Alloreactive t cells and cytokines in murine graft versus host disease 4

_ Discussions CHAPTER DISCUSSIONS The graft-versus-host disease (GVHD) is an in vivo indication of cell-mediated cytotoxicity The disease develops when immunocompetent lymphocytes are adoptively transferred into an allogeneic recipient whose immune system is compromised Because the donor and recipient are not genetically identical, the grafted lymphocytes target and damage host tissues but the host’s immunologicallycompromised state is incapable of responding against the graft In humans, GVHD often develops after bone marrow transplantation (BMT) in patients who have had radiation exposure or who have leukemia, immunodeficiency diseases, or autoimmune hemolytic anemia (Norton et al., 1992, 1994) Similarly, GVHD also develops when immunocompetent lymphocytes are transferred into an allogeneic severe combined immunodeficiency mouse or X-irradiated mouse (Theobald, 1995) The recipient mice exhibit poor activity, weight loss, lesions of skin, liver, and intestine (Hakim et al., 1998) The grafted lymphocytes generally home to a number of lymphoid organs, including lymph nodes and spleen, where they begin to proliferate in response to the allogeneic MHC antigens of the host (Gretz et al., 1996) 4.1 The use of SCID mouse as a model for studying experimental acute GVHD The congenital syndrome known as severe combined immune deficiency (SCID) is characterized by a loss of both B and T cell immunity due to a defect in the rearrangement of their antigen receptor genes (Schuler et al., 1986) The SCID syndrome was first reported in a mutant strain of mice (C.B-17), called SCID mice 136 _ Discussions (Bosma et al., 1983) The discovery of the SCID mutation resulted from a chance observation in the course of quantitating serum levels of IgG1 and IgG2a allotypes in specific-pathogen-free mice of the immunoglobulin heavy chain (IgH) congenic strains, BALB/c and C.B-17 allotype (Bosma et al., 1983) Mice homozygous for this mutation generally lack mature, functional lymphocytes and are highly susceptible to recurring infections, but otherwise appear normal Because SCID mice are severely compromised immunologically, they must be housed in a sterile environment They cannot fight off microorganisms of even low pathogenicity The absence of functional T and B cells enable these mice to accept foreign cells from other strains of mice without rejection This finding has made SCID mice a valuable model for the study of GVHD because their immunological state resembles that of patients who are immunologically compromised or have undergone irradiation before BMT C57BL/6 mice (H-2b) which are allogeneic to SCID mice were employed as donor mice in our study These animals were originally derived from strains of W L Russell at The Jackson Laboratory in 1938 and designated as C57 black (Smith et al., 1994) GVHD prevents potentially curative allogeneic stem cell transplantation from being offered to cancer patients who lack a suitably matched donor New methods to prevent GVHD are required to allow successful transplants across major histocompatibility complex barriers Based on the characteristics of the SCID mice, a model GVHD in C.B-17 SCID mice was developed to allow the study of allo-activated donor T cells without the confounding effects of host lymphocytes or complications induced by high-dose irradiation In our study, GVHD was induced by injecting each recipient C.B-17 SCID mouse with 10×106 or more mononuclear cells isolated from the spleen or LNs of week old C57BL/6J donor mice Using SCID mice as the GVHD model to study the progress of the disease is reliable as they never fail to 137 _ Discussions develop the disease when the allogeneic lymphocytes are transferred into these immunodeficient mice SCID recipient mice manifested consistent symptoms of GVHD and predictable progress of disease upon the induction of GVHD In general, their body weight starts to drop on day post-transplantation, and other symptoms such as hunched back at rest, severe diarrhea, decreased activity and ruffling of fur texture start to appear from the 3rd weeks after cell transfer Scaling of paws, lesions on tail skin and denuded skin only manifest at the later stage of the disease Changes in body weight and percentage of survival were selected to be the most objective indicators for GVHD in our studies 4.2 Target organs involved in acute GVHD and the roles played by chemokines in directing the infiltration of donor T cells into the host target organs The inflammatory reaction in acute GVHD begins with T cell infiltration Although all nucleated cells express on the cell surface major histocompatibility complex class I molecules which are major antigens for activating alloreactive T cells, T cell infiltration in acute GVHD does not occur evenly in the body T cells preferentially infiltrate certain organs, such as liver, skin and intestines, and cause serious damage to these organs, but other organs such as heart, muscle and the neural system are apparently unaffected (Nikolic et al., 2000; Hakim et al., 1998; Ferrara et al., 1991) This discrepancy may be due to the factors that govern T cell migration Of these factors, chemokines play a central role by determining the direction and T cell subpopulation in the trafficking and infiltration process Chemokines are secreted by various cells, and form a local high concentration to recruit inflammatory cells Inflammatory cells usually express several chemokine 138 _ Discussions receptors on the cell surface, and each cell type with distinct effector functions express different chemokine receptors CD4+ effector T cells generated in vitro express a broad range of chemokine receptors, some of which are preferentially expressed on Th1 cells (CXCR3, CCR5) or Th2 cells (CCR3, CCR4, CCR8) (Baggiolini et al., 1998; Rollins et al., 1997; Serody et al., 2000; Bonecchi et al., 1998; Sallusto et al., 1998; Nelson et al., 2001) This suggests that distinct chemotactic signals direct the localization of Th1 and Th2 cells to the sites of inflammation In the spectrum of chemokines we investigated, chemokine receptors CCR1 and CCR5 are specific for MIP-1α, CXCR2 for MIP-2, CXCR3 for Mig, CCR2 for MCP-1, and CCR1, CCR2 and CCR3 for MCP3 (Bonecchi et al., 1998; Sallusto et al., 1998) From the results generated by real time PCR and ELISA on various organs harvested from mice after injection of donor cells, we found that in the spleen and liver, the target organs of acute GVHD, MIP-1α, MIP-2 and Mig were the predominant chemokines expressed In another target organ, the skin, MIP-1α, MIP-2, MCP-1 and MCP-3 were all highly expressed In a nontarget organ of acute GVHD, the heart, the predominant chemokines expressed were MCP-1 and MCP-3 This distinct pattern of chemokine expression in these organs may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD The predominantly expressed chemokines MIP1α and Mig in the spleen, liver and skin recruit Th1 cells, which have been shown to play the central role in mediating alloimmune response in acute GVHD (Lu et al, 2001; Ferrara et al, 1991) Recent studies by Murai et al (1999) characterized the liver infiltrating CD8+ T cells They found a large population of donor CD8+ T cells in the liver, causing both portal hepatitis and non-suppurative destructive cholangitis These cells expressed CCR5 In contrast, the chemokines expressed in the heart of SCID 139 _ Discussions mice, after injection of donor cells, were mainly MCP-1 and MCP-3 These two chemokines not attract the acute GVHD mediating CCR5+ and CXCR3+ helper or CD8+ effector T cells MIP-2 was another chemokine that was up-regulated in target organs but peaked later than MIP-1α and Mig as shown in the serum and tissue ELISA results Its receptor, CXCR2, is predominantly up-regulated on neutrophils, monocytes and eosinophils (Nelson et al., 2001; Wolpe et al., 1989; Mercer-Jones et al., 1999) This chemokine might be responsible for recruiting these inflammatory cells into the target organs at a later stage of acute GVHD In fact, FACS analysis on the cells isolated from the spleen and LNs on day 21 post-transplantation revealed that the percentage of infiltrating T cells had decreased dramatically compared to day samples Instead, these tissues were infiltrated with many cells of high granularity (showing high side scatter in FACS, data not shown) There is a high possibility that these late infiltrating cells are the polymorphornuclear cells attracted by MIP-2 Nonetheless, further studies on their cell surface markers are required to confirm this observation Serody et al (2000) demonstrated that the recruitment of CD8+ T cells to acute GVHD liver relies heavily on MIP-1α However, using MIP-1α knockout splenocytes as donor cells failed to block CD4+ cells infiltration into acute GVHD liver Murai et al (1999) demonstrated that CD4+ T cells expressed much lower CCR5 than CD8+ T cells These observations indicate that other chemokines other than MIP-1α play a critical role in the recruitment of CD4+ T cells into the target organs It was recently reported that CXCR3 was up-regulated on CD4+ T cells during allogeneic activation (Ebert et al., 2001), and the use of anti-Mig Ab blocked the infiltration of CD4+ cells into skin allograft and significantly delayed the rejection (Koga et al., 1999) These 140 _ Discussions results correlate with our distinct chemokine expression patterns in the target and nontarget organs of murine acute GVHD Blocking antibodies directed at CCR1, CCR5 and CXCR3 or chemokine receptor antagonists have been used in preventing kidney allograft rejection (Grone et al., 1999; Hancock et al., 2000) and acute GVHD (Murai et al., 1999) in animal studies These treatments could dramatically reduce the infiltration of CCR5 or CXCR3 expressing T cells, and consequently reduce the damaging inflammatory activity In summary, we have documented the chemokine spectrum in target/non-target organs of mice with acute GVHD A distinctive pattern of chemokines expressed in target/non-target organs has been observed Although each chemokine may bind more than one receptors, and the complexity of the selective infiltration of certain organs in acute GVHD remains to be elucidated, at least the chemokine spectrum preferentially expressed in different organs may play an important role Based on the difference of chemokine expression observed, it is possible to modulate acute GVHD by changing micro-environmental chemokine repertoire in reducing the recruitment of alloreactive cells The simultaneous upregulation of multiple chemokines during acute GVHD as demonstrated in our study suggests that the blocking of or more chemokines or chemokine receptors might be required in order to effectively ameliorate the disease 4.3 Current approaches in purging the alloreactive cells from the donor T cell pool to prevent GVHD after transplantation As shown in our previous study, once the donor T cells enter an allogeneic host system, the alloreactive T cells will proliferate extensively in the lymphoid organs of the recipient On day post-transplantation, flow cytometric analysis based on CFSE 141 _ Discussions intensity showed that there were about 20% of the donor T cells that did not go into division (Fig 10) These T cells are supposed to be of other specificities Theoretically, if they are purified and re-injected into another immunodeficient recipient of same MHC background, they should not induce GVHD but protect the host from viral infection and leukemic relapse Following along this line of thinking, various ways to selectively deplete alloreactive T cells while preserving T cells of anti-viral or leukemic cell antigens are currently being explored by many researchers The activation of allogeneic donor T cells is usually achieved by co-culturing them in vitro with irradiated recipient cells (PBMC, spleen cells, etc) Activated T cells will upregulate surface markers such as CD25, CD69, CD95, and also increase in metabolism They can then be removed by anti-IL-2 receptor immunotoxin (Cavazzana-Calvo et al., 1990 & 1994; Amrolia et al., 2003) or anti-CD5 immunotoxin (Filipovich et al, 1990), immunomagnetic bead depletion of CD25+ and/or CD69+ population (Fehse et al 2000 & 2000b; Garderet et al., 1999; Koh et al., 2002), induction of activation-induced cell death with anti-CD95 mAb (Hartwig et al., 2002), photodynamic cell purging (Chen et al., 2002), or separation through density gradient centrifugation (Palathumpat et al., 1992a & 1992b) Nonetheless, many still observed that in subsequent assay after the depletion step, the initially non-activated T cells population still exhibited some residual proliferation or anti-recipient cytotoxicity, albeit to a lower extend compared to the untreated group (Garderet et al., 1999; Amrolia et al., 2003; Fehse et al., 2000 & 2000b; Cavazzana-Calvo et al., 1990 & 1994) Some researchers also reported that injecting the T cells that had undergone selective depletion could not completely prevent GVHD (Cavazzana-calvo et al., 1994; Koh et al., 2002; Harris et al., 1999) We felt that the limiting steps might be the efficiency of the ex-vivo depletion methods employed and the in vitro activation of 142 _ Discussions alloreactive T cells via MLC T cells respond differently to different types and strength of activation (Caruso et al., 1997) The in vitro stimulation via MLC may not be optimal to activate all allogeneic T cells, and some T cells might not get sufficient stimulus to drive them into activation Besides, the source of alloantigens employed in MLC usually comes from a single cell type, which might not represent the whole lot of alloantigens or minor histocompatible antigens that the donor T cells might encounter upon entering the host system Therefore, these allogeneic T cells remain quietscent and escape the ex vivo purging process, but can induce GVHD when injected into the recipient subsequently To study the T cell behaviour and the allogeneicity of the initially non-activating T cells more closely, we employed an in vivo process to stimulate the alloreactive T cells Our approach should have provided the optimum condition for T cells to be activated and proliferate, and allowed them to encounter all possible alloantigen during the process In our study, donor T cells were pre-labeled with CFSE and then injected into the primary recipient mice During each round of cell division, the relative intensity of the dye in each cell decreases by half This provides a clear and accurate visual differentiation between proliferating and non-proliferating cells These populations of cells were then separated by fluorescence activated cell sorting based on CFSE intensities This is an elegant and novel approach to separate responding from non-responding cells, and of being able to separate cells to very high purity 4.4 The dual characters of the initially non-proliferating (CFSEhi) T cells As expected, the sorted non-proliferating (CFSEhi) T cells failed to proliferate and secrete IFN-γ in vitro in the presence of alloantigen stimulation, indicating that the cell sorting process was successful and most of the proliferating (CFSElo) T cells were 143 _ Discussions removed However, these CFSEhi T cells were able to proliferate, induce IFN-γ secretion and cause acute GVHD in the secondary recipients Although these sorted CFSEhi T cells contained a small number of previously activated cells in the primary recipients, the proliferating T cells in the secondary recipients did not appear to be solely from these already activated cells, considering the high percentage of the proliferating T cells in the secondary recipients Also, to rule out the possibility that there might be some allogeneic T cells that have not entered division cycle by day 5, we performed the same study on LN cells isolated from primary recipients on day and even day 21 post-transplantation These cells induced a similar GVHD kinetic in the secondary recipients (data not shown) In fact, early in 1974, Bowers (1974) made a close observation He co-cultured donor T cells with fibroblast expressing the recipient’s MHC After 3-5 days of culture, the activated T cells were separated from the resting cells by zonal differential sedimentation through continuous Ficoll He then assessed the alloreactivities of the highly purified small lymphocytes (characteristics of resting T cells) and found that although their in vitro anti-recipient cytotoxicity was very much lower than their activated counterparts, their capability of inducing an in vivo GVH response did not differ much Similarly, the small lymphocytes, consisting mainly resting T cells isolated in our preliminary study using discontinuous Percoll gradient centrifugation, were also capable of inducing severe GVHD in the secondary recipient mice (data not shown) Our observation is further supported by more recent studies which demonstrated that the remaining T cells after ex-vivo activation and selective depletion process still exhibits relatively high alloreactivity (up to 20%) (Garderet et al., 1999; Amrolia et al., 2003; Fehse et al., 2000 & 2000b; CavazzanaCalvo et al., 1990 & 1994), and GVHD in the recipient mice could not be completely aborted (Koh et al., 2002, Harris et al, 1999) Cavazzana-calvo et al (1994) depleted 144 _ Discussions ex vivo activated alloreactive T cells with anti-IL-2 immunotoxin prior to injection into the recipient mice Similar to our results, the remaining cells did not exhibit detectable alloreactivity in vitro, but the mortality rate of the recipient mice was not improved much compared to those injected with untreated donor cells Besides, Caruso et al (1997) examined some known T cells activation markers (CD25, CD40L, CD69, CD71, HLA-DR, CD95L) expression on T cells under different stimulations and at different time points after stimulation He found that their expression kinetics were different, inferring that different clones of T cells might respond to antigens at different times with varying scales of activation and expression of surface marker Collectively, other researchers’ and our observations indicate that here is a T cell population that were different from those T cells that can proliferate in vitro in response to alloantigen stimulation and mount a rapid response in the primary SCID mouse recipients This led us to suggest that at least two populations of alloreactive T cells existed, and here we name them separately as rapid responders and delayed responders Rapid responder T cells become activated once they meet alloantigen stimulation, and proliferate into a large number of effector cells These cells are probably the cause of early acute rejection of organ transplantation or early acute GVHD in bone marrow transplantation Rapid responder T cells can be activated in vitro by alloantigen stimulation, and can be detected by MLR Delayed responder T cells somehow are regulated either by other cells or by their own intracellular events not to respond initially when they meet the alloantigen stimulation in vivo, but will mount an alloreactive response at a later time 145 _ Discussions proliferation assay, indicating the inability of the traditional MLR to reveal the whole repertoire of alloreactive T cells is not limited to the omission of the activated T cells that not proliferate in the assay period This assumption is supported by our results which show that some alloreactive T cells not respond in vitro but will mount a response towards alloantigens in vivo, or that some alloreactive T cells not respond in the time period of MLR but will respond at a later time if the alloantigen stimulation remains there Therefore, the existence of these cells helps explain a clinically observed phenomenon that acute rejection of transplanted organs can occur years post transplantation while the MLR shows donor specific non-responsiveness (Goulmy et al., 1989; Steinmann et al., 1994; Reinsmoen, 2002; Mikelson et al., 1993 & 1996; Segall et al., 1996) These so-called delayed responder T cells may be responsible for inducing the late-occurring acute rejections of organ transplantation or acute GVHD post BMT In conclusion, we have described the existence of the delayed responder alloreactive T cells that not respond to in vitro alloantigen stimulation, and therefore could not be detected by traditional MLR The discovery of delayed responder T cells may turn out to be important for clinical design of immunosuppressive therapeutic modalities Maintenance therapy is probably always needed until it is proven that all alloreactive T cells are either depleted or under the control of T suppressor cells Furthermore, prevention of acute GVHD by in vitro depletion of activated alloreactive T cells only may not be sufficient, because the delayed responder T cells will not be depleted This assumption is supported by studies showing that in vitro selective depletion of alloreactive T cells is only partially effective in preventing acute GVHD (Cavazzana-Calvo et al., 1994; Harris et al., 1999; Koh et al., 2002) Further characterization of these delayed responder T cells is needed 147 _ Discussions for their intracellular signaling pathways after antigen stimulation and their response to immunosuppressive agents 4.6 The rationale behind the search of Treg cells in the early proliferating (CFSElo) T cell population In our previous study on the alloreactivities of various fractions of T cells, we were surprised to find that the sorted CFSEhi LN cells can induce aGVHD (Fig 15) It was equally surprising to notice that CFSElo LN, which contained >98% activated and proliferating allogeneic donor T cells, actually induced a more delayed form of GVHD compared to the CFSEhi LN cells (Fig 18) More interestingly, besides a brief period of body weight drop, secondary recipients of spleen cells (which contained >95% CFSElo T cells) did not exhibit any obvious symptoms of aGVHD These observations were totally unexpected We had repeated the experiment a few times to confirm that our observations were reproducible and consistent In fact, in our preliminary experiment using Percoll for separating proliferating from non-proliferating cells, we had already observed that the cells isolated from the lower density fraction (contained mostly CFSElo cells) induced a delayed aGVHD compared to those isolated from the higher density fraction (enriched in CFSEhi cells) Strober and his co-workers published several papers reporting very similar observations as ours – after fractionation with Percoll, the donor spleen cells isolated from the high density fraction (60%/70% interface) could induce GVHD in the irradiated allogeneic recipients while cells from low density fraction (40%/50% interface) conferred protection against GVHD when mixed with unfractionated spleen cells (Palathumpat et al., 1992a & 1992b; Schmidt-Wolf et al., 1992) The only difference was that their spleen cells were obtained from healthy donor mice while ours were from inflamed 148 _ Discussions primary donors which were starting to develop aGVHD Nonetheless, their report had led us to suspect that the cells isolated from the low density Percoll fraction in our preliminary experiment might be enriched in regulatory T (Treg) cells Moreover, FACS analysis performed on these isolated cells revealed that the majority of the cells were CD45RBlo and CD25+ This corresponds to the characteristics of Treg described by Singh et al (2001) and Sakaguchi et al (2001) In addition, Taylor et al (2002) and Cohen et al (2002) had reported that the depletion of CD4+CD25+ Treg cells from donor T cells accelerated aGVHD induced in the allogeneic recipient mice, suggesting that Treg cells naturally present in the donor cell inoculums have an unforeseen effect in modulating the disease We thought it would be very interesting if we could demonstrate that the Treg cells were enriched in a certain fraction of donor cells, in this case most probably the early proliferating fraction, during the development of acute GVHD in the recipient mice This could be a rich source for us to study their characters and roles in acute GVHD, and probably be developed into a therapy for transplanted organ rejection and GVHD treatment 4.7 Characterization of the early proliferating (CFSElo) T cell population The results obtained from our study confirmed that these early proliferating cells are the result of alloantigen-driven proliferation of allogeneic donor T cells Besides, these cells are fully viable and immunologically competent – they proliferated and secreted various cytokines in in vitro MLC, and induced a brief body weight drop on around day 7, a typical symptom observed on mice induced with acute GVHD, upon injection into the secondary recipient mice Naturally, the anticipation that followed was whether there could be, due to some unknown mechanisms, an enriched population of Treg cells present in this early proliferating cell population However, although these 149 _ Discussions CFSElo cells showed increased expression of CD25, CD38, CTLA-4 and the majority are CD45RBlo compared to the CFSEhi cell population, these markers were shared by activated T cells, making the identification of Treg cells under such inflammatory condition difficult αEβ7 expression was reported to be exclusive on Treg cells, but in our study, their expression did not differ much between CFSEhi and CFSElo cells It is insufficient to determine the existence of Treg cells just base on the surface marker expression alone Powrie’s group had demonstrated, using mouse colitis model, that while adoptive transferring of CD4+CD45RBlo Treg cells could inhibit the disease induced in mice injected with only CD4+CD45hi cells, the administration of anti-IL-10 receptor mAb abrogated the ability of the Treg cells to inhibit colitis (Hara et al., 2001; Singh et al., 2001) Besides, adoptive transfer of Treg cells from IL-10 (-/-) donor also failed to inhibit colitis (Asseman et al., 1999) In addition to IL-10, Lan et al (2001) also reported that high level of IL-4 from Treg cells was required to protect mice from GVHD in mouse BMT model These data indicated that IL-4 and IL-10 might play an important role in mediating the suppressive function of Treg cells By performing in vitro MLR using irradiated Balb/c (H-2d) spleen cell as stimulating cells, we observed that unsorted spleen cells from primary recipient secreted a much higher IL-4 and IL10 levels than other cell fractions, followed by sorted CFSElo and unsorted LN cells (Fig 27) In contrast, CFSEhi LN cells did not produce any detectable amount of both cytokines Unfortunately, none of these cytokines could be detected in the serum of secondary recipient mice injected with either fraction of cells This suggests that either the CFSElo cells behave differently in in vivo and in vitro environment, or the Th2 cytokine levels secreted by these cells might be too low to give an effect in an in vivo environment Indeed, a rough comparison between the IL-4 or IL-10 levels with the 150 _ Discussions IFN-γ levels secreted by the same fraction of cells showed that the latter could be a hundred times higher This inferred that IL-4 and IL-10 might not play a pivotal role in delaying GVHD in the recipient mice of CFSElo cells Treg cells potently suppress the activation/proliferation of other T cells in a dose-dependent manner (Kiniyasu et al., 2000; Takahashi et al., 1998; Thornton & Shevach, 1998; Itoh et al., 1999) in in vitro MLC by inhibiting the IL-2 production by normal T cells directly or indirectly at gene transcription level (Takahashi et al., 1998; Thornton & Shevach, 1998) This might be achieved via cell-to-cell cognate interactions between Treg and normal T cells, or by the Th2 cytokine secretion from Treg cells Nonetheless, Treg cells themselves not proliferate to in vitro antigenic stimulations such as A, anti-CD3 Ab, antigenic peptides or alloantigens Therefore, the proliferative response of CFSElo cells upon alloantigenic stimulation and their ability to suppress IL-2 production were examined in our study Unfortunately, the results contradicted with what we had expected – spleen and CFSElo LN cells proliferated actively when co-cultured with irradiated Balb/c spleen cells, and high levels of pro-inflammatory cytokines such as IFN-γ, MIP-1α and IL-2 levels were detected (Figs 21, 27) This suggested that the CFSElo cells proliferated under alloantigenic stimulation and did not inhibit the secretion of inflammatory cytokines Co-transfer of Treg cells together with normal T cells could suppress autoimmune diseases such as colitis and GVHD Sakaguchi et al (1995) found that the transfer of CD4+CD25- T cells into nude mice led to the development of autoimmune disorders that could be prevented by the co-transfer of CD4+CD25+ Treg cells On the other hand, Read et al (2000) reported that as few as 5×104 CD4+CD25+ Treg cells were able to inhibit colitis induced by 4×105 (8 times more) CD4+CD45RBhi T cells 151 _ Discussions Moreover, co-transfer of CD4+CD25+ Treg cells together with CD4+CD25- T cells at 1:1 ratio could protect the recipient mice from GVHD (Cohen et al., 2002; Hoffmann et al., 2002) Using the same approach, we mixed CFSElo LN or spleen cells at different ratio with GVHD-inducing CFSEhi LN cells If there were any Treg cells in the early proliferating population, the GVHD in the recipient mice should at least be delayed, if not prevented It was disappointing that, the results were again in contrary to our expectations since the recipient mice injected with the highest CFSElo to CFSEhi LN cells ratio developed the most drastic disease The lifespan of recipients injected with other ratios of cell mixture did not improve much either (Fig 29) On the other hand, mixing unsorted spleen cells with CFSEhi LN cells appeared to give some positive effect on the survival rate of the recipient mice Nonetheless, while the recipients of spleen cells alone did not showed any noticeable symptoms of GVHD, those injected with these mixed cells did, albeit milder than the control mice injected with CFSEhi LN cells alone To further confirm if there might be true Treg cells among the spleen cell population, we injected the unsorted spleen cells into the secondary recipients a week earlier before injecting CFSEhi LN cells This should help the Treg cells, if any, to repopulate and establish the SCID recipients’ lymphoid environment better and thereby more effectively modulating the activities of the late coming cells This did not turn out as desired since all recipient mice developed excessive diarrhea and wasting syndromes, and the mortality rate did not differ much from the control mice (Fig 29c) The results implicated that the CFSElo population did not have much suppressive activity Taken together with the results we observed earlier, our study showed that few cells, if any, in the early proliferating population were Treg cells 152 _ Discussions 4.8 The mystery of why CFSElo T cells induced a delayed GVHD remains Despite all the experiments performed, we are still not able to explain why the proliferating LN cells induce a delayed GVHD compared to non-proliferating LN cells, and why unsorted spleen cells failed to induce disease at all Curious as to why GVHD was aggravated when the CFSEhi LN cells were injected into the secondary recipients a week later than the unsorted spleen cells, we investigated the proliferation kinetics of the CFSEhi LN cells under this condition We found that by day 4, there were only 0.2% and 0.6% of CFSEhi T cells in the LNs and spleen of the secondary recipient mice respectively, and hardly any were observed by day (Fig 30) On the other hand, mice that received the same number of CFSEhi T cells alone still had around 15% of non-dividing CFSEhi T cells by day in the LN (Fig 13) The higher diminution rate of the CFSEhi T cells in mice that were pre-injected with spleen cells might suggest that the CFSEhi T cells were proliferating at a faster rate in these mice Our initial idea was the hope that the early proliferating cells could inhibit the proliferation and establishment of the disease-inducing CFSEhi LN cells, but the opposite results were observed, i.e the early proliferating cells might actually be some helper or cytokineproducing cells which mediate the proliferation of effector T cells They themselves could not induce GVHD when injected alone, but could aggravate the disease when co-injected with effector cells One strong support for our hypothesis had been demonstrated in our earlier study which showed that by day post injection, most of the effector cells would have infiltrated into the target organs such as liver, intestine and skin (Figs 2-5) Cells that did not migrate out of the lymphoid tissues by day might not play an essential role in disease induction or tissue destruction These cells might be some helper T cells, or some non-alloantigen specific T cells that proliferated due to bystander effect and hence only induced a delayed disease when transferred into 153 _ Discussions secondary recipients Nonetheless, our hypothesis has to be further confirmed by more in depth studies For instance, in the experiment as shown in Fig 30, we should use donor cells of same genetic background but with different surface marker expression (eg Thy-1.1 and Thy-1.2) in order to better differentiate between the cells that proliferated from the first batch of cells and that from the second batch In this way, the proliferation of CFSEhi LN cells would not be masked by that of spleen cells More experiments are also required to characterize the early proliferating T cells from the primary recipient mice for their cytotoxicity, allogenicity and cytokine expression profiles 4.9 The use of diphtheria toxin in the construction of immunotoxins Diphtheria toxin is highly efficient at crossing eukaryotic cell membranes and initiating cell death Therefore, it has been used to construct immunotoxins (ITs) to target and kill specifically selected cells (Murphy et al., 1995; Neville et al., 1989; Nicholls & Youle, 1992; Youle et al., 1988) ITs were initially generated by chemically conjugating the toxin to a targeting protein (such as a monoclonal antibody) However, the ITs made in this mode are heterogeneous chimeric molecules, and it is difficult to produce ITs in large amounts because the antibody and toxin must be purified separately and then conjugated in a reaction that often resulted in a low yield More recently, ITs have been constructed by fusing IT toxin genes with those genes encoding targeting proteins (Murphy et al., 1986; Thompson et al., 1995; Vallera et al., 1996; Jean et al., 1991; Batra et al., 1991; Chan et al., 1995; Chaudhary et al., 1990; Williams et al., 1987) and expressing the recombinant fusion protein in E coli This genetic engineering procedure is cheaper and has the potential of optimizing the construct with respect to efficacy and minimizing side effects However, the IT is 154 _ Discussions usually synthesized in the form of inclusion bodies (Vallera et al., 1996) and then refolded in vitro Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these ITs have been generally limited to single-chain monovalent structures These additional steps to purify this protein and re-nature it into the functional conformation could be complex and costly if the IT is to be prepared on a large scale Several DT390 fusion IT targeting at T cells have been generated Hu et al (1997) constructed an anti-monkey CD3 IT by splicing the light and heavy chain variable regions of anti-monkey CD3 monoclonal Ab cDNA obtained from a hybridoma and ligating to DT390 gene Administering the purified protein synthesized from this IT recombinant plasmid can transiently deplete T cells to 1% of initial values in both the blood and lymph node compartments of the treated rhesus monkey, and thereby inducing long-term tolerance to mismatched renal allografts and delaying or preventing the T-cell-driven experimental allergic encephalomyelitis (EAE) Vallera et al (1996) also synthesized an identical IT by replacing the targeting moiety with a spliced cDNA from anti-mouse CD3 mAb hybridoma Using a mouse GVHD model same as ours, Vallera et al demonstrated that injecting 2µg of DT390-anti-CD3 IT in daily doses for days post-transplantation significantly improved the survival rate of the recipient mice, although survivors still showed histopathologic signs of GVHD These results showed that DT390-anti-CD3 IT has potent cytotoxicity against T cells and could be employed as a therapy to induce transplantation tolerance or treat autoimmune diseases and T cell leukemia In contrast with ricin and Pseudomonas exotoxin-based ITs, there is a potential problem using DT-based ITs in the treatment of human diseases Most people have been immunized against DT Therefore, these people have a pre-existing anti-DT 155 _ Discussions antibody titer which could potentially inhibit or alter the efficacy of these ITs This is the reason why a truncated form of DT, designated as DT390, was generated - to remove the target site of the anti-DT Ab from the DT protein Thompson et al (1995) investigated the inhibitory effect of the pre-existing anti-DT Ab in human serum to an native DT-anti-human CD3 and a truncated DT390 single-chain IT The DT390 IT was only moderately inhibited by anti-DT Ab in human serum under conditions resulting in complete neutralization of its native counterpart, suggesting the feasibility of employing this truncated form of IT in the treatment of human diseases The DT390 moiety is also reported to have the full enzymatic activity compared with the native DT (Vallera et al., 1996) 4.10 Amelioration of GVHD by in vivo expression of DT390-IL-2 Although GVHD is initiated by immunocompetent donor T cells present in the allogeneic bone marrow graft, total T cell depletion from the graft often results in engraftment failure, leukemia relapse, immunodeficiency and viral infection of the patient (Maraninchi et al., 1987; Aversa et al., 1994; Papadopoulos et al., 1994) Therefore, it is desirable to selectively deplete only the alloreactive T cells while preserving the rest which have other specificity This led us to employ DT390-IL-2 instead of DT390-anti- CD3 in our study to treat GVHD The IL-2 moiety of this IT will selectively target and bind to IL-2R expressing alloreactive donor T cells, while the toxin moiety kills the targeted cells once it is internalized This IT plasmid was constructed by reverse-transcribing the IL-2 cDNA from PHA-activated mouse T cells mRNAs and ligating it to the DT390 gene The whole gene construct was then cloned onto a mammalian expression vector to allow its expression in the mouse cells Instead of administrating purified IT proteins, we adopted the gene therapy approach in our 156 _ Discussions study by delivering the DT390-IL-2 plasmid into the parenchymal cells of the various organs in the recipient mice using an in vivo transfection kit This kit has proved itself efficient as the spleen and liver cells isolated from mice transfected with a plasmid encoding a GFP-reporter gene expressed the green fluorescent protein (Fig 33) The expression levels of GFP varied with different amount of GFP-encoding plasmid delivered – percentage of liver cells expressing GFP increased when the amount of plasmid delivered increased from 10 µg to 20 µg, but the expression reduced when the amount of plasmid was increased further This suggests that there is an optimal dosage of plasmid to be used in order to achieve the highest expression level This dosage may vary depending on the different types of cells and plasmids used Besides, according to the manufacturer of the in vivo transfection kit, the highest levels of transgene expression are often found in the liver, with lower levels of expression found in many other organs including spleen, kidney, lungs and heart Indeed, we observed a much higher GFP expression level in cells isolated from liver than spleen in our experiment (Fig 33) Since liver is one of the main target organs in GVHD, we hope that a high level of DT390-IL-2 expression in this organ can more effectively eliminate the infiltrating alloreactive donor T cells and thereby reduce the damage to the organ Recipient mice were transfected on either day 4, day or both days posttransplantation Gene delivery was given on day post-transplantation because it was found that the donor alloreactive T cells expressed the highest levels of IL-2R at around day 5, and according to the manufacturer, there is a latent period of 8-24 hours before the transfected gene reaches its optimal expression level Another dose was given on day post-transplantation because most alloreactive T cells would have infiltrated into the target organs by this time (Figs 2-3), and we were worried that the expression of the DT390-IL-2 plasmids transfected on day could not sustain until day 157 _ Discussions Indeed, recipient mice receiving only a single injection of DT390-IL-2 plasmid, either on day or 7, did not show any difference in the survival rate compared to the mice receiving control plasmids or just the delivery solution (data not shown) While all the control mice died before day 50 post-transplantation, three out of four of the DT390-IL2 treated mice still survived by day 90, although one of the surviving mice showed obvious symptoms of GVHD These results reflected that not only the dosage of DT390-IL-2 administered was important, but also the timing and frequency of its administration There were some apparent advantages of this technique over the conventional treatment using purified IT protein The production of this IT was simple as only gene cloning and plasmid isolation were required Since no complicated process for extracting, purifying and re-naturing proteins was involved, the cost of production could be kept to a minimum Also, DNA is much more stable than proteins, so the handling process is less demanding In addition, the procedure of administration is simple – just a single or two injection of the plasmid into the recipient mouse, and the gene expression level will sustain for a short period of time No multiple and frequent injection is required Despite this, the expression of the gene is transient and not permanent Therefore, after the initial expression of the transgene has subsided, the remaining T cells could mount a normal response against foreign antigens without being eliminated This is also a better choice over the viral-based gene transfection method as one does not need to worry about the possibility that the viral vector may recombine with some latent virus genes within the genome of the host cells This strategy is not without its problems The serum level of the IT is critical in determining to its efficacy in treatment A level that is too high could be toxic to the normal host cells and organs, whilst too low levels of IT may not be efficacious 158 _ Discussions Unlike the administration of purified IT proteins of which the dosage can be accurately manipulated, the in vivo expression level of the transfected gene could be difficult to control Different plasmid, when delivered into different cells, could be expressed at different levels Besides, according to the manufacturer of this in vivo transfection kit, the expression levels of the transfected gene might not be equal in different organs and tissues This could result in unequal localized concentration of this IT in different parts of the body Lastly, the administration of IT plasmids using the current in vivo transfection strategy requires the injection of an amount of delivery solution equals to 1/10 of the recipient’s body weight! Therefore, new non-viral based transfection methods need to be developed in order to deliver genes into human body 4.11 Conclusions and future directions The inflammatory reaction in acute GVHD begins with T cell infiltration Although all nucleated cells express MHC class I molecules on the cell surface which are major antigens for activating alloreactive T cells, T cells preferentially infiltrate certain organs, such as liver, skin and intestines, and cause serious damages to these organs Our study found that the distinctive pattern of chemokines expressed in target/nontarget organs correlate with the infiltration kinetics of the donor T cells into these organs Our previous study demonstrated that blocking certain adhesion molecules such as L-selectin and α4-integrin can change the donor cell homing pattern and ameliorate murine acute GVHD (Li et al., 2001) Similarly, in vivo neutralization of chemokines or blocking their respective receptors might delay the infiltration of donor T cells into the target organs, and thereby modulate the progression of GVHD However, our data suggest that targeting more than one chemokines or their receptors might be necessary to be effective 159 _ Discussions Upon entering the host system, alloreactive donor T cells undergo vigorous proliferation before infiltrating into the target organs Theoretically, those T cells which remain quiescent and non-reactive against the alloantigens should be of different antigenic specificities, and would not induce GVHD upon transplantation into another immune-compromised host of the same antigenicity as the primary host However, this is not often the case as many researchers found that that the injection of these purified resting T cells stimulated via the in vitro MLC could not completely prevent GVHD in the host Also, in clinical practice, a negative mixed lymphocyte reaction (MLR) of recipient T cells responding to donor cells does not necessarily mean that a donorspecific tolerance has been established In our study, we employed an in vivo alloantigenic system to fully stimulate the allogeneic donor T cells We observed that allogeneic donor T cells that did not proliferate even under this stimulation for more than days were still able to induce GVHD upon transferring into secondary recipients This implies that there exists an alloreactive T cell population that does not respond to in vitro and initial in vivo alloantigenic stimulation, but can mount a delayed alloresponse in vivo This helps explain a clinically observed phenomenon that acute rejection of transplanted organs can occur years after transplantation while MLR shows donor specific non-responsiveness Another surprising finding in our study is that the proliferating T cells that remain in the lymphoid organs days after transplantation, when isolated and re-injected into secondary recipients, induced only a much delayed form of GVHD The mechanism for this observation is still not clear We feel that these cells might be some helper T cells which aids the true allogeneic effector T cells to activate and proliferate, or just some bystander cells that were stimulated into proliferation during the “cytokine storm” period at the initial phase of GVHD They themselves might not involve directly in the killing of host cells This is 160 _ Discussions supported by our previous observation that most alloreactive donor T cells would have migrated out of the lymphoid organs and infiltrated the target organs by day posttransplantation Amelioration of GVHD was attempted using DT390-IL-2 recombinant IT plasmid with encouraging results DT390-IL-2 has been demonstrated to be highly potent against PHA-activated T cells, and its specificity is restricted only to IL-2R expressing cells in MLC When administered in vivo into the recipient mice twice after allogeneic T cell transfer, three of the four treated mice were still surviving by day 90 post-transplantation, while all the control mice and other mice treated with different IT constructs died before day 50 However, further investigations are required to confirm the DT390-IL-2 secretion level in the MLC supernatant and serum in the treated mouse using western blot and ELISA Besides, the in vivo localized expression of this IT will also be studied using an anti-DT Ab in IHC Although recipient mice treated with DT390-IL-2 showed delayed or ameliorated GVHD, spleen and LN cells of these recovered mice should be isolated to determine if engraftment is successful, and if the T cells conserve their immunocompetency Experiments to manipulate the dosage, timing and frequency of gene delivery are also essential in optimizing the expression level of DT390-IL-2 plasmid without causing toxicity to normal host cells and organs 161 ... may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD The predominantly expressed chemokines MIP1α and Mig in the spleen,... CD8+ T cells These observations indicate that other chemokines other than MIP-1α play a critical role in the recruitment of CD4+ T cells into the target organs It was recently reported that CXCR3... just some bystander cells that were stimulated into proliferation during the “cytokine storm” period at the initial phase of GVHD They themselves might not involve directly in the killing of host