whereas GBS patients in nations with higher than average income died
during the recovery phase. A major reason for this difference may be because the intensive and
critical care facilities in Bangladesh are inadequate, and unaffordable to many. Additionally, as
some care centers are unable to provide constant cardiac monitoring for patients, the patient’s
conditions may worsen without appropriate, and sometimes urgently required, treatment, which
leads to a higher rate of fatality of GBS patients in Bangladesh.

1.3Preceding infections in GBS

1.3.1 Major preceding infections

In some recent studies, GBS has been presented as a spectrum disorder which varies among
regions and have significant heterogeneity including clinical presentations, electrophysiology and
preceding infections [22]. Among all the patients with GBS, around two-third has been reported
with past infections before the commencement of neurological signs and among the infectious
agents, the causative microbes of GBS is thought to be Campylobacter jejuni, Epstein-Barr virus,
cytomegalovirus, and Mycoplasma pneumoniae [23]. It's also been noted that certain previous
infections were linked to various GBS subtypes. Generally, Campylobacter jejuni contains
gangliosides-like lipo-oligosaccharides (LOS) structures which acts as molecular mimicry with
the host and involve in the AMAN subtype pathogenesis [24]. Typically, infection by CMV is
considered to be related to severe deficits in motor sensory, demyelination process, and antibodies
concerning to the GM2 ganglioside [23]. Infection by Mycoplasma pneumoniae is thought to be
linked with anti-galactocerebroside antibodies and child patients with GBS [25]. Moreover, there
are evidences that suggest prior Zika virus (ZIKV) infection can lead to neurologic disorders such
as GBS in adult patients and micro-cephaly in infants [26]. Some previous review papers noted
that during or immediately after the ZIKV infection, some patients showed neurological symptoms
 Page |5

which suggests a para-infectious rather than post-infectious pattern that is typically seen in GBS
incidences [27]. According to CDC, there is a strong linkage amongst GBS and Zika virus
infection, but only a tiny percentage of patients with recently Zika virus infection develop GBS.
GBS caused by Zika virus is predicted to be 0.24 in 1000 infections [26]. Additionally, latest
studies reported progressive upsurge of GBS cases in SARS-CoV-2 infected patients with a raised
concern over a possible association [28]. While the chief complaints involve respiratory problems
during COVID-19 infection, some studies concerning the outcome of the disease have already
explored the possible involvement of secondary neurological effect of the infection affecting at
least 36% of all patients signifying the neurotrophic potential of the virus [29]. Although the world
is still struggling to deal with the COVID-19 pandemic and complications associated with it,
association of this deadly virus with a serious neurological disorder such as GBS need to be
explored.

1.3.2 Infection by C. jejuni following GBS development:

Since start of gastrointestinal microbe culture, Campylobacter has been detected 2-7 times
higher than Shigela, Salmonella [30]. In USA, C. jejuni infection cases about 2.4 million, including
other causal bacteria (such as Salmonella or Shigela spp), were discovered, making it the most
frequent enteric pathogen [31]. Epidemiological studies related to GBS stated C. jejuni is an
eliciting causative bacteria. Kuroki et al. [32] detected presence of C. jejuni in 30% of patients
with GBS, while Rees et al. [33] isolated it from 8% of patients GBS. The association between
GBS disease development after infection with C. jejuni infection was firstly described in late
twentieth century when an older male developed GBS after pervious gastroenteritis by C. jejuni
infection [34]. The C. jejuni seropositivity percentage varies between 26 to 76 percent, with the
highest reported from China in AMAN as well as within AIDP with 42 percent, signifying that
infection by C. jejuni affects AMAN more often than AIDP [35]. However, in an investigation of
86 GBS patients in Japan, it was discovered that among 20 C. jejuni positive patients, AMAN
reported 70 percent and AIDP 15 percent [36]. According to cases mentioned, axonal and
demyelinating both GBS subtypes can be caused by infection with C. jejuni.
 Page |6

1.3.2.1 Risk related to C. jejuni infection and GBS

Following exposure to specific C. jejuni serotypes, the risk of getting GBS increases.
Penner subtype O:19 is more commonly linked to GBS in USA, while Penner subtype O:41 being
more common in South Africa. Despite the fact that C. jejuni pathogens are more common among
people, the cases reported GBS after C. jejuni infection is relatively few with an incidence of 1 in
1000, implying that other genetic variables are involved in the disease's development [37]. It has
been demonstrated that GBS followed by C. jejuni might be more severe than other factors
associated in causing the disease.

Figure 1.1: Sources and route of transmission of C. jejuni [38]

 Page |7

1.3.2.2 Molecular similarity in host gangliosides and C. jejuni

Molecular mimicry stated as double recognizing incident in which a separate B cell or T

cell receptor and an antigen of the host have similarity, and infection triggers antibody or T cell
cross-reactivity, causing autoimmunity similar to GBS. A vulnerable host may produce
autoantibodies that target all bacterial gangliosides like lipo-oligosaccharides (LOS) and
gangliosides of host peripheral nerve, causing axon degeneration and peripheral nerves get
demyelinated, according to several theories. When the immune system attacks the Schwann cells
that cover the nerves, it might result in paralysis or muscle weakness, it also permits enzymes to
injure the myelin covering of axons which allows nerve impulses to be received and processed
more quickly [39]. The Cst-II gene is thought to code the formation of ganglioside alike LOS
components GM1 and GD1 in C. jejuni, which are presently the only biomarkers linked to GBS
[40]. The kind of mimicry of ganglioside in C. jejuni determines the anti-ganglioside antibodies
and links with the GBS variants. GM1 are the most common cross-reactive antibodies related to
ganglioside but the process has not yet been fully explained.
 Page |8

Figure 1.2: Schematic structure of ganglioside and its corresponding LOS of C. jejuni and
antibody mediated autoimmunity [41].
 Page |9

1.4 Clinical Features of GBS:

In majority of the cases, the commencement of weakness in the lower limbs takes place
within several days or weeks, at a maximum of less than 4 weeks, which then ascends to the upper
limbs. The presence of sensory disturbances also noted but in a lesser extent. Development of
respiratory failure and requirement of mechanical ventilation is being needed in around 25% of
GBS patients [3]. It takes around 2-4 weeks to reach maximum state of weakness after the onset
of symptoms with recovery development within weeks to months [40, 42]. Respiratory failure
might occur early in some cases and in others there might be no respiratory failure at all, meaning
the extent of respiratory involvement varies among individuals with GBS. In 50 – 70% of cases,
weakness is facial muscles occurs and is bilateral [43, 44]. There are similarities in clinical features
of axonal forms of GBS with the classical demyelinating GBS. However, the severity and rapidity
of the disease is more often linked to axonal type with respiratory and cranial nerve involvement
[45-48]. In comparison to AIDP, AMAN subtype is more likely to be linked with past infection
with C. jejuni.

1.5 GBS Variants:

Subsequent subcategories of GBS were discovered depending on the neurological
functional blocks. Several variants have been reported since early cases and reports of GBS which
differ from the most prevalent type of the disease and its presentation known as acute inflammatory
demyelinating polyradiculoneuropathy (AIDP), Acute Motor Axonal Neuropathy (AMAN),
Acute Motor-Sensory Axonal Neuropathy (AMSAN), and Miller Fisher Syndrome (MFS), and
the Pharyngeal-Cervical-Brachial presentation are all GBS variants. Among the variants, AMAN
is solely based on motor involvements without the presence of sensory indications and signs.
Whereas, AMSAN is alike AMAN other than the sensory involvement. In late phase of the disease
progression, it significantly differs from AMAN as it clinically involves sensory fibers, includes
broader geographic distribution, more prolonged course, and slow improvement in prognosis [49].
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Figure 1.4: Schematic presentation of major GBS subtypes AIDP at the left and AMAN or
AMSAN at the middle along with a normal motor nerve at the right [50].

Miller Fisher Syndrome, on the other hand, is described as an abrupt or chronic demyelinating
polyradiculoneuropathy with clinical characteristics that differ significantly from conventional
GBS [52-54]. In around 85% of GBS patients, NCS studies discloses that demyelination is
consistent with the AIDP subtype of GBS [55, 56]. It is not only important to detect the
abnormalities in motor nerves in identification of features of demyelination, but also in
differentiation of AMAN subtype from AMSAN subtype of GBS [57-59].
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1.6 Immune-pathogenesis of GBS

GBS is an autoimmune condition that resembles an improper response in the immune
system, in which the immunological attacks on neurons appears to arise because neuronal targets
are cross-reactive with infectious organism targets. In a susceptible individual, a cascade of
immunological events leading to immune system activation can result in an autoimmune reaction
on brain cells. In figure 1.5, illustration related to the sequence of events and severity of this
response is schematically presented. During the initial infection or immunization, cross-reactive
epitopes are provided together with signals of danger. The adaptive system was not intended to
react towards those with self-homology, but it was supposed to respond to other contagious non-
self-epitopes, according to theory. Nonetheless, new or previously suppressed lymphocytes
activate APCs in response to potent danger signals with local effects, and regrettably favorable
host conditions initiates an adaptive response to antigens [60].

Figure 1.5: Schematic representation of the sequence of events and severity of immune
responses in GBS: The first phase with red part indicates primary triggering incidents (such as
infection), later part in green represents a specific immune reaction against the causative agent.
Throughout this phase, the onset of GBS starts where natural epitopes with cross-reactivity results
in immunologic response. Next, blue represents myelin from Schwann cell precursors are
regenerated and lastly, Chevrons specify axon’s with slow regrowth [60]

1.6.1 Innate immunity in GBS:

Alike in other disease events, in the course of the early phase of the immunological
reactions in GBS, the innate immune system tends to recognize the molecular patterns present in
most pathogens such as pathogen associated molecular patterns or bacterial lipopolysaccharide
along with molecules endogenous in nature are released by injured cells called damage associated
molecular patterns with the help non-specific pattern receptors, for example TLRs or NLRs.
Attaching to these molecular pattern receptors initiate the immune cascade causing cytokine
release, cellular accumulation surrounding the infected area and eliminate the pathogen or infected
cell via a process called apoptosis as well as enters in the adaptive phase of the immune response
 P a g e | 12

[61]. Damian proposed in 1964 that invading organism antigens are similar to host antigens,
resulting in dual identification of TCR or antibodies [62]. Microorganisms have evolved to avoid
host immune detection by imitating both microbe as well as host antigens, which can lead to auto-
antigen recognition and an immunological response against host tissues. As a result, the concept
of autoimmune disease involving molecular mimicry has gained traction.

Figure 1.6: Schematic presentation of intricate network of innate immune cells and adaptive
immune cells playing role in GBS pathogenesis [63].

1.6.2 Adaptive immunity in GBS:

There are numerous indications that cell mediated immunity involved in the GBS
pathophysiology (64). Adaptive immunity engaged in GBS pathogenesis, with lymphocytes and
macrophages infiltrated in the peripheral nerve system. Neural biopsies of GBS patients detected
the presence of cellular infiltrates by macrophages, CD4+ and CD8+ T lymphocytes suggesting
involvement of CMI in the disease's pathophysiology (65). Furthermore, some GBS patients'
peripheral blood contained T-cells responsive to neural antigens P2 and P0, indicating systemic T-
cell activation (66). Previous research has found Campylobacter DNA in healthy people and GBS
 P a g e | 13

patients, antigens are delivered by MHC class II to T cells via the route for antigen recognition
(67). T-dependent IgG1 and IgG3 antibody responses against gangliosides GM1 and GT1a have
also been identified in recent research, proposing involvement of T-helper cells in the immune
reaction in patients with GBS. Experiments EAN, an animal GBS model of AIDP subtype,
revealed the role of T-cells in the GBS pathogenesis, as rodents lacking T-cells couldn’t create
EAN although they were immunized using the neuritogenic protein P0 or P2 (68). Some latest
experiments with mouse deficient with CD4, CD8 and CD28 by Zhu and colleagues also provided
evident that T-cell possess a vital role in the development of EAN [69]. Previous studies in patients
with GBS has found IgG type antibodies predominantly, indicating association of T helper cell
[70].

Figure 1.6: Role of lymphocytes in damaging the nerve cells when triggered by the immune-
agents during autoimmune neuropathies [70].
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1.6.3 Role of cytokines and chemokines in GBS:

Cytokines have a significant role in initiation, propagation and modulation of damages

specifically associated with tissues, as well as act as signal molecules between immune system
cells. In GBS, IFN- γ has a twofold effect: it can cause inflammation in EAN and GBS, and
evidences suggests that it can transform into CD4+CD25+ suppressor T cells from outer
CD4+CD25 T cells [71].

Figure 1.7: In the pathophysiology of experimental autoimmune neuropathies, cytokines and

chemokines function as signal molecules engaged in natural and acquired immunity.

Similarly, TNF-α, likewise, has a double role in GBS, functioning as both in causing inflammation
and a protective agent. Since IL-17 and IL-22 are released by Th17 cells, remains crucial in
inflammatory disorders, previous studies have found high level of IL-17A and IL-22 in plasma
 P a g e | 15

throughout the severe stage of GBS but concentration of IL-17A was reduced subsequently next
to IVIg therapy [72]. Furthermore, because several other cytokines are involved in some other
autoimmune disorders, which could be explored as GBS biomarkers. To discover more about their
potential involvement in GBS, more research is required.
Chemokines are low molecular mass cytokines that drive cell movement in response to
facilitated diffusion and have a role in immune cell activation. They are categorized into four sub-
groups based upon the arrangement of two homologous cysteines. Several investigations on the
relationship between chemokines and GBS have revealed that chemokines are also associated in
GBS patients' immunological responses. CCL2 levels in the blood were elevated during the severe
stage of GBS, peaked during the plateau phase, and then returned to normal throughout the
recovery stage [73]. Endoneurial macrophages produced higher amounts of chemokines such as
CCR1 and CCR5, with CCL5 co-localizing to axons on EAN and GBS sciatic nerves [74].

1.6.4 Role of CD4+ and CD8+ T cells in GBS pathogenesis:

T cells have a vital protagonist in the pathogenic cascade of immune driven nerve injury.
Activated CD8+ suppressor T cells contribute in direct destruction of both myelin and axons,
whereas CD4+ T cells mediate local inflammatory responses. Research focused on CD4+ T cells
exist since decades, with EAN being used as an animal model to observe autoimmune distal
polyneuropathy caused mostly by CD4+ T cells. CD8+ T lymphocytes have a vital role in biological
events such tumors, virus-related infection, prolonged inflammation, and autoimmune diseases
[75-78]. It was shown that CD8+ T lymphocytes which are activated aggregate around necrotic
secretory cells, and that effector particles of cytotoxic CD8+ T lymphocytes like perforin,
Granzyme B, IFN-γ, and TNF-α may also be found [79]. The presence of active CD8+ T
lymphocytes with cytotoxic effects indicates that the cells are able to destroy the surrounding cells,
resulting in cell death or apoptosis. It was discovered that IFN-γ have a pivotal role in initiating
CD8+ T lymphocyte mediated illness specific to MBP, since its inhibition lowered severity.
Furthermore, a breach in peripheral tolerance followed by virus related infection was shown to
cause CNS autoimmunity mediated by CD8+ T cells [80].
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Figure 1.4: Schematic representation of the probable pathophysiology of GBS as a paradigm of

autoimmune disease where both conventional auto-reactive T cells directed against self-proteins
as well as group 1 CD1-restricted T cells targeting self-lipids of the PNS myelin may contribute
to causing the disease [81].

The relocation of macrophages through the blood brain barrier triggers cytokines production,
which increases vascular penetrability and facilitates the entry of stimulated T lymphocytes.
Lymphocytes from GBS patients grown in myelinated axons often degrade myelin directly, as seen
in Figure 1.5 [82]. In mouse models, the role of immune response at cellular level mediated by T
lymphocytes has been shown [83]. As a result of these cells passing over the permeable blood
nerve barrier, activated CD8+ T cells can be produced. They become active once inside, stimulating
macrophages to release substances that contribute to the immune disorder related to autoimmunity
[84].
 P a g e | 17

Figure 1.5: immunological mechanism linked to APC, MHC I, MHC II involving CD8 T cell and
CD4 T cell respectively, antibody (Ab) for the destruction of self-tissues [85].

1.7 Diagnosis and Detection of GBS

In suspected GBS patients, a lumbar puncture is almost always performed typically

showing increased protein with normal cell count. During the first week, GBS patients have been
observed to have typical CSF protein concentrations, although by the second week this increases
in 90 percent patients [86]. Electromyography may be beneficial in clinically challenging
situations to endorse diagnosis, particularly in cases of acute pain, and to subcategorize GBS into
AMAN and AIDP [87]. The diagnosis of GBS is often seems difficult, especially in patients with
asymmetrical weakness and weakness only in the arms or rapidly progressive respiratory
dysfunction with relative protection of muscle strength in the extremes, or with severe pain or
autonomic dysfunction [88]. To confirm GBS cases, Nerve conduction and CSF protein
concentration investigations are two important diagnostics [89].
 P a g e | 18

1.8 Treatment of GBS

Until present, two clinically proven therapeutics of Guillain-Barre Syndrome have been
described, namely plasma exchanging (PE) and Intravenous immune globulin G (IVIG) treatment
approaches. However, the optimal dosage and duration of the treatment for both option is still
being explored in research studies conducted worldwide [90, 91]. The execution of these
treatments in clinical setting is complex as the disease presentation and severity greatly varies
among patients with GBS. The therapeutic trials by both PE and IVIg mainly focused on patients
unable to walk independently and adult [92]. It still requires further study whether these available
treatments are effective on a later stage of disease progression among patients with GBS.

1.8.1 Future Treatments:

Current and future studies will reveal new approaches towards treating GBS and managing
the patients in a better way. Study shows that one of the promising way to arrest progression of
AMAN subtype is inhibition of complement activation, as AMAN subtype is associated with an
anti-ganglioside antibody mediated neuropathy [93, 94]. Drugs that stop T cells from crossing the
blood-brain barrier, as well as agents that disrupt T lymphocyte cytokine-induced macrophage
priming, have been shown to be useful in treatment. Several studies suggest that pro-inflammatory
cytokines including IL1, IL6, IFN-γ, and TNF-α develop disease, while cytokines concerning anti-
inflammatories restrict and modify the inflammatory response; however, the specific mechanism
is unknown. As a result, cytokine-based therapies that focus on controlling their pathways remain
vital and potentially effective. However, before such rational targeted therapies are applied to
humans, further investigations are needed and specific optimal timing for the treatment need to be
determined.

1.9 Hypothesis

In terms of etiology and pathogenesis, it's still unknown how distinct GBS subtypes are
related to one another. It is evident through previous studies that T-cell subsets may have a major
role in causing the disease and its progression [115]. The study of cytotoxic T-cell subsets in
 P a g e | 19

relation to the GBS subtypes can greatly impact the current understanding of the intricate network
of immune cells and their involvement in the disease pathogenesis and progression.

1.10 Study Objectives:

GBS Patients and healthy individuals are included in our prospective study in finding:
1. The percentages of CD4+ and CD8+ T cells in peripheral blood of patients with GBS and
healthy controls, and connection with disease progression,
2. The association of CD4+ and CD8+ T cells with GBS disease severity
3. The association between CD4+ and CD8+ T cells percentages and subtypes of GBS
(AMAN & AIDP).
4. CD4+/CD8+ T cell ratios in exploring probable association between them.
5. The correlation between of CD4+ and CD8+ T cells percentages and serum albumin levels
in patients.
 P a g e | 20

Materials and Methods

 P a g e | 21

2.1 Study subjects

Our study population included 26 patients with GBS (18 males, and 8 females, median age
33.5 years, [interquartile range, IQR; 19-40 years]) and 10 healthy controls (8 males, and 2
females, median age 31.5 years, [IQR; 24-40 years]) from Bangladesh. Based on the National
Institute of Neurological Disorders and Stroke (NINDS) criteria stated by Asbury and Cornblath,
our study patients were enrolled from Dhaka Medical College and Hospital (DMCH), Dhaka. [95,
96]. Every healthy individuals enlisted in our study were racially and hereditarily distinct, and
none of them had past neurological or chronic inflammatory illnesses or serious sickness. Before
collecting the data and samples, all study participants including patients and healthy individuals
signed a written informed permission form for being enlisted in our study.
At entry and weekly follow-ups, collection of blood samples were performed in every study
participants to count cells by FACS analysis. We obtained comprehensive medical and
demographic data, including sex, age, cranial nerve involvement, antecedent events,
electrophysiological features, MRC score on baseline, GBS DS (disability score), symptoms, and
prior infections (diarrhea, any respiratory tract infection, any sort high temperature, or any other
diseases in the past). At study entrance and weekly follow-ups, the degree of disease severity was
assessed using the Medical Research Council (MRC) total score at base (maximum level of
weakness). Patients with GBS were considered as seriously affected who had MRC total score
between 0-20, mildly affected having MRC sum score 21-40 and less affected having MRC sum
score of 41-60. Patients with GBS had their outcomes graded according to the GBS disability score
(GBS-DS) at 6 months. Progressive outcome is characterize by the capacity to move unassisted
where GBS-DS was recorded 0, 1, or 2, while a poor prospect is regarded as the failure to stand
and walk unaided, or death, according to the GBS-DS (GBS-DS of 3, 4, 5 or 6). We included
follow-up patients as well as healthy controls in comparing our results and for a conclusion. Blood
samples were collected from entry patients, follow-up patients and healthy control groups.
Following progress of all the patients were monitored and follow-up blood samples were collected
at standard and fixed time intervals (2 weeks, 4 weeks, 13 weeks, and 26 weeks). The Institutional
Review Board along with the ethics committee of the icddr,b, Dhaka, Bangladesh, examined and
approved the entire work.
 P a g e | 22

2.2 Work-outline:

Patients with GBS Diagnosis and enrollment

Clinical baseline data collection including demographic information,

antecedent infection and MRC Score

Baseline blood collection in separate

Ethylenediamine tetraacetic acid (EDTA) tubes

Density gradient centrifugation with

ficoll paque and PBMC separation

PBMC count following adjustment

of cells by adding cell culture media

Flow cytometry was used to detect CD4+ and CD8+ T

cells after surface labeling with particular antibodies.

Acquisition of data is done by BD

Accuri C6 Software; data analysis
by FlowJo Software

Statistical analysis
 P a g e | 23

2.3 Sample collection:

For isolation of peripheral blood mononuclear cells, 8-13 ml blood was taken on average
from study participants in various EDTA commercial tubes. Then the collected blood was kept
undisturbed at room temperature for more than 30 minutes.

2.4 Separation of peripheral blood mononuclear cells (PBMC):

 Firstly, collected blood was mixed gently inside the tube with the help of a serological
pipette and the volume of the blood was noted.
 Then, the blood was taken in a 15 ml falcon tube and under laid on ficoll paque where the
ratio of blood and ficoll was kept 2:1.
 After that, the blood was taken to a density centrifuge machine and run for 25 minutes at
1700 rpm.
 The plasma layer on the top was separated carefully by a micro-pipette to separate
eppendorf tubes. Then the layer of PBMC bands were collected by micro-pipette and
transferred to a 15ml falcon tube and filled up to 10 ml with D-PBS and then taken to
centrifuge machine and allowed for centrifuge at 2000 rpm for a duration of 5 minutes.
 Afterwards, removed the supernatant, and cells were re-suspended in 5 mL D-PBS. Using
a microscope, 20l was taken for cell counting in a hemacytometer.
 The remaining suspension was taken to centrifuge machine again and allowed to centrifuge
again at 2000 rpm for another 5 minutes.
 Based on the cell count, the pellet of PBMC was re-suspended in cell culture media with
the count 10x 106 cells per ml.

2.5 Surface staining

1) 1 X 106 cells were taken in a FACS tube and 1 ml of FACS staining buffer was added. It
was taken for centrifugation at 500 g at room temperature for a duration of 5 minutes.
2) The cells were re-suspended in 100 µl FACS staining buffer after the supernatant was
removed.
3) Recommended amount (3µL) of anti CD8 antibody was added to cells for the analysis of
CD8+ T cells. Pulse vortex was done to gently mix.
4) Then it was then kept at 2-8°C for at least 30 minutes, covered from light.
 P a g e | 24

5) After incubation, the samples were washed with 100 µl FACS staining buffer, left for
centrifugation for a duration of 5 minutes at room temperature at 400-600 x g, and then the
supernatant was removed.
6) The cell samples then re-suspended in FACS staining buffer in a volume of 300 µl.
7) Recommended amount (3µL) of anti CD4 antibody was added to cells for the analysis of
CD4+ T cells. Pulse vortex was done to gently mix.
8) Steps 4-6 was carried out in a similar way for the samples coated with CD4 antibody.
9) Acquisition of data is done by BD Accuri C6 Software.
10) FlowJo Software is used for Data analysis and statistical analysis is done by GraphPad
Prism software.

2.6 Flowcytometry panel designing and gating strategy:

The BD Accuri C6 Plus flow cytometer is the platform's most recent generation. Flow
cytometry is now much more accessible to novice and veteran flow cytometry researchers with
improved sensitivity, reliability, and capabilities. It can detect up to 4 fluorochrome at a time.
Before running the experiment, we did compensation by single staining to subtract the unwanted
spillover on filter. For everyday experiment, we also performed formula minus one (FMO) for
each of the fluorochrome to fix the gate. In the experiment, we first locate the lymphocyte by
considering the side scatter, and forward scatter optical detector. Then we isolate the singlet cells
by exclusion the doublet cells considering forward scatter (FSC-A) and forward scatter (FSC-H).
CD8+ populations were determined from the singlet cells gate. Accordingly for CD4+ populations,
we first located the lymphocyte by considering the side scatter (SSC-A) and forward scatter (FSC-
A) optical detector. Then we isolate the singlet cells by exclusion the doublet cells considering the
side scatter (SSC-A) and forward scatter (FSC-A). Lastly, CD4+ populations were determined
from the singlet cells. The data was acquired by BD Accuri C6 software and converted to fcs
format for further analysis through FlowJo.
 P a g e | 25

Figure 2.1: 1) SSC-H vs FSC-H density plot. On the plot, each dot or point represents a single particle
that has traveled past the laser. (2) Singlet cells were isolated by excluding the doublet cells. (3) To
determine the CD8+ cells, a gate was used.

Figure 2.2: 1) SSC-A vs FSC-A density plot. On the plot, each dot or point represents a single
particle that has traveled past the laser. (2) Singlet cells were isolated by excluding the doublet
cells. (3) To determine the CD4+ cells, a gate was used.

2.7 Statistical analysis:

The percentages of CD4+ and CD8+ T lymphocytes in patients with GBS and healthy
controls were assessed in this study. The patient’s demographic information, antecedent illness,
clinical and electrophysiological features were analyzed by Microsoft Excel. The mean ± standard
deviation are used to express continuous variables. Unpaired t-test were used to assess the mean
differences of CD4+ and CD8+ T cell percentages of the patients and healthy controls, treated &
non-treated patients, and patients with AMAN and AIDP subtypes. By analyzing the percentages
of CD4+ and CD8+ T lymphocytes using simple one-way ANOVA, we were able to calculate the
patients' weekly progression from the outset of disease. The associating between percentage
 P a g e | 26

differences of CD4+ and CD8+ T cells and disease severity (GBS-DS) were measured using simple
linear regression of correlation. The CD4+/CD8+ T cell ratio was determined by ratio paired t-test.
The profiling of serum albumin level and the percentage variations of CD4+ and CD8+ T
lymphocytes were determined by one sample t and Wilcoxon test. At p <0.05, results were
deliberated to be significant.
 P a g e | 27

Results
 P a g e | 28

3.1 Demography of the study participants:

In this study, in total 26 GBS patients along with 10 healthy controls (HC) were studied

where their median age was 33.5 years and 31.5 years respectively. The percentage of male patients

was dominant in both case (69.2%) control (83%). Among 26 patients, 15 patients (57.6%) had

antecedent illness where major illness includes gastroenteritis (19.2%), common cold (19.2%),

RTI (11.5%), others (15.3%). Around 31% patients had no antecedent illness. Cranial nerve

involvement was found in 11 (42.3%) patients among them 10 (38.4%) had bulbar nerve

involvement and 2 (7.7%) had facial nerve involvement. At enrolment, autonomic dysfunction was

observed in 11 (42.3%) patients. During the enrollment, 19 (73%) patients had MRC sum score

between 0-20, 3 (11.5 %) patients had MRC sum score between 21-40, 4 (15.5%) patients had

MRC sum score between 41-60. At the time of enrollment, 12 (46.1 %) patients were chair bound

or bed ridden (GBS disability score of 4), 10 (38.5 %) patients were admitted in ICU for ventilation

support (GBS disability score of 5), and 4 (15.4 %) 3 patients unable to walk independently (GBS

disability score). Electromyography (EMG) test was performed on 23 patients with GBS where

the axonal subtype (AMAN, 69.2%) prevailed, followed by the demyelinating subtype (AIDP,

19.2%) and 3 (11.6%) patients were unclassified.

 P a g e | 29

Table 3.1 Demographic features and antecedent illness of GBS patients (N= 26)

Characteristics Number of patients (%)

Sex Male 18 (69.2 %)
Female 8 (30.8 %)
Median Age; IQR 33.5; 19-40 years
Antecedent illness (Total) 15 (57.6 %)
Gastroenteritis 5 (19.2 %)
Common cold 5 (19.2 %)
RTI 3 (11.5 %)
Other 4 (15.3 %)
None 8 (30.7 %)
Cranial Nerve Involvement 11 (42.3 %)
Bulbar 10 (38.4 %)
Facial 2 (7.7 %)
Autonomic dysfunction 11 (42.3 %)
41-60 4 (15.5%)
MRC Sum Score 21-40 3 (11.5%)
(at enrolment) 0-20 19 (73%)
Pain (at enrolment) 19 (73 %)
0 or 1 (healthy or minor syndrome) 0
GBS disability score 2 (capable to walk independently) 0
(at enrolment) 3 (unable to walk independently) 4 (15.4 %)
4 (chair bound or bed ridden) 12 (46.1 %)
5 (ventilation) 10 (38.5 %)
Electrophysiology
Classification AMAN 18 (69.2 %)
(N=24) AIDP 5 (19.2 %)
Unclassified 3 (11.6 %)
 P a g e | 30

3.2 Prognosis of GBS patients

At the time of enrolment, disease severity was high in majority patients. The GBS DS was
used to determine the severity of the disease. During follow-up at 2 weeks, the severity seems
moderate with 46.2 % patients having GBS disability score of 4. During follow-up at 4 weeks, the
severity score did not have any significant change compared to the first follow-up. During the third
follow-up at 13 weeks, the severity score in patients dropped, with around 23% of patients having
a GBS DS 0 or 1 and 15.4% has a GBS DS 2.
GBS disability score (GBS-DS)

0 or 1

0 5 10 15 20 25 30 35 40
Number of patients
GBS-DS-Enrolment GBS-DS-F1 GBS-DS-F2 GBS-DS-F3

Figure 3.1: GBS disability score (DS) at different time points (F1 means first follow-up at 2 weeks,
F2 means second follow-up at 4 weeks and F3 means third follow-up at 13 weeks)
 P a g e | 31

3.3 Illustrative Analysis:

3.3.1 Determination of CD4+ and CD8+ T cells percentage in patients with GBS and healthy

controls:

The baseline percentages of CD4+ and CD8+ T Lymphocytes in 26 patients with GBS and

10 healthy controls were assessed. T cell distribution throughout the bloodstream of patients with

GBS and HC is stated in percentage of PBMC. A graphical presentation of this variation is given

in the figure 3.2.

Figure 3.2: Comparative study of CD4+ and CD8+ T cells in patients and healthy controls. Values
are presented as box plot diagram with median, upper, and lower quartiles defining the maximum
and minimum value. The mean value is expressed as mean ± standard deviation.

The percentage of CD4+ T cells was lower in patients in comparison to healthy control at the onset

of disease. The mean value of CD4+ T cells in patients with GBS was 27.72 ± 4.283 (mean ± SD)

and in HC was 30.34 ± 4.116 (mean ± SD). The P value that defines there was no significance of

the result and the value, P= 0.3537. The percentage of CD8+ T cells was higher in patients in
 P a g e | 32

comparison to healthy control at the onset of disease. The mean value of CD8+ T cells in patients

with GBS was 22.50±10.47 (mean ± SD) and in HC was 19.71±8.702 (mean ± SD). The P value

that defines there was no significance of the result and the value, P=0.4802.

3.3.2 Correlation of CD4+ and CD8+ T cell percentages with disease progression:

Mean number of CD4+ T cell percentage was lower in follow-up patients from the onset of

disease. At the time of enrolment the mean percentage of CD4+ T cells were observed 27.71 ±

4.006 (mean ± SD) and the coefficient of variation was 14.46%. At two weeks follow-up, the mean

percentage of CD4+ T cells were observed 29.68 ± 4.700 (mean ± SD) and the coefficient of

variation was 15.83%. At four weeks follow-up, the mean percentage of CD4+ T cells were

observed 24.77 ± 2.665 (mean ± SD) and the coefficient of variation was 10.76%. During the

thirteen weeks follow-up, the mean percentage of CD4+ T cells were observed 25.23 ± 2.892 (mean

± SD) and the coefficient of variation was 11.46%.

 P a g e | 33

Figure 3.3: The percentage of CD4+ and CD8+ T cells in patients at different time interval. Values
are presented in mean/ median & error plot where the dots signifies the mean values.

The mean number of CD8+ T cell percentage showed gradual increased from onset of the disease

to thirteen weeks follow-up. At the time of enrolment the mean percentage of CD8+ T cells were

observed 22.50 ± 10.47 (mean ± SD) and the coefficient of variation was 46.51%. At two weeks

follow-up, the mean percentage of CD8+ T cells were observed 20.88 ± 7.032 (mean ± SD) and

the coefficient of variation was 33.67%. At four weeks follow-up, the mean percentage of CD8+

T cells were observed 25.49 ± 7.124 (mean ± SD) and the coefficient of variation was 27.94%.

During the thirteen weeks follow-up, the mean percentage of CD8+ T cells were observed 27.49 ±

7.781 (mean ± SD) and the coefficient of variation was 28.31%.

 P a g e | 34

3.3.3 Association of CD4+ and CD8+ T cells with disease severity:

The GBS-DS of were compared with the CD8+ T cells percentages in figure 3.4. In fig. A,

it can be seen that there is no correlation between the CD4+ T cells percentage with the GBS-DS

ranging from 0 to 6. The p value is 0.0782 which shows no significance. The r value is 0.3323

which indicates no significant correlation between this two. In fig B, it can be seen that there is a

positive correlation between the CD8+ T cells percentage with the GBS disability score ranging

from 0 to 6. The p value is 0.7093 which shows no significance. The r value is 0.05853 which

indicates the positive relation between this two.

Figure 3.4: Association of CD4+ and CD8+ T cells with GBS disease severity. Values are
presented in XY plot with each bar representing the mean values.

The curve line passing through the bar with the value of CD4+ T cell percentages and GBS

disability score was seen quite flat with the meaning of no significant trend between the two

variables. On the other hand, the curve line passing through the bar with the value of CD8+ T cell

percentages and GBS disability score was seen to be a straight line with a significant increase of
 P a g e | 35

CD8+ T cell percentages with the GBS disability score meaning a significant correlation between

these two variables.

3.3.4 A comparative study between percentages of CD4+ and CD8+ T cells and GBS subtypes:
Mean number of CD4+ T cell percentage varied among AMAN and AIDP GBS subtypes
which is showed in fig 3.11. In patients with AMAN GBS subtype, the mean number of CD4+ T
cell percentage was 26.51 ± 3.748 (mean ± SD). Comparatively in patients with AIDP GBS
subtypes, the mean number of CD8+ T cell percentage was higher 28.64 ± 4.756 (mean ± SD). The
p value was 0.1850 which showed no significance of the result.

Figure 3.5: Comparative analysis of CD4+ and CD8+ T cell percentages in AMAN and AIDP GBS
subtypes. Values are presented in mean/ median % error plot containing the mean percentages with
standard deviations

Mean number of CD8+ T cell percentages varied among AMAN and AIDP GBS subtypes
is showed in figure 3.10. In patients with AMAN GBS subtype, the mean number of CD8+ T cell
percentage was 25.87 ± 10.32 (mean ± SD). Comparatively in patients with AIDP GBS subtypes,
the mean number of CD8+ T cell percentage was 21.77 ± 5.715 (mean ± SD). The p value was
0.1194 which showed no significance of the result.
 P a g e | 36

3.3.5 Comparative study of CD8+ and CD4+ T cells ratio in patients with GBS:

Proportion of CD4+ and CD8+ T cell in patients were analyzed where the percentage of
CD4+ T cells outnumbered CD8+ T cells in all our cases. p value was 0.0004 (p<0.05) showing
that this result is significantly correlated. The geometric mean of ratios (CD4/CD8) is 1.290 with
the standard deviation of log (ratios) 0.1692. The pairing shows significantly effective as the
correlation coefficient value, r= 0.4152 and p value is 0.0059.

Figure 3.6: Comparative ratio analysis of CD4+ and CD8+ T cells in patients with GBS. Values
are presented in column graph defining the mean ratio with minimum and maximum values.
 P a g e | 37

3.3.6 Association between CD4+ and CD8+ T cells and serum albumin level:

We have measured baseline serum albumin of 26 patients. The mean value of serum
albumin level was 37.94 g/liter which was in normal range (35-55 g/liter). Correlation between
serum albumin level and CD4+ and CD8+ T cells percentages were shown in the figure 3.7.

Figure 3.7: relative profiling of CD4+ and CD8+ T cell percentages with serum albumin level
during enrolment. Values are presented in XY column graph and the line represents the change
pattern between the two variables.

There was no significant correlation between serum albumin level and CD4+ T cells
percentage, which was statistically proven with one sample t-test and Wilcoxon test (Fig 3.7A).
We have found a trend of increasing serum albumin level among patients with higher percentages
of CD8+ T cells. Serum albumin level was positively correlated with CD8+ T cells (Fig 3.7A)
proven with one sample t-test and Wilcoxon test (p value was <0.0001).
 P a g e | 38

Discussion
 P a g e | 39

Discussion:
Humoural and cellular immune mechanisms are involved in the pathogenesis of GBS.
Previously, several studies have described the function of humoural factors. The present study was
performed on patients with GBS to investigate the cytotoxic effect of cellular immune responses
during the disease course. An increasing trend of CD8+ T cell was found in patients with GBS than
the control population. Elevated percentage of CD8+ T cell was positively correlated with
increasing disease severity, axonal GBS and elevated level of serum albumin. The percentage of
CD4+ T cells was showed decreasing trend in patients with GBS and had no correlation with the
disease severity and serum albumin level. In addition, our study explored a noteworthy correlation
of CD4+/CD8+ T cell ratio which might serve as an important insight in terms of finding the role
of T cells in GBS disease development and progression.
Increased levels of activated T cells in patients during the initial stages of the disease,
besides increased T cell activation indicators in the serum imply that T cells are involved in GBS
pathogenesis [97, 98]. Nerve biopsies of GBS patients also shown presence of CD4+ and CD8+ T
cell infiltrates [99]. In comparison to healthy controls, we noticed no substantial changes in CD8+
T cell percentages throughout the disease progression. Our findings are similar with that of Sindern
et al., who found no distinct change in the proportion of CD8+ T cells in patients and healthy
controls [99]. It was also proposed that in GBS patients, the form of the antecedent infection may
influence the aberrant alterations of the CD8+ T cell. In our study sample, we could not include
serological tests in determining the preceding infectious agents and so their association with CD4+
and CD8+ T cells.
Immunological mechanisms differ amongst GBS subtypes; for example, the axonal variant
is greater in frequency of positive anti-GM1 antibody compared to demyelinating variant [100]. In
the demyelinating form of the disease, infiltrating lymphocytes and demyelination mediated by
macrophage were identified, whereas macrophages were shown throughout the periaxonal zone in
the axonal form [101]. Our study found no substantial changes in CD8+ T cell percentages among
two major subtypes of GBS. As our study population was small, so a definitive conclusion about
the differences of this specific T cell in major GBS subtypes needs further extended study with
large population.
 P a g e | 40

Several studies indicated that demyelination is caused by immune mechanisms involving

cellular and humoral immunity. Several studies of the frequencies of circulatory T lymphocyte
types in GBS patients have been done, but the results have been inconsistent, indicating variations
from the typical pattern. In our study patients had higher CD8+ T cells percentage compared to
healthy control in the entry phase. During the enrolment of our study, however, we found no
change in CD4+ T cells percentage in patients compared to healthy control. The higher percentage
of CD8+ T cells may have role with the cytotoxicity and clearance of neural cells as macrophage
and natural killer cells become active. During the study, the mean number of CD8+ T cell
percentage showed gradual increased from entry to thirteen weeks follow-up However, the mean
number of CD4+ T cell percentage showed gradual decrease from entry to thirteen weeks follow-
up. We found a link connecting the CD8+ T cell percentage with GBS disability score as r value
indicates the positive relation between this two but no correlation between CD4+ T cell percentage
and GBS disability score. During the study, the mean CD8+ T cell percentage varied among the
healthy control, treated and non-treated patients.
Previous research stated the drop of CD4+ T cells in GBS patients while the rose of CD8+
T cells, especially during the development stage [101]. Other research, on the other hand, found
that in GBS patients, the CD4+ T cells percentages increased while the CD8+ T cells percentages
decreased [101]. Related to first study, we have observed that percentage of CD4+ T cells in GBS
patients dropped over time, whereas the percentage of CD8+ T cells rose. The significant change
in the ratio of these two T cell subtypes might have a role in disease pathogenesis and correlated
with disease progression. So further study related to the percentage of these two subsets and their
ratio analysis in a relatively large cohort can conclude the potential role and abnormalities related
to GBS development.
Studies stated hypo-albuminemia following IVIG treatment in patients with GBS linked to
an extra severity and even worst result [102]. One goal of this study was to evaluate serum albumin
levels and their involvement as a biomarker in GBS patients and if they have any relationship with
percentages of CD4+ and CD8+ T cells. We observed no substantial escalation or reduction in
serum albumin level among the patients with CD4+ T cell percentages and a trend following
escalation in serum albumin level among the patients along with the percentages of CD8+ T cells
were seen. The p value was <0.0001 which showed significance of both of the result. Albumin
level in serum explored to check for biomarker with prognostic effects in a variety of disease
 P a g e | 41

disorders, in addition to a possible relationship with immune cells and IVIg therapies. [103].
Furthermore, in the situation of acute phase, a low albumin level in the serum can be a powerful
risk factor for poor outcome [104, 105]. Our study findings can now be examined and explored in
further prospective studies, preferably with a significant number of study individuals.
In conclusion, the significance of CD4+ & CD8+ T lymphocytes in etiology and GBS
development is unclear, understanding their involvement in disease pathogenesis and progression
is critical yet important. In future studies with a significant number of patients and healthy controls
can confirm the trend observed in our study and explain underlying role with this T cell subset and
association in disease development and progression. Though there were some shortcomings and
some results were not significant due to small sample number, a further transparent and extensive
investigation of these respective cytokines of cytotoxic T cell subsets and also CD8+ T cells need
to be investigated to explain the functional role of the cytotoxic T cell. The samples for inspection
of the respective cytokines have preserved. The whole study may evolve a thread that will persuade
us to demonstrate the potential biomarkers and potent therapeutic targets in GBS for early
prognosis of the disease and necessity of therapeutic administration which will also reduce the
unnecessary burden of expensive treatment.

Future direction:

New evidences and findings may include any abnormality in CD4+ and CD8+ T cell
proportions between subtypes of GBS and can figure out whether normalization of this subset
takes place after immunotherapy given to patients with GBS. Determining CD4+ & CD8+ T cell
proportions might be helpful in distinguishing the GBS subtypes and serve as possible option to
monitor disease progression and therapeutic treatment during the course of the disease. So yet,
there haven't been enough studies in experimental animals of GBS or infections to draw any
conclusions. Animal models may contribute to explore underlying pathophysiology of GBS and
interaction between the cellular and humoral components that lead to GBS. Furthermore, these
animal studies will aid in our understanding of the effects of various cytokines as well as their
cumulative impact on immune system regulation and disease. Additionally, any imbalance
between T cell responses can be experimented and probable therapeutics can be aimed in restoring
the imbalance.
P a g e | 42
 P a g e | 43

References
 P a g e | 44

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Appendices
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Glossary

Abbreviated Term Full Form

AIDP Acute inflammatory demyelinating polyneuropathy
AMAN Acute motor axonal neuropathy
AMSAN Acute motor sensory axonal neuropathy
CIDP Chronic inflammatory demyelinating polyneuropathy
APCs Antigen presenting cells
HC Healthy control
MAC Membrane attack complex
C. jejuni Campylobacter jejuni
CSF Cerebrospinal fluid
i.e That is
ELISA Enzyme linked immunosorbent assay
FACS Fluorescent activated cell sorting
GBS Guillain-barré syndrome
GBS-DS GBS disability score
GR Glucocorticoid Receptor
IVIG Intravenous immunoglobulin
MFS Miller fisher syndrome
MRCSM Medical research council sum score
NINDS National institute of Neurological Disorders and Stroke
PBS Phosphate buffered saline
LOS Lipo-oligosaccharide
IgG Immunoglobulin G
MS Multiple Sclerosis
µl Microliter
 P a g e | 55

A. Common Laboratory Apparatus

Name Description Source

FACS Tubes 5 ml Polystyrene Round-Bottom BD Falcon
Tube
Eppendorf Tubes® Safe-lock tubes 1.5 ml Eppendorf AG, 22331
Hamburg, Germany
BD Falcon™ 15/50.0 ml high-clarity BD, One Bectone Drive,
polypropylene conical tube Franklin Lakes, NJ 07417
USA
Petri Dishes Sterilin Sterilin limited, U.K
Electric pipette Accu-jet® pro BRAND
Centrifuge Tabletop Refrigerated Centrifuge ESCO
Micro-pipettes FINNPIPETTE Thermo SCIENTIFIC
 P a g e | 56

B. Reagents
Number Reagent Source

1 D-PBS Gibco

2 Invitrogen™ Ficoll™ Plaque GE Healthcare

3 RPMI/ RPMI 1640 w/o L-Glutamine Gibco

4 PEST Gibco

5 L-Glutamine Gibco

6 Na-Pyruvate Gibco

7 Amphotericine B Gibco

8 HI-FBS Gibco

9 DMSO Sigma

10 Trypen Blue Gibco

 P a g e | 57

C. Buffers and Reagent solutions

Culture Media:
Reagents Amount
RPMI/ RPMI 1640 w/o L-Glutamine 87.4 ml
PEST 500 µl/0.5 ml
L-glutamine 1.0 ml
Na-pyruvate 1.0 ml
Amphotericine B 100 µl/0.1 ml
HI-FBS 10.0 ml

Freezing Media:
Reagents Amount
HI-FBS 45 ml
DMSO (Sigma Cat #D2650) 5 ml

The heat inactivated FBS (HI-FBS) was filtered and 45ml was taken into a sterile 50ml Falcon
tube. 5ml of DMSO (Sigma Cat #D2650) was added to make total volume 50ml. Then the
freezing media was stored at -20°C for regular uses.