REVIEW

Systemically Circulating Colitogenic Memory CD4+ T Cells
May Be an Ideal Target for the Treatment of
Inflammatory Bowel Diseases
Takanori Kanai,1 Mamoru Watanabe2 and Toshifumi Hibi1

Division of Gastroenterology and Hepatology, Department of Internal Medicine,
School of Medicine, Keio University, Tokyo, Japan2
Department of Gastroenterology and Hepatology, Graduate School,
Tokyo Medical and Dental University, Tokyo, Japan

1(Received for publication on February 12, 2009)(Revised for publication on April 17, 2009)(Accepted for publication on May 21, 2009)

Abstract
Inflammatory bowel diseases (IBD) are thought to be caused by a complex interaction of genetic, immunological, and environmental factors. Why is it that once an IBD develops it lasts along time? Considering this simple question, we propose that coliotogenic memory CD4+T cells
that remember the prototype of the disease in each patient are formed in IBD at the onset,
and, perceiving them as “benign T-cell leukemia”-like lifelong memory CD4+T cells that hematogenously spread throughout the body, we thus propose that systemic circulating colitogenic
memory CD4+T cells would be an ideal target for the treatment of IBD. Accordingly, selective
depletion of colitogenic memory CD4+T cells by leukocytapheresis and blockade of circulationof colitogenic memory CD4+T cells by a newly developed immunosuppressant, FTY720, may
be associated with dramatic efficacy and a marked reduction of inflammatory cytokines produced by activated leucocytes. We here describe the immunological pathogenesis focusing on
the generation of circulating colitogenic memory CD4+T cells and the possible logics of leukocytapheresis and FTY720 for the treatment of IBD. (Keio J Med 58 (4) : 203－209, December 2009)

Keywords: inflammatory bowel disease, colitogenic memory CD4+ T cells, leukocytapheresis, FTY720
Introduction
Inflammatory bowel diseases (IBD) are caused bychronic inflammatory responses in the gut wall, commonly take persistent courses, but in some patients relapse after remissions.1-5 Despite the advent of an age
when “malignant” leukemia is cured by bone marrow(BM) transplantation, “benign” IBD that are mediated by“benign” immune cells are still intractable diseases thatpersist throughout life. For example, even when the local
intestinal areas of inflammation are adequately resected
in Crohn’s disease (CD), it relapses in another part of thedigestive tract with a similar feature of the previous disease, such as longitudinal ulcers and “cobblestone” ap-

pearance. It is also known that extra-intestinal complications, such as primary sclerosing cholangitis (PSC) andpyoderma gangrenosum, develop in some cases of IBD
after surgical remission, and that ileal pouchitis develops
after total colectomy in patients with ulcerative colitis(UC).6, 7 These findings provided clues that led us to therecent hypothesis that colitogenic memory CD4+T cells,
which are capable of reproducing colitis, continuously
circulate throughout the body, not just the intestine, andthat they actively circulate in peripheral blood and are
involved in the intractability and persistence of IBD.
Moreover, we demonstrated the paradox that eventhough IL-7 is essential as a factor for the maintenanceand proliferation of colitogenic memory CD4+T cells,8-11

Reprint requests to: Takanori Kanai, MD, PhD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, Keio
University, 35 Shinanomachi, Shinjyuku, Tokyo 160-8582, Japan, Tel: +81-3-3353-1211, Fax: +81-3-3341-3631, E-mail: takagast@sc.itc.keio.ac.jp
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Kanai T, et al: Strategy for the Treatment of IBD

204

however, intestinal IL-7 decreases in tandem with thedecrease in goblet cells in accordance with the severityof intestinal inflammation in IBD.1 2 Thus, we demonstrated that other sites outside inflamed intestine act as
reservoirs of disease-specific colitogenic memory CD4+T
cells in chronic colitis.1 2 In this review article we describe the persistence of circulating colitogenic memory
CD4+T cells that are able to permanently maintain colitis
and the mechanism of the maintenance, and also we provide an outline of the immunological assessment of thelogics of leukocytoapheresis and FTY720 treatments thatblock the circulation of colitogenic memory CD4+T cells
for IBD.
Are IBDs Autoimmune Diseases?
In understanding IBD as chronic, “autoimmune-like”,
diseases, an important point is the presence of autoantibodies. Unlike systemic lupus erythematosus (SLE) andmyasthenia gravis, autoantibodies involved in UC and
CD have not been characterized sufficiently to reveal thepathogenesis of IBD, although some markers, such as
antineutophil cytoplasmic antibodies (ANCA) and antiSaccharomyces cerevisiae antibodies (ASCA) are used
for the diagnostic tool.13 The autoantibodies in this caserefer to the target molecules that are specific to mucosal
compartments, such as epithelial cells and interstitialcells, in the intestines. However, these must be proved
through various approaches, such as (a) the identificationof autoantibody molecules and epitopes, (b) the presenceof autoantibodies against self-antigens, (c) the development of animal models for conditions resembling human
IBD associated with hyperimmunization using purified
antigens, (d) the isolation of T-cell receptors (TCR) reacting to autoimmune epitopes, and (e) the experimental
development of conditions resembling human IBD intransgenic mice with the TCR genes. However, the pathology of IBD assuming an autoimmune mechanism isfurther complicated by the presence of intestinal bacteria. Several animal models for chronic colitis were established in the 1990s, particularly using gene-manipulated
mice. Of note, these mice models, such as IL-10–/– andTCRα–/– mice, require the presence of indigenous bacterial flora, and do not develop morbidity in a germ-freeenvironment.3 Also, it has been reported that antibiotics
are effective in some patients with IBD.1 4 These facts
suggest the possibility that the autoantigens involved in
the autoimmune mechanism for IBD may be the antigens
derived from symbiotic intestinal bacteria, rather than
the antigens inherent to the human body. In view of thehistory of symbiosis starting before the evolution of anthropoid apes, intestinal bacterial flora seems to be a partof self with respect to the immune response of the humanbody.

How Are Colitogenic CD4+ Effector
T Cells Generated?
As mentioned before, it is thought that aberrant reactivity of CD4+ helper T cells to antigens derived from intestinal bacteria is important in the immune mechanismof chronic intestinal inflammation.3, 4 The immune response in the actual living body consists of the following
processes. First, precursor T cells move from BM into
the thymus. After entering the thymus, precursor T cells
undergo selective removal of the cells that are reactive toself-antigens (negative selection), achieving eliminationof autoreactivity and induction of self-tolerance (central
tolerance). However, negative selection is not perfect. A
small number of autoreactive clones move out of the thymus, mixed in the large majority of clones that recognize
non-self antigens possibly including antigens derived
from intestinal bacteria. The clones that recognize nonself-antigens extensively proliferate in the thymus (positive selection), and move out of the thymus in the formof mature naive T cells. Of note, there are special clones
called CD4+CD25+Foxp3+ regulatory T cells (TR),1 5-1 7
which probably recognize self-antigens in the thymus
without being eliminated and differentiate to memorytype (CD44high) CD4+T cells within the thymus. Thesecells seem to develop following a completely different
pathway than that of the naive CD4+T cells that can recognize self and non-self. The CD4 +CD25 +Foxp3 +T R
cells normally exert suppressive control over autoreactive clones in the periphery and monitor the aberrant activity of autoreactive clones at the peripheral level (peripheral tolerance).
Once moved into the periphery, the naive CD4+T cells
that recognize intestinal bacterial antigens continuously
monitor the invasion of intestinal bacterial antigens, patrolling the blood and dropping in mesenteric lymph
nodes (MLN). On the other hand, foreign antigens arecontinuously taken up by professional antigen-presenting
cells (APC), dendritic cells (DC), in intestinal lymphoidtissues, Peyer’s patches, isolated lymph follicles, andprobably lamina propria, and are processed by DC occurring there. 1 8- 2 1 In this process, importantly, DC
themselves remain to be inactivated in the absence of local inflammation (in case of uptaking normal commesal
bacteria without inflammation) and migrate to regional
MLN as immature DC.22 In contrast, if the intestinal inflammation occurs in form of activation of epithelial
cells and resident NK cells and macrophages by innate
immune system through Toll-like receptors (TLR) and/or
other molecules (e.g. NOD2/CARD15) (in case of uptaking commesal or pathologic bacteria with inflammation),
DC themselves are activated, and migrate to regional
MLN as mature and activated DC. When the naive
CD4+T cells recognizing intestinal bacterial antigens, incidentally encounter the activated DC undergoing antigen presentation, the naive CD4+T cells are activatedKeio J Med 2009; 58 (4): 203－209

quickly with the help of various proinflammatory cytokines and TLR signaling via activated APC, and differentiate to colitogenic effector CD4+T cells. In this process, the effector CD4+T cells acquire the expression of
gut-homing receptors (e.g. CCR9 and integrin α4ß7)needed for the movement into the intestinal mucosa.23, 24
It is now well known that the acquisition of these guthoming receptors can be specifically performed by mucosal DC, but not by DC in other sites.23, 24 As a result,the effector CD4+T cells activated in MLN re-enter the
blood flow via the thoracic duct and eventually enter theintestinal mucosa, where MAdCAM-1, the ligand for integrin α4ß7, and CCL19/CCL21, the chemokines for
CCR9, are expressed. It seems that both gut-homing receptors are critically involved in homing to small intestine in steady state of intestine, but still unknown whether these molecules are also important for gut-homing in
inflammatory condition. Also, integrin α4ß7, but not
CCR9, seems to be involved in homing to large intestine.
This area is now extensively under investigation for understanding the differences of gut-homing between small
and large intestines.
The lymphocytes in the intestinal mucosa have a special characteristic.18 Unlike the cells in peripheral bloodand lymph nodes, these cells consist of an overwhelmingly large number of memory T cells, express characteristic homing receptors, which are not expressed outside
the intestine, and are poorly proliferative. Strangely,
most lymphocytes in the mucosa do not return to theblood flow and are believed to undergo apoptosis in themucosa as is a well-known case of HIV-infected CD4+T
cells.25 However, the possibility remains that a small minority of intramucosal lymphocytes may return to theblood flow via an unknown mechanism and contribute topersistence of intestinal inflammation.
Are Colitogenic CD4+T Cells Effector or
Memory T Cells?
When considering the basic treatment of IBD, we areconfronted with the very important question of whether
colitogenic CD4+T cells are simple short-lived effector T
cells or long-lived memory T cells.26, 27 If they are effector T cells, even if a large number of effector CD4+T
cells were to cause mucosal damage, since the cells areshort-lived, all that would be needed is a treatment strategy that soundly attacked and destroyed them. However,
if some of the colitogenic CD4+T cells have developed
to memory T cells, cure becomes a knotty problem. The
reason being that memory CD4+T cells that remember
the prototype of diseases are retained hidden for long periods, and are capable of immediately supplying large
numbers of new effector CD4+T cells for each recurrence
from colitogenic memory CD4+T cells. That is because
an immune mechanism exists whereby, unlike the pathway from naive to effector CD4+T cells, memory CD4+T

205

cells that have acquired antigen specificity are easily reactivated by antigens and also by bystander proinflammatory cytokines, without requiring opportunities to acquire antigen-specificity, and they produce large numberof effector CD4+T cells. Expressed in a different manner,
if we consider the reason why IBD are intractable andpersist lifelong, we eventually arrive at the hypothesisthat colitogenic CD4+T cells in IBD are memory CD4+T
cells acting as “memory-stem cells”.
We investigated the above question by using the
CD4+CD45RBhighT cell transfer model of chronic colitis. 2 8, 2 9 The model is created by transferring naive
CD4+CD45RBhigh T cells from spleen or MLN into SCID
or RAG-deficient (RAG-1–/– or RAG-2–/–) mice. Whenhoused in a SPF-environment, the mice develop weightloss and diarrhea approximately 3 to 4 wk after transfer,and histologically they develop chronic colitis accompanied by marked CD4+T cell infiltration along with activated macrophages and DC.2 9 The important advantageof this model is that it is a transfer model in which there
is no supply of new naive CD4+T cells, and the fact thatthe repeated immune responses that naive CD4+T cells
from the thymus give rise to in healthy mice are eliminated makes it possible to track the immune responseover time after the transfer.3 0 As mentioned before, because this model also does not develop in germ-free recipients like other models of chronic colitis, enteric bacterial antigens appear to be the responsible antigens thatcause the colitis.3, 4 Although there is critical criticism
against this adoptive transfer model of IBD, which istransferred into “lymphopenic” animals,3 1 we hypothesize that the lymphopenic condition is exactly one ofcauses for the development of IBD. Indeed, the reason
that the incidence of IBD is now extremely increasing in
the developed countries recent years may be due to thehygiene environment, which easily induces the lymphopenic condition.3 2 As another example, it has been reported that autoimmune diabetes model of NOD micehas originally a lymphopenic condition, and thus develop
the disease.33
By 4 to 6 wk after transfer of CD4+CD45RBhigh cells,
CD4+T cells in inflamed mucosa had markedly proliferated, and FACS analysis showed that they wereCD44 highCD62L -IL-7Rα high cells, so-called effectormemory-type memory T cells (TEM).11 Again, such characteristics of our colitis model raise another importantquestion whether the colitogenic CD4+CD44highCD62LT cells can be defined as TEM cells rather than just effector T cells in the persistent presence of intestinal bacteria. In general, immunological memory has evolved to
warrant rapid and efficient elimination of microbial
agents that repeatedly enter the organism.2 6 As a rule,immunological memory builds up, following successful
elimination from the organism. In contrast, persistent ofAg, like in chronic infectious diseases, often leads to theexhaustion of the immune response.3 4, 3 5 In immune re-206

sponses in mice with chronic colitis, the target commensal bacteria are never eliminated, but persist throughout
life. Thus, would the colitogenic CD4 + T cells in
CD4 +CD45RB high-transferred colitis model build up
memory against antigens? Against this issue, we firstfound that the colitogenic CD4+T cells highly expressedboth CD44 and IL-7Rα. It is generally thought that highly expressed IL-7Rα is one of accepted memory, but noteffector, T cell markers. Second, memory, but not effector, CD4+T cells are critically controlled the survival by
IL-7.36, 37 Consistent with this, we found that the colitogenic LP CD4+T cells were markedly decreased in IL-7deficient RAG-1–/– (IL-7–/– x RAG-1–/–) mice transferredwith the colitogenic CD4+T cells as compared with thetransferred control IL-7-sufficient RAG-1 –/– (IL-7+/+x
RAG-1–/–) mice, indicating that the maintenance of colitogenic CD4+T cells is dependent on IL-7, and thus they
are memory T cells rather than effector T cells. Furthermore, to assess the longevity of the colitogenic CD4+
cells, we performed sequential transfers of LP CD4 +cells originally obtained from colitic CD4+CD45RBhighcell-transferred SCID mice into new SCID mice, andshowed that SCID mice transferred with colitic LP CD4+
cells stably developed colitis until at least the sixth transfer, indicating that colitogenic LP CD4+T cells would bequite long-lived memory CD4+T cells.38
Is Intestinal IL-7 Essential for the Maintenance ofColitogenic Memory CD4+T Cells?
Since several groups including us have previously reported that IL-7 is produced by intestinal epithelia, especially by goblet cells,3 9, 4 0 we obviously hypothesizedthat intestinal IL-7 should be a key molecule for the development and maintenance of chronic colitis. However,
it is widely known from pathological diagnosis that IBD
are characterized by a decrease in goblet cells when they
become chronic (‘goblet depletion’). 4 1 Actually, we
showed in our hands that here was also a marked decrease of goblet cells at the sites of the chronic colitis lesions in the CD4+CD45RBhigh T cell transfer model, andwe found that there was a correlated decrease in IL-7
production by the epithelial cells.12 We thus reconsidered
and hypothesized that intestinal IL-7 is not required tosimply maintain chronic colitis, and that colitogenic
memory CD4+T cells are maintained by IL-7 outside theintestine. To solve this, we performed parabiosis surgery
that connected colitic RAG-2–/– mice and new IL-7–/– x
RAG-1–/– mice. Even though the intestinal epithelial
cells of the IL-7–/– x RAG-1–/–host mice do not produce
IL-7, as a result of the shared hemodynamics they can beviewed as mice that produce IL-7 outside the intestine.
Surprisingly, despite the deficiency of intestinal IL-7, severe colitis also developed in the IL-7–/– x RAG-1–/–hostside, the same as in the control IL-7+/+ x RAG-1–/–hostmice.12 These findings demonstrated that intestinal IL-7

Kanai T, et al: Strategy for the Treatment of IBD

is actually not essential for the maintenance of colitogenic memory CD4+T cells in mice with chronic colitis.
Do Colitogenic Memory CD4+T Cells Continuously
Circulate throughout the Body?
In the previous section we showed that IL-7 is essential to maintain colitogenic memory CD4+T cells, butthat IL-7 at the intestine is not essential. How then do thecolitogenic memory CD4+T cells gain access to IL-7 outside the intestine? There would be no problem if the soluble IL-7 simply arrived in the intestine via the circulation, but, in reality, that is hard to imagine, because homeostatic cytokines, such as IL-7 and IL-15, are thoughtto act by cross talk with lymphocytes and in the networks of certain microenvironments.3 6, 3 7 Instead, it isimportant to bear in mind that the memory system ofcolitogenic memory CD4+T cells is critically different
from that of neurons that have true memory activity, andwhereas neurons extend roots in one place, memory
CD4+T cells are mobile and capable of moving throughout the body. We therefore hypothesized that colitogenic
memory CD4+T cells constantly circulate throughout the
body seeking out the IL-7 that is essential in order tomaintain chronic colitis.
Indeed, we showed not only marked infiltration of theinflamed large intestine by colitogenic CD4+T cells in
the established chronic colitis, but the constant presenceof CD4+T cells in their peripheral blood and thoracic
duct (Fig. 1). 4 2 In other words, colitogenic memory
CD4+T cells were found to be capable of constantly circulating in the blood and thoracic duct to maintain thecolitis even after the establishment of chronic colitis. We
therefore isolated the CD4+T cells from the peripheral
blood of the colitic SCID mice, and performed adoptive
retransfer into new SCID mice. Expectedly, we foundthat similar and equivalent chronic colitis developed as aresult of the transfer of the colitic, but not normal, peripheral blood CD4+T cells.4 2 Actually, however, it appears impossible to demonstrate a similar phenomenon
in IBD in human, because even though they may be IBD
patients, CD4+T cells that recognize a much greater diversity of antigens are present in human peripheral bloodthan in this simplistic model of colitis in mice.
Logics of the Blocking the Circulation of Colitogenic
Memory CD4+T Cells for the Treatment of IBD
As stated above, once CD patients develop manifestations of the disease, they endure it lifelong through repeated remissions and relapses. The variety of promisingtreatments that have become available have never been
curative, and CD relapses again even after all of the sitesthat have been identified as lesions, including the regional lymph nodes, have been resected. If we assume thatpatients have such a pathological condition, treatments

Keio J Med 2009; 58 (4): 203－209

207

Fig. 1 Systemic circulation of colitogenic memory CD4+ T cells in IBD and their blockade strategies as the treatment of IBD.
Colitogenic memory CD4+ T cells continuously circulate in the body to migrate to IL-7-producing sites, such as BM, skin and liver.
Leukocytapheresis may remove systemic circulating colitogenic memory CD4+ T cells from the peripheral blood. FTY720 may inhibit
the egress of colitogenic memory CD4+ T cells from regional LN, resulting in the blockade of their systemic circulation. Administrationof anti-integrin α4 mAb (natalizumab) may suppress the migration of colitogenic memory CD4+ T cells to inflamed mucosa. MLN;
mesenteric lymph node, BM; bone marrow.

that target intestinal inflammation by local surgery may
be nothing more than treatment methods expected tohave temporary efficacy. We therefore like to devise atreatment strategy that would suppress the systemic circulation of colitogenic memory CD4+T cells.
In recent years leukocytapheresis has been aggressively used in Japan as a means of treatment, primarily for
UC and thereafter CD in the active phase.4 3 How does
leukocytapheresis contribute to suppressing the pathology in the hemodynamics of colitogenic memory CD4+T
cells described in the previous section? The principal
methods of leukapheresis used clinically are granulocyte
and monocyte adsorption apheresis (GCAP; Adacolumn),44 which chiefly removes granulocytes and monocytes, and leukocytapheresis (LCAP; Cellsorba),45 whichremoves lymphocytes, monocytes and granulocytes as awhole. We recently reported that LCAP therapy selectively removed CD4+CD45RO+CD62L-TEMcells, whichhave a similar phenotype of murine colitogenic memory
CD4+TEM cells without affecting CD4+CD25+Foxp3+ TR
cells, which suppress immunity. 4 6 The results of theabove clinical research in humans and the hemodynamics of colitogenic memory CD4+T cells described in theprevious section suggest that the hemodynamics inchronic colitis are more dynamic than previously imagined, and that the continuous circulation of colitogenic
memory CD4 +T cells plays an important role in themaintenance of the IBD. Another important readout isthe assessment of the role of leukocytapheresis therapy,
in other words, that the removal of large numbers of theabove-described intestinal TEM-type colitogenic memory
CD4+T cells that constantly circulate from the peripheral
blood over a series of weeks itself may exert a therapeutic effect in the sense of blocking the vicious cycle ofchronic colitis (Fig. 1).
In terms of the leukocyte circulation, it has been developed very unique immunosuppressant, FTY720 as mentioned before.47, 48 FTY720 is a sphingosine-1-phosphate(S1P) receptor modulator, which induces prolonged
down-modulation of the surface expression of the S1P
receptor, and thereby inhibits the egress of lymphocytes
from thymus, lymph nodes (LN) and Peyer’s patches
leading to peripheral blood (PB) lymphopenia.47, 48 Fromthe view of clinical application, in animal models,
FTY720 has been shown to prevent autoimmune diseases,4 9, 5 0 or graft rejection after allo-transplantation. 5 1
Moreover, it was recently shown that FTY720 reduced
the number of lesions and clinical disease activity of patients with multiple sclerosis in a phase II, placebo-controlled trial.52 Although little was known about how coliotogenic memory CD4+T cells in IBD are controlled by
FTY720, we recently demonstrated that FTY720 suppresses the development of colitis induced by adoptive
transfer of colitogenic memory CD4+T cells of colitic
CD4+CD45RBhigh T cell-transferred SCID mice. Furthermore, we found that FTY720 treatment induced marked

Kanai T, et al: Strategy for the Treatment of IBD

208lymphopenia of colitogenic memory CD4+T cells in the
periphery.53, 54 In addition, we recently demonstrated thatFTY720 is able to suppress the development of chronic
colitis by modulating the trafficking of colitogenic memory CD4+T cells in BM in addition to the well-known effect to control the egress and sequestration of lymphocyte in LN.54 Collectively, FTY720 treatment may offer
the potential not only to prevent the onset of disease but
also to treat colitogenic memory CD4+T cell-mediatedautoimmune diseases including IBD (Fig. 1). The
FTY720 study would provide another impact in terms ofa characteristic of colitogenic memory CD4+T cell trafficking for the maintenance of IBD. Although some investigators suggested that LP T cells do not migrate outof the gut,55 our results indicated that colitogenic memory LP CD4+T cells are needed to constitutively recirculate into MLNs, and are re-stimulated by Ag-bearing
dendritic cells in MLN to maintain the colitogenic memory CD4+T cells for sustaining chronic colitis.
Furthermore, similarly treatment of IBD with antibodies that inhibit integrin α4 (Natalizumab) (Fig. 1), a celladhesion molecule that is essential for lymphocyte guthoming, has been tried as well.56 In view of the similarity between treatment strategies with FTY720 and antiintegrin α4 antibody, which block the circulation ofpathological lymphocytes, on the one hand, and leukocytapheresis, which removes circulating pathological lymphocytes from the peripheral blood, on the other, in recent years a movement that recommends more frequent
leukocytapheresis has been advocated for the treatmentof ulcerative colitis, but blocking their circulation itself
may be what is really important in terms of the mechanism. The results of further analysis in the future are being awaited.

2.
3.
4.
5.
6.

7.
8.

9.

10.

11.
12.

13.

Conclusion
We propose that once chronic colitis develops, systemically circulating colitogenic memory CD4+T cells may
be retained in the body and be involved in the colitis becoming permanent. Complete cure of IBD by conventional local resection or anti-inflammatory therapy may
be difficult. We view IBD as systemic diseases, which
pathogenic memory CD4+T cells circulate in the body,suggesting the need and potential for systemic therapy
that postulates the blockade of their circulation, including leukocytapheresis and FTY720, with the aim of removal of the colitogenic memory CD4+T cells that remember the disease and are retained in the body longterm.
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