TRENDSin
`Immunology
`
`Fel_)ruary 2005
`· -· ,Vf?t 26, No. 2
`pp,·65-118
`
`Editor Christine Ferguson
`
`Assistant Editor Morag Robertson
`
`Editorial Coordinator Simon Bradbrook
`
`Illustrations The Studio
`
`Publishing Manager 0. Claire Moulton
`
`Editorial Enquiries
`Trends in Immunology
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`Advisory Editorial Board
`G. Anderson, Birmingham, UK
`J.D. Ashwell, Bethesda, USA
`F.H. Bach, Boston, USA
`F.R. Carbone, Melbourne, Australia
`M.F. Flajnik, Baltimore, USA
`S. Grabbe, Munster, Germany
`B.A. Imhof, Geneva, Switzerland
`P.W. Kincade, Oklahoma City, USA
`M. Krone,nberg, La Jolla, USA
`J.D. Lambris, Philadelphia, USA
`A. Mantovani, Milan, Italy
`L.M. Moretta, Genova, Italy
`G.J.V. Nossa!, Melbourne, Australia
`P.S. Ohashi, Toronto, Canada
`L.A.J. O'Neill, Dublin, Ireland
`R.M. Ransohoff, Cleveland, USA
`K.B.M. Reid, Oxford, UK
`P. Ricciardi-Castagnoli, Milan, Italy
`C. Terhorst, Boston, USA
`K. Wood, Oxford, UK
`C.Y. Yu, Columbus, USA
`R.M. Zinkernagel , Zurich, Switzerland
`
`MMl--11
`I Research Focus
`65 Rapid control of foot-and-mouth disease
`outbreaks: is RNAi a possible solution?
`Marvin J. Grubman and Teresa de los Santos
`
`68 Gfi-1: another piece in the HSC puzzle
`Sonia Cellot and Guy Sauvageau
`
`@ji,jj.j,-
`72 Old lysosomes, new tricks: MHC II dynamics in DCs
`Amy Y. Chow and Ira Mel Iman
`79 Chronic antigenic load and apoptosis in immunosenescence
`Lia Ginaldi, Massimo De Martinis, Daniela Monti and Claudio Franceschi
`85 Vaccination against encapsulated bacteria in humans: paradoxes
`Sandra Weller, Claude-Agnes Reynaud and Jean-Claude Weill
`· ·
`90 Activation-induced .deaminase: controversies and open questions
`Vasco M. Barreto, Almudena R. Ramiro and Michel C. Nussenzweig
`
`i,t¥Mil'II.
`97 Merging complexes: properties of membrane raft assembly during lymphocyte
`signaling
`William Rodgers,-Darise Farris and Sudha Mishra
`104 The mannose receptor: linking homeostasis and immunity through sugar
`recognition
`Philip A. Taylor, Siamon Gordon and Luisa Martinez-Pomares
`111 Sinks, suppressors and antigen presenters: how lymphodepletion enhances
`T cell-mediated tumor immunotherapy
`.
`1
`,
`Christopher A. Klebanoff, Hung T. Khong, Paul A. Antony, Douglas C. Palmer and
`Nicholas P. Restifo
`
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`L G~aca, T:: ·
`IL-7: maint~ining.T-cell m_emory and achieving ·homeostasis ··
`L.M. e·radley, L j-tay~·es and S.L. ·swain
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`A stromal address ·code_ defineq by fibroblasts
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`lni_mune
`·
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`TRENDS]
`www.trends.com
`
`Cover: The intracellular events invo lved in the assembly of pMHC II complexes occu'r in the endocytic pathway'of professional APCs.
`In this issue rnmft-lft:f). Ira Mell man and Amy Chow put forward their view thal production of, and escape by, p0MHCUC9mple-;.es in
`an otherwise 'normal' complement of endocytic organelles ocurs because these .events ~r<;_tightly link~d t_o t/]e devi lpp'Tjent ~rid
`maturation status of the APCs during an immune response. Cover ir11age by David ~oonay. <;over de0sigl) blf K~te .)onjls. '.....,l w
`
`Miltenyi Ex. 1032 Page 1
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`

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`ELSEVIER
`
`Sinks, suppressors and antig·en ..
`presenters: ho\N lymphodepl~tion
`enhances T cell-mediated t·u,mor
`immunotherapy
`
`Christopher A. Klebanoff1-2, Hung T. Khong 3, Paul A. Antony2, Douglas C. Palmer2·*
`and Nicholas P. Restifo2
`
`1Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD 20814, USA
`2Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1502, USA
`3 University of South Alabama Cancer Research Institute, 307 N. University Blvd./M SB 2015, Mobile, AL 36688-0002, USA
`
`Lymphodepletion followed by adoptive cell transfer
`(ACT) of autologous, tumor-reactive T cells boosts
`antitumor immunotherapeutic activity in mouse and in
`humans. In the most recent clinical trials, lymphodeple(cid:173)
`tion together with ACT has an objective response rate of
`50% in patients with solid metastatic tumors. The
`mechanisms underlying this recent advance in cancer
`immunotherapy are beginning to be elucidated and
`include: the elimination of cellular cytokine 'sinks' for
`homeostatic Yc-cytokines, such as interleukin-7 (IL-7),
`IL-15 and possibly IL-21, which activate and expand
`tumor-reactive T cells; the impairment of co4+co25 +
`regulatory T (Treg) cells that suppress tumor-reactive
`T cells; and the induction of tumor apoptosis and
`necrosis in conjunction with antigen-presenting cell
`activation. Knowledge of these factors could be
`exploited therapeutically to improve the in vivo function
`of adoptively transferred, tumor-reactive T cells for the
`treatment of cancer.
`
`Introduction
`Adoptive cell transfer (ACT) of large numbers of auto(cid:173)
`logous tumor-reactive T cells into a tumor-bearing host
`represents a promising therapy for the treatment of
`metastatic cancer in humans [l]. This exciting therapy
`uses the rapid ex uiuo expansion of tumor-infiltrating or
`-reactive lymphocytes (TILs), which are subsequently
`transferred in conjunction with the administration of a
`high-dose of a stimulatory cytokine, in particular inter(cid:173)
`leukin-2 (IL-2). Although other forms of immunoth erapy,
`such a s tumor-antigen (AgJ vaccination ur the admini(cid:173)
`stration of immune-stimulatin g eytokin es a lone, are
`capable of 1·aising tumor-r ,active T cells in vivo, they do
`not reliably indu ce the 1· gression ofla1·ge establis hed solid
`tumors (reviewed in Ref. 12] ). ACT is capable of mediating
`
`Corresponding author: Palmer, D .C. 1pal rncrd@ma il.nih .gov1.
`• Written in par tial fulfillm enL ot' a P hlJ in Biochemisu-y at the George Washi ngto n
`University, Washington , DC 20052, USA.
`Available online 23 December 2004
`
`tumor regression [3-61, howevet',) these effects are even
`more pronounced in the absence of host lymphocytes [7,8].
`This approach has resulted in the most consistent and
`dramatic clinical responses observed in the treatment of
`metastatic cancer [7] (Figure la).
`These clinical responses are associated with auto(cid:173)
`immune manifestations in sites that express shared
`melanocyte or melanoma Ags, such as the skin, in the
`form of vitiligo. With lymphodepletion, we have also
`observed melanocyte destruction at immune privileged
`sites, such as the eye (Figure lb,c). Although skin-de(cid:173)
`pigmentation has been previously associated with immu(cid:173)
`notherapies, inflammation of the anterior segment of
`the eye has not been previously observed, and might be
`indicative of a more potent activating stimulus. Fortu(cid:173)
`nately, these eye manifestations are treatable with local
`steroids, which do not detract from the antitumor immune
`destruction.
`I
`The specific mechanisms that contribute to this
`enhanced state of immunity remain poorly understood.
`Recent insights in two rapidly expanding fields, the
`cytokine-mediated homeostasis of mature lymphocytes
`by Yc-cytokin.es, such as IL-7, IL-15 and IL-21 , and the
`control of autoreactive T cells by CD4 +cn25 + regulatory
`T (Treg) cells, provide the foundation for what might be
`occurring after lymphodepletion. The removal of lympho(cid:173)
`cytes that compete for homeostatic cytokines or suppress
`tumor-reactive T cells might contribute to the enhance(cid:173)
`ment and subsequent tumor destruction by the adoptively
`transferr:ed T cells. The less described role of antigen(cid:173)
`presenting cell (AFC) activation by lymphodepletion
`might also have a role in T-cell activation. Knowledge of
`these factors presents th e potential for therapeutic
`exploitation in the treatment of metastatic cancer in
`humans.
`
`A link between lymphodepletion and augmented
`immune function
`It has long been observed that transfer of small numbers
`of T cells into lymphopenic hosts results in T-cell
`
`www.sciencedire<:t.com 1471 -49061$ · see fro nt mailer i>ublis hed by Elsevie r ltd , doi:10. 1016/j.it.2004.12.003
`
`Miltenyi Ex. 1032 Page 2
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`

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`Figure 1. Potentiation of immune response to non-mutated shared self-antigens and tumor antigens by lymphodepletion. Following adoptive cell transfer therapy with
`tumor-infiltrating lymphocytes (Tlls). in the setting of chemotherapy-mediated lymphoablation: (al Computed tomography (CT) scan of liver metastases in a melanoma
`patient. Metastatic lesions progressed ra pidly before th e treatment (from day -45 to day - 25) then regressed dramatically after adoptive T-cell transfer (day + 34).
`(bl Exten sive destruction of normal melanocytes (vitiligol in a patient who experienced a rem arkable clinical response. (c) Anterior uveitis (i nflammation of the eye) in a
`patient who exhibited >99% tumor reduction Photographs co urtesy of S..A. Rosenberg.
`
`expansion, a process described as homeostatic prolifer(cid:173)
`ation [9-16]. As the T cells proliferate, they assume an
`Ag-experienced or memory phenotype, which is indicated
`byupregulation ofCD44, Ly6C and CD122 (IL-2-IL-15R~)
`[12,15]. This T-cell expansion and acquisition of a memory
`phenotype is also associated with enhanced effector
`functions, determined by ex vivo analysis of interferon-y
`(IFN-y) release and cytolysis [12,14,15]. In a recent
`autoimmunity study, King et al. showed that the inherent
`lymphopenia in non-obese diabetic (NOD) mice and
`increased expression of IL-21 drives the homeostatic
`expansion of autoreactive cells, precipitating self-tissue
`destruction [17] . Furthermore, adoptive transfer of
`nai've lymphocytic choriomenigitis virus (LCMV)-specific
`CDS + T-cell receptor (TCR) transgenic splenocytes into
`LCMV-infected lymphodepleted, but not replete, hosts
`rapidly reduced viral titers to below detection limits [13].
`Analogous results have also been observed in another
`model, based on the 2C T-cell receptor (TCR) transgenic
`model. 2C TCR transgenic T cells are specific for an
`Ld-restricted a:-ketoglutarate dehydrogenase epitope or
`the SIYRYYGL peptide in the context MHC I H2-Kb_
`These cells expand homeostatically in Rag_ 1_ mice and
`specifically treat tumors, at a comparable level to the
`T cells derived from Ag-specific vaccination [15]. Dummer
`et al. observed that adoptive transfer of a polyclonal
`population of T cells into a Rag-I- or sublethally
`irradiated mouse specifically inhibits tumor growth [18].
`It is interesting to note that in these models, lympho(cid:173)
`penic-induced activation and pathogen or tumor clearance
`are generated in the absence of Ag-specific vaccination.
`Ag-specific CDs + T cells are rnquired for pathogen or
`tumor clearance, and the need for subsequen t immuniz(cid:173)
`ation might be negated [19,20]. These model Ags have
`given us great insighL into homeostatic proliferation
`but focus primarily on Ags that are of a foreign OTigin.
`Perhaps using a self~ OT tumor-antigen might provide a
`setting that is more analogous to the human condition.
`A recently described tumor model, Pmel-1, which uses a
`transgenic CDS+ T-cell specific for both the native and
`altered peptide ligand of the melanocyte and melanoma
`
`www.sciencedirect.com
`
`differentiation molecule gplO0, might enable us to better
`model unmutated self-antigens in the clinical setting [5,21].
`-
`~
`.
`Evidence for the presence of homeostatic cellular
`· cytokine 'sinks'
`The proliferation ofadoptively transferred Tcells in lympho(cid:173)
`penic hosts can be reduced in a dose-dependent manner,
`either by increasing the total number of Ag-specific cells
`transfused or by co-transferring an 'irrelevant' population of
`Tcells [9, 15,16,22]. Host CDS + T cells have a dominant role
`in modulating both donor CDS + and CD4 + T-cell expansion
`in lymphopenic hosts [16]. In the srupe study, host CD4 +
`cells inhibited donor CD4 +, and to a lesser extent CDs +
`T-cell proliferation.
`· The ability of the host to inhibit the proliferation of
`adoptively transferred T cells could be the result of
`physical contact between T cells or competition between
`T cells for self-MHC-peptide complexes and supportive
`homeostatic cytokines (Figure 2): Nai've T-cell prolifer(cid:173)
`ation is a result of self-MHC-peptide interactions in a
`lymphopenic environment (reviewed in Ref. [23]), whereas
`memory T cells proliferate independently of these inter(cid:173)
`actions [12,16]. This is suggestive of Ag-independent
`activation, a notion that has recently received a great
`deal of attention. Some studies have focused on the role of
`host-derived IL-7 and IL-15, and more recently IL-21, Ye
`cytokines known to stimulate and induce the proliferation
`of mature T cells [24-26].
`In transgenic mice that overexpress IL-7 or IL-15, there
`is a substantial increase in the absolute number ofT cells
`[27 ,28]. This increase is driven largely by the expansion of
`CDS+ CD44 high memory T cells. Exogenous administration
`of IL-15 or induction of host expression of IL-15 in
`wikltype mice by reagents, such as poly I:C, lipopolysac(cid:173)
`charicle (LPS) and type I-IFNs, causes a selective increase
`in the proliferation of CDs·r c1144high T cells in vivo
`[29-31]. Exogenous administration of IL-7 to mice
`enhances T-cell number and function [32,33] and, in both
`intact and immunodeficient non-human primates, causes
`a considerable yet reversible increase in the circulating
`levels of naive and Ag-experienced CD4 + and CDs+
`
`Miltenyi Ex. 1032 Page 3
`
`

`

`(a)
`
`(b)
`
`~
`
`-
`
`•IL-21?
`
`• -■ IL-15
`
`♦ IL-7
`
`. .
`
`Lymphoablation ~ rJJ ' •
`and
`• • ♦ -~
`•
`'>-· ■
`I-----------+-
`transfer of tumor-
`Ag-specific tumors
`
`II
`
`Horneostptic
`-expansion ·ot
`transferred t ce1ls--
`
`Endogenous lymphocyte repertoire
`under normal homeostasis
`
`Repopula.tion with tumor-Ag-
`9pecific T cells
`
`TRENDS in Immunology
`
`Figure 2. Removal of consumptive cytokine 'sink' by lymphoablation. (a) Under normal homeostasis, a smaller quantity of cytoki nes [IL-7 (yellow), IL-15 (green) and possibly
`IL-21 (red)] are available owing to consumption by the endogenous lymphocyte population. (b) After lymphodepletion preconditioning by chemotherapy or irradiation, the
`co nsum ptive cellular cytokine 'sinks' are removed, enab ling the adoptively transferred T cells a less competitive environment a·nd greater acdess to IL-7, IL-15 and IL-21.
`(cl This is followed by preferential homeostatic expansion and re-population of the peripheral lymphoid compartments with the adoptively transferred, tumor-reactive T cells.
`
`T cells [34] . Although similar studies have yet to be
`conducted in humans, there is evidence to support an
`inverse correlation between serum levels of IL-7 and the
`severity of lymphopenia caused by conditions, such as
`AIDS and cytotoxic drug therapy (reviewed in Ref. (35]) .
`Currently, there are no data available that evaluate the
`in vivo effects ofIL-15 in primates or humans.
`Studies that examine mice deficient in IL-7 or IL-15
`have demonstrated that these cytokines have a supportive
`role in the survival and/or proliferation of adoptively
`transferred T cells in lymphodepleted hosts [36-40]. The
`absence of IL-7 inhibits nai:Ve CD4 + and CD8 + T-cell
`homeostatic proliferation and survival in a lymphopenic
`environment. The proliferation of donor cells in these
`IL- 7-t- hosts is rescued by the administration of
`exogenous IL-7 [36,41]. IL-7 has a significant role in
`na'ive T-cell homeostatic proliferation but has little impact
`on memory T-cell expansion and survival in a lymphopenic
`host [37,38]. In contrast to IL-7, IL-15 does not contribute
`to naive T-cell homeostasis but does have a pivotal role in
`memory CD8 + T-cell proliferation and durability (38] . The
`homeostatic expansion of memory CD8+ T cells does not
`rely solely on IL-7, however, these cells do constitutively
`express high levels of both IL-7Ra (CD127) and the
`components of the IL-15-IL-2 heterodimeric complex,
`IL-2-IL-15R~(CD122) and Ye (CD132), and thus might be
`sensitive to IL-7 [30,38,42].
`In summary, it is clear that cytokines can have a
`profound impact in regulating the homeostasis ofT cells in
`
`Box 1. Unresolved questions regarding the use of Ye
`cytokines
`
`• Does the absence of endogenous IL-15 impair the early or late in
`vivo pro liferation, functionality, and maintenance of adoptively
`transferred tumor-specific T cells, and thus their ability to mediate
`tumor destruction?
`• What infl uence does host-derived IL-7 alone, or in combination
`with IL-15, have on tumor-reactive T cells and their function?
`• What is the role of IL-21 in homeostatic proliferatio n, and does
`exogenous administration improve the ability of adoptive trans(cid:173)
`ferred tumor-specific T cells to kill tumor?
`
`www.sciencedirect.com
`
`both the normal and lymphopenic settings. Thf expression
`of IL-7 and IL-15 receptors on 9Uemory T cells might
`indicate that the administration of these cytokines could
`enhance both the survival and proliferation of adoptively
`transferred, tumor-reactive T cells in vivo. In a recently
`described murine tumor model [5] 1 it appe~rs that the
`long-term tumor treatment efficacy of adoptively trans(cid:173)
`ferred, tumor-specific CD8 + T cells is compromised in
`whole-body irradiated IL-15- 1- but not wildtype hosts
`[43]. Endogenously produced IL-15 might contribute to a
`sustained antitumor treatment response by adoptively
`transferred CD8+ T cells in a lymphopenic setting. It is
`likely that non-tumor-specific T cells and other immune
`cells, such as natural killer (NK) cells, consume IL-15 in
`the wildtype host. Thus, there could be competition for
`homeostatic cytokines in the replete host. It is also
`important to mention the newly discovered cytokine
`IL-21, which shares homology with IL-2 and IL-15 and
`binds their common ye receptor [44,45]. This :cytokine
`might augment the function of adJptively transferred
`tumor-specific CD8+ Tcells (17,46] and is the subject of
`ongoing investigations. Many unresolved ; questions
`regarding the use of ye receptor cytokines remain to be
`answered (Box 1).
`
`Role of regulatory cells in antitumor immunity
`Naturally occurring and induced CD4 + CD25 + Treg cells
`can potently suppress immune responses to self~Ags and
`foreign Ag~ in both humans and mice. The phenotype and
`function of Treg cells have been reviewed in detail
`elsewhere [47,48]. There are some features of Treg cells
`that might be of particular interest to tumor immuno(cid:173)
`logists. CD4 + CD25 + Treg cells express high levels of cell(cid:173)
`surface molecules typically associated with activation;
`these include CD25 (IL-2Ra), glucocorticoid-induced
`tumor necrosis factor (TNF)-receptor (GITR) and cytotoxic
`T lymph ocyte-associated Ag-4 (CTLA-4). I n addition,
`they also express a unique intracellular protein, Foxp3.
`Treg cells are completely absent in mice that are deficient
`in the genes encoding IL-2, IL-2Ra, IL-2R~ and Foxp3,
`suggesting a crucial role for both IL-2 signaling and Foxp3
`
`Miltenyi Ex. 1032 Page 4
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`

`

`expressinn in Treg-cell ontogeny and survival [49-51]. The
`role of Treg cells in maintaining immunological tolerance
`to self-Ags has been illuminated, in part, through adoptive
`transfer experiments. A spectrum of tissue-specific auto(cid:173)
`immune diseases, including thyroiditis, oophoritis, gas(cid:173)
`t ritis and inflammatory bowel disease, occurs when
`immunodeficient mice are transfused with T cells depleted
`of CD4 + CD25 + cells [52,53]. The concomitant transfer of
`Treg cells abrogates development of these autoimmune
`diseases. In some mode1s, transfer of Treg cells after the
`onset of disease can even be curative [54]. The close
`correlation between autoimmunity an_d tumor immunity
`[5,7,55-59] suggests that Treg cells have a crucial role in
`T-cell tolerance to tumor [60--62].
`Although the significance of Treg cells in human
`cancers has only recently begun to be explored, many of
`the findings in mice could be recapitulated in humans
`[61,63,64]. Wang et al. recently reported on the isolation of
`CD4 + CD25 + TIL clones derived from a melanoma patient
`(65]. These clones displayed many of the phenotypic and
`functional properties associated with naturally occurring
`Treg cells and were antigen-specific. Another report has
`shown that Treg cells present in the circulation of patients
`vaccinated against melanoma Ags can suppress the
`proliferation of a polyclonal population of CD4 + CD25 -
`T cells [66], however, the Ag-specificity of these cells was
`not determined. Treg cells capable of suppressing the
`in vitro function of tumor-reactive T cells in humans has
`also been found in other tumors besides melanoma (67,68].
`In one instance, tumor deposits taken from patients
`with lung cancer reportedly contained large numbers of
`CD4 +cn25+ Treg cells capable of suppressing the
`proliferation of autologous TILs [68]. In contrast to these
`murine studies, no conclusive data link the in vivo
`function of tumor-reactive Treg cells and the progression
`of cancer in humans, although some recent evidence
`suggests that Treg tumor infiltration might be associated ·
`with survival of cancer patients (69]. However, removal of
`these regulatory elements (Box 2), perhaps in conjunction
`with cytolrine sinks, could reverse or prevent the tolerance
`of adoptively transferred tumor-reactive T cells (Figure 3).
`
`Effects of lymphodepleting radiation and chemotherapy
`on APC function
`Lymphodepletion before ACT uses total body irradiation
`(TBI) or cytotoxic drugs. Although these modalities were
`initially intended to deplete the lymphoid compartment of
`r ecipients, they can also facilitate the presentation of
`t wnor antigens by triggering t umor cell death and antigen
`release . Subsequently, these antigen s can be t aken up and
`presented by APCs to enhance the activation of th e
`adoptively transferred tumor-reactive cells (70].
`Indirect evidence indicates that there might be other
`beneficial effects of lymphodepletion in our current ACT
`model. Irradiation antl/ot chemotherapy leads to acti(cid:173)
`v ation of host cells, resulting in the release of prn(cid:173)
`infian.unatory cytokines, such as TNF-a., IL-1 and IL-4
`[71-74], and upre6'1.l.1ation of co-stimulatory molecules,
`such as CDS0 [75). ln a recent study, activation markers
`I-Ab (class II) and CD86 were upregulated on splenic DCs
`as early as 6 h after TBI. Furthermore, ex vivo IL-12
`WW'.V,3Ciencedirect.com
`
`Box 2. Eradication of !regs• c-
`
`Although ·Food and Drug Administration-approved drugs are
`available that can selectivel-y deplete CD25-expressing cells in
`humans, including humanized anti-Tac (anti-CD25) and ONTAK'·"
`(IL-2 conjugated !o diphtheria toxin), it ls cur(ently unknown what
`effect these reagents ·mi.ght·have on Treg cells as well as on activated
`tumor-reactive T ce·us:·
`·
`~-·
`Until a unique cell -surl'ace, 11rfe_a·ge marker capable of 'discriminat·
`ing between Treg . and activai~ci.:r cells can be .discovered, non·
`specific lymphodepletion r-night _pe the only practical approach to
`removing Treg cells from patient; to·r the purpose of augmenting
`their in vivo immune reactivity.to a tumor.
`·
`
`production was significantly higher from DCs isolated 6 h
`· after irradiation than DCs from non-irradiated mice [76].
`Consistent with this finding, serum levels of IL-12 were
`also increased in these rnice. Elimination of competitive
`T cells through lymphodep1etion might improve donor
`T-cell access to, and activation by, antigen-bearing APCs
`[77), although other workers have found that competition
`might not play an influential role [78].
`In addition to enhanced APC function and- availability,
`the preconditioning regimen can\1.amage the integrity of
`mucosa! barriers through radiation-induced apoptosis of
`the cells lining these organs. Damage inflicted on the
`intestinal tract might permit the translocation ofbacterial
`products, such as LPS, into the systemic circulation [79] .
`LPS activates T cells in vivo [31] and might enhance the
`antitumor response. Thus, proinfiammatory cytokines
`and microbial products provide crucial 'danger signals'
`for the activation and maturation of DCs, thus enhancing
`T cell-mediated tumor treatment.
`Although initially beneficial, lymphodepletion can
`depress the absolute number of host APCs. As reported
`in several nmrine studies, the total number of mono(cid:173)
`cytes, macrophages and DCs are only slightly reduced at
`6 and 24 h but are significantly reduced 5 days after TBI
`[71,76]. This could be detrimental in the case of the
`current tumor therapy model, which requires an active
`vaccination for successful tumor therapy [5]. Interest(cid:173)
`ingly, late clearance did not hinder 1the activation of the
`donor T cells [76). This corroborates with several reports
`that demonstrate antigenic stimulation, befqre APC
`clearance, is all that is required to initiate activ ation
`and proliferation of T cells [80,81].
`
`Therapeutic implications and future directions
`It is clear t h at lymphodepletion before adoptive transfer of
`tumor-r eactive T cells into animals and humans with
`cancer augments in vivo function of th e transferred cells
`and the therapeutic outcome. Increased access to t he
`homeostatic cytokines, such as IL-7 and IL-15 , th rough
`elimination of cytokine sinks, eradication of the suppres(cid:173)
`sive influence of Treg cells and enhancement of APC
`activation and availability appear to be the underlying
`mechanisms involved in this paradigm (Figure 4). The
`mechanisms are complex, however, elucidation through
`the use of targeted cytokine administr ation, add-back of
`sink or suppressor elements and selective deletion or
`addition of APCs might explain how these mechanisms
`interact.
`
`Miltenyi Ex. 1032 Page 5
`
`

`

`(a)
`
`Lymphoreplete
`
`(b)
`
`Lymph nodes
`
`Melanoma
`
`~
`
`·-~J.o, .
`t
`
`T~mor
`excised ·
`\
`
`Administration of
`ex vivo expanded Tlls
`
`'
`
`Tumor sites
`
`~p MHC I
`~ MHC II
`tl IL-2R
`
`1:i T helper
`.
`Treg
`tji CTL
`
`~ TAA
`
`•
`IL-2
`♦ IL-7
`♦ IL-10
`
`111
`
`ll-15
`
`♦ TGF-~
`
`TRENDS in Immunology
`'
`Figure 3. Removal of the inhibitory effect of Treg cells and activation of DCs by lymphoablation. (al Under normal homeostasis, Treg cells (red) i'n the tumor draining lymph
`node (TDLN), might exert inhibitory effects on the transferred tumor-specific T cells !blue). In add ition, Treg cells m ight prevent CD4.,. Th cells (green) from providing
`supportive cytokines, such as IL-2 (blue). OCs (pink) in the tumor, wh ich cross-present tumor-associated antigens (TAAs), m ight induce tolerance in the absence of
`inflammation and in the presence of immunosuppressive cytokines, such as IL-10 (brown) and TGF-13 (purple). The net o utcome of these inhibitory factors cou ld abrogate an
`effective antitumor response. (bl After t he induction of lymphopenia, ex vivo expanded tumor-reactive Tlls are adoptively transferred (blue). This preconditioning before cell
`transfer effectively eliminates Treg cel ls and other cytokine-competing cells, thus removi ng i nhibition and freeing supportive cytokines, such as IL-2, IL-7 {yellow) and IL-15
`(green). The ablative conditioning might also activate DCs that are capable of cross-presenting TAAs in the presence of danger signals. The effect of ablation is a 'permissive'
`and activating environment that augments tumor-specific T cell-induced tu mor destruction.
`
`Other modalities that mimic or enhance the positive
`effects of lymphodepletion could have great therapeutic
`potential. The exogenous administration of supportive
`cytokines, such as IL-2, IL-7, IL-15 or IL-21, either alone
`or in concert, could stimulate the adoptively transferred
`T cells and enhance their tumor-killing ability. Ways to
`selectively deplete or inhibit CD4 + CD25+ Treg cells
`with the use of antibodies against CD25 or GITR remain
`the pursuit of many workers, although clear effects have
`been less then forthcoming. Modulation of adoptively
`transferred cells either through overproduction of
`cytokines, such as IL-7, IL-15 (43] or IL-21, pr cytok.ine
`receptors, such as IL-7Ra and IL-15Ra., are currently
`being explored as possible means to enhance 'l'-cell
`www.sciencedirect.com
`
`function
`in vivo. Reagents that stimulate host
`expression of IL-15 and IL-15Ra. [30,31,82], including
`type I IFNs, CpG DNA, LPS, dsRNA and dsRNA(cid:173)
`imitator s, such as polyLC, might enhance the function(cid:173)
`ality of adoptively transferred cells.
`Finally, although IL-2 has been the preferred
`cytokine for the ex vivo [83J and in vivo [7,81 activation
`and expansion of tumor-reactive T cells, it also drives
`Treg prolifemtion and activation [84J and triggers
`aJ)Optosis in activated T cells (85]. Exploration of
`other cytokines that can drive tumor-reactive 1.1 cells,
`such as IL-7, IL-15 and IL-21, might prove to be more
`efficacious in the activation of adoptively transferred
`tumor-reactive cells.
`
`Miltenyi Ex. 1032 Page 6
`
`

`

`Eliminate
`cytpkine sinks
`
`Eradicate
`regulatory
`elements
`
`Enhance APC
`activation and
`availibility
`
`TRENDS in Immunology
`
`Figure 4. An interactive model for the mechanisms underlyi ng the impact of
`lymphodepletion on adoptively transferred T cells. (i) Elimination of cytokfne sinks:
`lymphoablation, either by chemotherapy or irradiation, reduces the consumptive
`cytokine 'sinks' created by the endogenous lymphocyte repertoire, thus enabling
`the transferred T cells a less competitive access to homeostatic cytokines, such as
`IL-7 and IL-15. (ii) Eradication of regulatory elements: Treg cells, such as the
`Co4+co25 + cells, that would otherwise exert an inhibitory effect o n th<\ transferred
`T ce ll s are diminished
`in number and
`function afte r
`lymphoabla tion.
`(iii) Enhancement of APC activation and availability: lymphoablatio n, either by
`irradiation or chemotherapy, can induce tumor apoptosis and necrosis, resulting in
`uptake and presentation of tumor antigens by APCs. Furthermore, there is some
`evidence that suggests that after lymphodepletion, APCs become activated, th us
`enhancing stimulation of the adoptively transferred T cel ls. The transferred T cel ls
`also have less competition with irrelevant T cel ls for APCs, which m ight improve
`activation. Th ese mechanisms can act independently or in synergy to augm ent the
`function of the tumor-reactive T cells.
`
`Acknowledgements
`We would like to thank Crystal L. Mackall for critical review of the
`manuscript and Steven A. Rosenberg for his support of translational
`research at the National Cancer Institute.
`
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