Fully MHC-Disparate Mixed Hemopoietic Chimeras Show Specific Defects in the Control of Chronic Viral

This information is current as Brent H. Koehn, Matthew A. Williams, Keshawna Borom, of October 3, 2021. Shivaprakash Gangappa, Thomas C. Pearson, Rafi Ahmed and Christian P. Larsen J Immunol 2007; 179:2616-2626; ; doi: 10.4049/jimmunol.179.4.2616 http://www.jimmunol.org/content/179/4/2616 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Fully MHC-Disparate Mixed Hemopoietic Chimeras Show Specific Defects in the Control of Chronic Viral Infections1

Brent H. Koehn,* Matthew A. Williams,2* Keshawna Borom,* Shivaprakash Gangappa,* Thomas C. Pearson,* Rafi Ahmed,† and Christian P. Larsen3*

The establishment of mixed allogeneic chimerism can induce donor-specific transplantation tolerance across full MHC barriers. However, a theoretical disadvantage of this approach is the possibility that the state of mixed chimerism might negatively affect the recipient’s immune competence to control pathogens. Previous studies using murine models have not supported this hypoth- esis, because they indicate that acute viral infections are cleared by chimeric animals with similar kinetics to that of unmanipulated controls. However, chronic or persistent viral infections often require a more complex and sustained response with cooperation between CD4 Th cells, CTL, and B cells for effective control. The current study indicates that profound defects become manifest in the control of chronic pathogenic infections in MHC-disparate mixed allogeneic chimeric mice. Furthermore, we show that ineffective priming of Downloaded from the donor-restricted CTL response leads to persistence, as well as severe T cell exhaustion. Our results further suggest that either T cell adoptive immunotherapy or selected MHC haplotype matching partially restore immune competence. These approaches may facilitate the translation of mixed chimerism therapeutic regimens. The Journal of Immunology, 2007, 179: 2616–2626.

he establishment of hemopoietic chimerism shows con- using transient blockade of T cell costimulatory pathways in siderable promise as a strategy for the treatment of several conjunction with donor bone marrow and minimal or no my- T hematologic diseases and as a method to induce specific elosuppression to establish a state of stable mixed hemopoietic http://www.jimmunol.org/ immunologic tolerance to allogeneic organ transplants (1–4). Ide- chimerism in mice (4, 11–13). In concept, by selectively delet- ally, the tolerant state induced by therapeutic hemopoietic chimer- ing donor-reactive cells in the periphery and maintaining toler- ism should not only confer durable and imperturbable bidirectional ance by ongoing deletion of donor-reactive T cells developing unresponsiveness to donor and recipient Ags in the absence of in the thymus (14, 15), it was hoped that this approach would ongoing , but also allow the maintenance of better preserve a broad T cell repertoire and thus maintain pro- robust protective immunity against the panoply of pathogens and tective immunity. opportunistic infections that may be confronted by the recipient. Mixed chimerism is thought to offer an additional advantage

Furthermore, in order for therapeutic mixed chimerism approaches over complete chimerism for preserving protective immunity in by guest on October 3, 2021 to enjoy wide application for tolerance induction to organ trans- that the mixed chimera harbors the full complement of donor- and plants, it is important that these approaches be applicable across recipient-derived APC, B cells, and T cells, conceptually allowing varying degrees of MHC disparity. for effective generation of either recipient- or donor-restricted im- Early studies of chimerism induction used myelo- and lymphoa- mune responses (5, 16). Early experiments to assess the specificity blative approaches, which resulted in complete replacement of the of tolerance, and by implication immune competence, through the recipient’s hemopoietic system. Although these strategies success- application of skin grafts from third-party donors provided evi- fully achieved robust tolerance, they resulted in significant defi- dence for at least some degree of immune competence (16, 17). In ciencies in immune competence when performed across major other studies, MHC-disparate mixed chimeras rapidly controlled MHC disparities (5–8). As a result, emphasis shifted toward ap- infections when challenged with vaccinia virus or the Armstrong proaches to establish a state of mixed chimerism in which there is strain of lymphocytic choriomeningitis virus (LCMV),4 which nor- coexistence of donor and recipient hemopoietic elements in the mally causes an acute that is rapidly cleared in CD8ϩ T hopes of maintaining pathogen-specific immunity (5). cell-dependent manner (18–20). However, a different picture Initial approaches to mixed chimerism induction involved near emerged when mixed chimeras were challenged with a LCMV complete, nonselective depletion of the existing T cell repertoire variant (clone 13) (18) that produces a chronic infection in normal (9, 10), but more recently several protocols have been devised mice, requiring participation of both CD4 and CD8 T cells to clear viremia over a 2- to 3-mo period (21–25). Importantly, Armstrong and clone 13 strains are know to share 99.8% sequence homology, *Emory Transplant Center and Department of Surgery, and †Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322 including all defined CD4 and CD8 T cell epitopes (24, 26). These Received for publication October 11, 2006. Accepted for publication June 6, 2007. results suggested that mixed chimeras may have subtle, but im- The costs of publication of this article were defrayed in part by the payment of page portant immune defects that become manifest during chronic/per- charges. This article must therefore be hereby marked advertisement in accordance sistent infections, requiring a more complex and sustained immune with 18 U.S.C. Section 1734 solely to indicate this fact. response to achieve viral control. To date, the underlying immune 1 This work was supported in part by Research Grants AI44644 and AI040519 from mechanisms for these observations have not been defined. Given the National Institutes of Health and by the Carlos and Marguerite Mason Trust. 2 Current address: Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195. 4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; NP, 3 Address correspondence and reprint requests to Dr. Christian P. Larsen, Emory nuclear protein. Transplant Center, 5105 WMB, 101 Woodruff Circle, Atlanta, GA 30322. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 2617

the clinical importance in transplantation of persistent pathogens Flow cytometry and tetramer staining such as virus, polyoma BK virus, CMV, EBV, and MHC class I H-2Db,Kb,orLd tetramers complexed with LCMV nuclear more recently LCMV (27–33), coupled with the desire to apply protein (NP)396–404, gp33–41, gp276–286, or NP118–126 were pro- mixed chimerism approaches in both living and deceased donor duced, as previously described (39). Biotinylated complexes were tet- organ transplantation with varying degrees of MHC disparity (5, ramerized using allophycocyanin-conjugated streptavidin (Molecular 34), it is imperative that studies be undertaken to interrogate the Probes), and staining was conducted directly ex vivo. All Abs were pur- chased from BD Pharmingen, except the mAb clone H113 (anti-LCMV- immune competence of allogeneic mixed chimeras, to understand NP), which was purified and conjugated with the AlexaFluor 647 protein- mechanisms of immunodeficiency, and to develop strategies to labeling kit, per the manufacturer’s instructions (Molecular Probes). maintain or restore protective immunity. In the current study, we Splenocytes were RBC lysed, stained with the indicated probes at 4°C, and demonstrate that there are profound defects in the control of acquired using a FACSCalibur flow cytometer (BD Biosciences). FlowJo was used for analysis (Tree Star). For intracellular cytokine analysis, 106 spleno- chronic pathogenic infections in fully MHC-disparate mixed chi- cytes were cultured in the presence of a given peptide (0.1 ␮g/ml) and brefel- meric mice. Additionally, we show that the deficiency arises from din A for 5–6 h, at 37°C. Following the surface Ag staining, cells were stained inadequate development of donor-restricted T cells within the chi- for intracellular cytokines using the Cytofix/Cytoperm kit (BD Pharmingen), meras’ peripheral repertoire. As a result, despite the initial gener- according to the manufacturer’s instructions. The anti-IFN-␥ clone XMG1.2 ation of a functional host-restricted response, donor-derived cells was used for intracellular cytokine detection. within the chimera are a relative immunologic blind spot allowing active viral replication and promoting severe immune pathology. ELISA Furthermore, the sustained viremia promotes a profound degree of At given time points, serum was collected from individual mice, and T cell exhaustion of the recipient-restricted response, further com- LCMV-specific serum Ab titers were determined by a solid-phase ELISA, pounding the immune incompetence. Finally, we provide evidence as described previously (40). Analysis for allotype-specific responses was Downloaded from performed in duplicate using biotinylated anti-mouse IgG2aa- or IgG2ab- that immune competence can be largely restored by T cell adoptive specific detection Abs (BD Pharmingen). The LCMV-specific Ab titer was immunotherapy or selective MHC matching. These considerations determined by normalizing to background binding (OD492), and is ex- should be the focus of future translational and clinical investiga- pressed as the reciprocal of the highest dilution showing a reading Ͼ2 SDs tions to enhance the safety and effectiveness of strategies to pro- from background. mote tolerance through chimerism induction protocols. Serum cytokine quantification http://www.jimmunol.org/ Serum levels of TNF-␣, IL-12p70, and MCP-1 were measured in a mul- Materials and Methods tiplex assay at the indicated time points by Cytometric Bead Array (BD Mice and viral infections Biosciences), according to the manufacturer’s instructions (41, 42). C57BL/6J (CD45.2ϩ), B6.SJL-PtprcaPep3b/BoyJ (CD45.1ϩ), BALB/cJ, CB6.F1/J (BALB/c ϫ B6), MHC II⌬/⌬ (deletion of entire class II region, In vivo cytotoxicity assay C57BL/6 background) (35), and B6-nu/nu mice were purchased from The In vivo epitope-specific killing was measured using a protocol developed Jackson Laboratory. KbDb-deficient-B6 mice were maintained at the by Barber et al. (43), with modifications. Target splenocytes from naive Emory Division of Animal Research (36). Crosses were generated and CB6.F mice were differentially costained with CellTrace Far Red maintained at Emory University’s Division of Animal Resources. All an- 1 by guest on October 3, 2021 DDAO-SE (2 ␮M; Molecular Probes) and CFSE (0.0025, 0.25, or 5 ␮M; imals were male and between 4 and 8 wk of age when experiments were Sigma-Aldrich) so as to identify five unique populations. Each population begun. Mice were infected with 1 ϫ 106 PFU LCMV clone 13 injected i.v. was then pulsed with the indicated peptide, washed, then recombined and to initiate chronic infection, or 2 ϫ 105 PFU LCMV Armstrong i.p. to adoptively transferred i.v. in equal quantities to chronically infected recip- initiate acute infection (37). All experiments were conducted in accordance ients. Five hours later, the spleens were harvested and single cell suspen- with institutional guidelines for animal care and use. sions were acquired directly using a FACSCalibur. The relative survival of each target population was normalized to the unpulsed population, and the Induction of hemopoietic chimerism percentage of specific killing was calculated against the survival of that population in naive (n ϭ 3) control animals, as described previously (43): Four-week-old recipient (B6-CD45.1ϩ congenic) mice were pretreated on 100 – ((percentage of peptide pulsed in infected/percentage of unpulsed day Ϫ1 with 20 mg/kg busulfan, i.p. (Busulfex; Orphan Medical). Bone in infected)/(percentage of peptide pulsed in uninfected/percentage of marrow from donor mice was flushed from tibiae and femurs, resuspended unpulsed in uninfected)) ϫ 100. at 2 ϫ 107 cells/500 ␮l sterile saline, and injected i.v. into busulfan-treated recipients. On days 0 and 2 postinfusion, recipients were also given 0.5 mg Thymic reconstitution each of hamster anti-mouse CD40L mAb (MR1; Bioexpress) and CTLA- 4.Ig (Bristol-Myers Squibb) administered i.p. (4). Recipients of BALB/c Recipient B6-nu/nu (athymic) mice were treated as above for the induction marrow were also given 0.2 mg of anti-NK1.1 (PK136) on day Ϫ1, to of hemopoietic chimerism, using T-depleted bone marrow. Additionally, overcome the NK cell barrier (38). Biweekly monitoring for levels of donor thymic fragments from the indicated donors were transplanted under the chimerism was done by flow cytometric measurement of donor CD45.2 kidney capsule, under general anesthesia (44). Animals were allowed to expression on PBL. reconstitute for 90 days, at which time mixed chimerism and T cell recon- stitution were confirmed. Viral quantification T cell enrichment and adoptive transfer ␮ Total RNA extraction was conducted on 100 l of whole blood using a ϩ ϩ QIAmp blood RNA extraction kit (Qiagen), and first-strand cDNA was T cells were enriched using the mouse CD8 or CD4 T cell isolation kit synthesized using an ABI high capacity cDNA archive kit (Applied Bio- and an AutoMACS separator (Miltenyi Biotec). Purity was confirmed to be Ͼ92% CD8ϩ or CD4ϩ by FACs analysis, with Ͻ0.5% contamination due systems), according to the manufacturer’s instructions. The LCMV primer ϩ ϩ sets and probe for TaqMan RT-PCR were designed using MGB Eclipse to CD4 or CD8 T cells, respectively. Forty-eight hours before infection, 12 ϫ 106 of the indicated enriched population was adoptively transferred Design 2.3 Software and synthesized by EPOCH Biosciences with se- ϩ ϩ quences, as follows: forward primer, GCAATCGTATTACCTCTTATCG i.v. For mice receiving both CD8 and CD4 T cells, sorted cells were 6 CAG; reverse primer, CAACCATCGTCATCGTCAGGAAAC; and probe, pooled to deliver 12 ϫ 10 of each population per recipient. 5ЈFAM-GGCAAAGTCCCATCGTT-3ЈMGB. Real-time PCR was per- formed in duplicate for each sample, on an ABI 7900H in a total volume Statistics of 15 ␮l(5␮l of cDNA template). The genome copy number was calcu- lated by fitting the threshold cycle to a standard curve of known copy Statistical analyses were performed using Student’s t test or one-way number. ANOVA (in vivo killing) with GraphPad Prism software. 2618 HEMOPOIETIC CHIMERAS AND CHRONIC INFECTION

FIGURE 1. Viral control is associated with shared MHC. Hemopoietic chimeras with varied degrees of MHC sharing were generated using the indicated donor bone marrow: BALB/c donor, CB6.F1 donor, or B6 congenic donor. Eight weeks postchimerism induction, animals were infected with 1 ϫ 106 PFU LCMV clone 13 to initiate chronic infection. A, Viral titers from PBL at indicated time points represent the mean for Ն9 an- imals. B, Viral titers from day 127 postinfection. Line indicates median value. C, Weight loss is represented as the mean percentage of preinfection weight; dashed line indicates a severe weight loss threshold requiring eu- thanasia. D, Serum cytokines for day 98 postinfection. TNF-␣, MCP-1, and IL-12p70 levels are shown; n ϭ 3. Data from each figure are representative of three inde- pendent experiments. All error bars indicate SEM. Downloaded from http://www.jimmunol.org/

Results congenic and semi-MHC-disparate mixed chimeras began to con- Fully MHC-disparate mixed hemopoietic chimeras show trol viremia, the fully MHC-disparate mixed chimeras exhibited impaired control of chronic infections relative to semiallogeneic progressive weight loss associated with their high-level viremic or congenic mixed chimeras state (Fig. 1C). Analysis of serum from the infected MHC-dispar- ate mixed chimeras 98 days postinfection revealed the presence of To rigorously evaluate the immune competence of mixed hemo- a systemic inflammatory state, characterized by high levels of poietic chimeras with varying degrees of MHC disparity, stable the inflammatory cytokine TNF-␣ and the chemokine MCP-1. mixed chimerism was established in C57BL/6 (B6-CD45.1ϩ)re- There was a trend toward increased IL-12p70, but this did not by guest on October 3, 2021 cipients using one of three defined bone marrow donors, as fol- reach statistical significance. This cytokine storm was not lows: fully MHC-disparate BALB/c (H-2d) donors; semi-MHC- present in the infected congenic or semi-MHC-disparate mixed disparate BALB/c ϫ B6-F donors (CB6F /J, H-2bxd), which 1 1 chimeras (Fig. 1D). express the same allogeneic MHC H-2d molecules as the BALB/c donors, but also express the recipient MHC class I and II H-2b alleles; and finally, congenic B6-CD45.2ϩ (H-2b) control donors. Fully MHC-disparate mixed hemopoietic chimeras exhibit Chimerism was established via a protocol consisting of a mini- accelerated and more pronounced functional exhaustion of mally myelosuppressive dose of busulfan and short-term CD28/ viral-specific T cells CD40 costimulation blockade, as previously described (4). Multi- To gain insight into the mechanisms that underlie the immune- lineage mixed chimerism developed in a predictable fashion (4), deficient state in fully MHC-disparate mixed chimeras, we ana- and to similar degrees in each group (45 Ϯ 5% donor chimerism). lyzed the number and functional properties of viral-specific T cells Eight weeks after chimerism induction, recipient mice were chal- generated in the three experimental groups using MHC tetramers lenged with LCMV clone 13 to initiate a chronic pathogenic chal- and detection of intracellular cytokines after ex vivo restimulation. lenge. Viral load and immune responses were assessed at 14 and In normal B6 mice, the prolonged exposure to high viral load after 30 days and at monthly intervals thereafter. LCMV clone 13 infection results in a distinct pattern of epitope As expected, all groups showed high-level viremia 2 wk after immunodominance and a spectrum of T cell responses. This ranges b d infection (Fig. 1A). The recipients of congenic bone marrow (B6- from deletion (D /NP396,L/NP118) to varying degrees of func- b b CD45.2gB6-CD45.1) gradually controlled the virus with kinetics tional impairment of viral-specific T cells (D /gp33, K /NP205), similar to unmanipulated B6 mice (37) (data not shown). In con- before viremia is ultimately controlled by T cells that recognize trast, chimeras given fully MHC-disparate BALB/c bone marrow subdominant epitopes (Db/gp276, Kd/gp283) (20, 37, 45). showed a considerable deficiency in viral control, maintaining high The mice in each of the three experimental groups were infected viral titers throughout the 4-mo period of observation (Fig. 1, A as in the previous experiment, and splenocytes were harvested for and B) (18). Interestingly, the recipients of the semi-MHC-dispar- analysis at monthly intervals. All groups showed transient expan- b d ate CB6F1 donor bone marrow effectively controlled the viral chal- sion, followed by rapid deletion of the D /NP396- and L /NP118- lenge with kinetics indistinguishable from the congenic chimeras specific T cells (data not shown). Similarly, there was expansion of (Fig. 1A). Db/gp33- and Db/gp276-specific T cells, as assessed by MHC tet- The differences in viral control were associated with significant ramer-peptide staining in each of the different groups (Fig. 2, A and clinical manifestations. All of the experimental groups experienced B). However, there were striking differences in the degree and a similar degree of early weight loss (ϳ15%) within the first 2 wk rapidity of functional impairment of the Db/gp33 (Ͼ90% exhaus- after infection. However, whereas body weight stabilized as the tion at day 14)- and Db/gp276 (Ͼ90% by day 30)-specific T cells The Journal of Immunology 2619

FIGURE 2. Lack of any MHC-shared alleles leads to a rapid and severe state of T cell exhaustion. Spleno- cytes from LCMV-infected chimeric mice were har- vested and stained with MHC tetramers, or intracellular IFN-␥ after ex vivo restimulation with indicated viral peptides. A, Representative flow plots from day 98 gated on CD8ϩ splenocytes showing the percentage staining positive for MHC tetramer Db/gp276 (top), or Downloaded from intracellular gp276-induced IFN-␥ production after ex vivo restimulation (bottom), as well as the activation marker CD44 (y-axis). B, The absolute number of splenocytes staining positive for MHC tetramer Db/ gp276 or Db/gp33 (open histogram) and the fraction ca- pable of producing IFN-␥ upon ex vivo restimulation http://www.jimmunol.org/ (shaded portion) for days 14 and 98. Each bar represents the mean of at least three mice; error bars represent SEM. C, Time course showing the mean fraction of Db/ gp276 tetramer-staining cells capable of IFN-␥ produc- tion after a 5-h restimulation for the indicated time points postinfection (n ϭ 3). Data are representative of two independent experiments. by guest on October 3, 2021

that was uniquely seen in fully MHC-disparate mixed chimeras and the H-2d donor-restricted peptides gp99 and gp283. The assay (Fig. 2, B and C). This was manifest as a dramatic decrease in the was internally controlled by inclusion of a reference target popu- percentage of these viral-specific T cells that were capable of lation that was not pulsed with peptide. Five hours after transfer of IFN-␥ production in response to peptide stimulation relative to the these targets to the infected chimeras, their elimination was com- other groups (Fig. 2C). pared with the unpulsed reference population and normalized to relative survival of the populations in a group of naive recipient Fully MHC-disparate mixed chimeras are unable to kill in a mice, as described (43). donor-restricted fashion The congenic chimeras showed highly efficient killing of gp276 The ability of CTLs to recognize and kill infected cells is critically peptide-pulsed cells with much more limited killing of targets important for the control of intracellular bacteria and (46, bearing the gp33 LCMV peptide (H-2b restricted) (Fig. 3). The 47). To measure CTL function in the various mixed chimeras, we lower levels of gp33-restricted killing may reflect the significantly bϫd ϩ compared their ability to eliminate CB6.F1 (H-2 ) target cells fewer absolute numbers of gp33 tetramer T cells relative to that had been pulsed with either donor (H-2d) or recipient (H-2b) gp276 tetramerϩ cells or the degree of impaired IFN-␥ production, MHC-restricted viral peptides in an in vivo killing assay. To gen- which was more pronounced for the gp33 epitope (see Fig. 2). erate targets for this assay, H-2dϫb splenocytes were differentially H2b-restricted killing in semidisparate and fully MHC-disparate labeled with both CFSE and CellTrace Far Red; each population mixed chimeras was similar to that seen in congenic chimeras. was then pulsed with one of four virally derived peptides. These However, there appeared to be a trend toward a diminished capac- included the H-2b recipient-restricted peptides gp33 and gp276, ity to kill in fully disparate chimeras, although the data did not 2620 HEMOPOIETIC CHIMERAS AND CHRONIC INFECTION Downloaded from

FIGURE 3. Mismatched chimeras are unable to kill in a donor-restricted fashion. Chronically infected chimeras were given differentially fluorescence-

labeled and peptide-pulsed splenocytes. Five distinct target populations are indicated (gated populations) and correspond with the peptide loading indicated http://www.jimmunol.org/ in the naive plot (far left), which was used as a reference to calculate the relative percentage of specific killing (see Materials and Methods). A, Representative flow cytometry plots indicating the percentage of specific killing of peptide-pulsed targets (indicated directly above) in 5 h, 104 days postinfection for individual animals from each experimental group. The upper right population in each plot represents an internal (no peptide) control. B, Summary data of recipient (Db)- and donor (Kd)-restricted killing for each experimental group are shown, for the four indicated peptide epitopes (n ϭ 3); error bars represent SEM; asterisk indicates p value of Ͻ0.05 for indicated epitope. Data are representative of two independent experiments.

reach statistical significance. Furthermore, it is noteworthy that Donor-restricted T cell help in chronically infected fully MHC- by guest on October 3, 2021 whereas the recipient-restricted response (Db/gp276) showed re- disparate mixed chimeras is impaired ␥ duced capacity for IFN- production (Fig. 2), there is a retained Next, we explored the status of the humoral immune response after capacity for host cell-directed cytolysis, suggesting that the chronic virus infection in our chimeras as an indicator of a Th- functional exhaustion is not complete. This is consistent with dependent immune response. The delivery of donor-restricted CD4 work from Wherry et al. (37) (our unpublished observations) T cell help in fully MHC-disparate chimeras was determined by showing distinct stages of exhaustion in the course of LCMV assaying for class-switched IgG2aa allotype Ab that is produced by clone 13 infection. d B cells bearing the allele derived from the BALB/c genotype and H2 -restricted killing in congenic chimeras was undetectable. was distinguished from B6-derived IgG2ab allotype (22, 48). The This is not surprising because these chimeras were never primed to d overall level of LCMV-specific Abs was not significantly different virus in the presence of H2 restriction elements. Interestingly, the among the chimeras 3 mo after infection (Fig. 4). As expected, the semi-MHC-disparate mixed chimeras showed a small, but repro- b d B6 congenic chimeras produced only B6-derived Ab (IgG2a ). In- ducible ability to kill targets pulsed with either of the K -restricted terestingly, despite having roughly equal numbers of donor- and peptides (gp283, 13.5 Ϯ 1.8%; gp99, 6.8 Ϯ 3.9% killing). Killing for gp283 was significantly greater in semi-MHC-disparate chime- ras vs the other groups ( p Ͻ 0.05); however, control of virus in these chimeras would not be absolutely dependent on this mode of killing. This is because both infected donor and recipient cells in these animals would also present H-2b restriction elements and would be susceptible to recipient-restricted killing as well. In con- trast, the fully MHC-disparate mixed chimeras, although capable of killing in a recipient-restricted fashion, showed no detectable ability to lyse targets in an H-2d-restricted manner (Fig. 3; Ld/

NP118-mediated killing was also undetectable; data not shown). Although there was no evidence for IFN-␥ production after re- stimulation with gp283 or gp99 peptides (data not shown), without FIGURE 4. Serum anti-LCMV Ab titers. Serum from LCMV-infected tetramer staining we were unable to determine whether T cells mice was analyzed at day 100 for class-switched LCMV-specific IgG2a directed against these epitopes have become exhausted or deleted. Ab. The scatter plot indicates the log10 dilution at which detectable LCMV- Nevertheless, the absence of killing toward these epitopes would specific IgG2a of allotype a (BALB/c derived, open symbols) or b (B6 be expected to limit the ability of CTL to control virus in infected derived, closed symbols) is detected. Horizontal line indicates mean value. donor cells that only express H-2d restriction elements. Data are representative of two independent experiments. The Journal of Immunology 2621

FIGURE 5. Viral persistence is associated with in- fection of donor-derived cells in MHC-disparate mixed chimeras. A, Representative flow plots indicating sur- face staining for the congenic marker CD45.2 (x-axis), which is present exclusively on donor-derived cells. A mAb specific for LCMV nucleoprotein (clone H113, y- axis) was used in an intracellular stain to indicate virally infected cells at day 34 (top) and day 104 (bottom). B, Summary data for virally infected cells that are either donor (CD45.2ϩ) or recipient (CD45.2Ϫ) derived

(n ϭ 3). Data are representative of two independent Downloaded from experiments. http://www.jimmunol.org/

recipient-derived B cells, the fully MHC-disparate mixed chimeras mixed chimeras that, at this time point, had low or undetectable almost exclusively produced recipient-derived (IgG2ab) Ab (Fig. levels of virus in the serum (Fig. 1) were negative for intracellular by guest on October 3, 2021 4). This indicates that donor-derived B cells are not receiving cog- LCMV-NP. In contrast, the fully MHC-disparate mixed chimeras nate donor-restricted help from CD4 T cells, demonstrating a def- that continued to have high viral loads (Fig. 1) showed that virus icit in the presence or function of T cells that recognize LCMV in was preferentially harbored in the donor-derived CD45.2ϩ popu- an I-Ad-orI-Ed-restricted manner. The Ab response in the semi- lation (Fig. 5). These findings support the notion that donor-de- MHC-disparate mixed chimeras was comprised of both donor- and rived cells in the fully MHC-disparate mixed chimeras are not recipient-derived Ab. In this setting, the donor B cells have a targeted effectively and act as a reservoir for viral replication and mixed genotype, expressing both recipient and donor MHC class II persistence. and Ig allotypes. As such, they do not absolutely require donor- restricted CD4-derived help to class-switch. Immune competence of MHC-disparate chimeras is partially restored by MHC class I matching Donor-derived cells are a reservoir for viral replication in fully Next we sought to define the requirement for CD4 and CD8 donor- MHC-disparate mixed chimeras restricted responses for immune competence of mixed chimeras. In light of the significant deficiency in the ability of the fully Viral control and immune responses were evaluated using selected MHC-disparate mixed chimeras to produce effector cytokines or MHC-deficient bone marrow donors that expressed the full com- kill in a donor-restricted fashion, we hypothesized that the high plement of H-2d MHC class I and II molecules, but expressed viral load in these animals might be the result of unimpeded rep- exclusively either the H-2b MHC class I or class II molecules, but lication of virus in donor-derived cells. To test this, we compared not both. Using this approach, we could control whether the chi- the level of virus in donor- and recipient-derived cells in the dif- meras did or did not have the ability to directly survey donor cells ferent chimeras at various times using a mAb that is specific to the bearing viral peptides using class I or class II recipient-restricted T LCMV nucleoprotein (clone H113) in an intracellular staining cells. To accomplish this, we generated mixed chimeras in B6

flow cytometric assay (49). At 34 days postinfection, when virus is mice using bone marrow from F1 donors that were a cross of systemically present in all experimental groups (Fig. 1A), LCMV BALB/c (H-2d) ϫ B6 class I deficient (KbDbϪ/Ϫ) mice, which nucleoprotein was detected in both donor (CD45.2ϩ)- and recipi- would restore recipient class II expression on the donor bone mar- Ϫ ent (CD45.2 )-derived cells, with no apparent preference (Fig. 5). row, class II matched. Conversely, bone marrow from F1 donors However, the congenic and semi-MHC-disparate mixed chimeras that were a cross of BALB/c (H-2d) ϫ B6 class II-deficient mice have fewer infected cells in comparison with the fully MHC-dis- (MHC II⌬/⌬) would restore recipient class I expression on the do- parate mixed chimeras at this time point, similar to results found in nor bone marrow, class I matched. Fig. 1. Subsequently, analysis at 104 days postinfection reveals The class II matched mixed chimeras showed a very similar clear differences in the levels and cellular localization of virus in degree of immunodeficiency to the fully MHC-disparate mixed the different groups. First, the congenic and semi-MHC-disparate chimeras, maintaining high viral titers for Ͼ4 mo postinfection 2622 HEMOPOIETIC CHIMERAS AND CHRONIC INFECTION

FIGURE 6. MHC class I matching par- tially restores immune competence. Mixed chimeras were set up with B6.CD45.1 recip- ients and CB6.F1 donors specifically defi- cient in either H2b class I or class II. Chi- meras were then infected with LCMV clone

13. A, Day 127 viral titers for experimental Downloaded from chiimeras; line indicates the median value. B,Db/gp276 tetramer staining at day 98 (top) and corresponding IFN-␥ cytokine response upon ex vivo gp276 peptide restimulation (bottom); plots are gated on CD8ϩ spleno- cytes. Summary data represent the percent- age of IFN-␥ function after ex vivo restimu- http://www.jimmunol.org/ lation for Db/gp276 tetramerϩ cells. C, Representative and summary (n ϭ 3) data for intracellular LCMV-NP staining in do- nor- vs recipient-derived splenocytes at day 104 postinfection. by guest on October 3, 2021

(Fig. 6A). Additionally, these animals exhibited a corresponding ras matched for MHC class I cleared virus, those that main- profile of epitope exhaustion, in which gp276-specific CD8 T cells tained high titers harbored virus equally in donor- and recipient- are present, but never recover a significant amount of function derived cells (Fig. 6C). Thus, it appears that MHC class II (Fig. 6B). Just as with the fully MHC-disparate mixed chimeras, matching alone offers little or no degree of protection, whereas the inability to kill targets in a donor-restricted fashion is defective shared MHC class I appears to improve immune function, fa- in class II matched allogeneic mixed chimeras (data not shown), cilitating the control of chronic infections. and as a result they harbor virally infected cells preferentially in their donor-derived cellular compartment (Fig. 6C). In contrast, MHC class I matched mixed chimeras show a partial restoration The inability to generate donor-restricted CD8 T cell responses of immune competence (viral clearance) when compared with is the result of impaired positive selection on recipient thymic the fully MHC-disparate mixed chimeras ( p Ͻ 0.05) (Fig. 6A). epithelium This was associated with maintenance of body weight and lower Although the hemopoietic compartment of the mixed chimeras is serum levels of inflammatory cytokines (data not shown). In composed of approximately equal numbers of recipient and donor addition, consistent with the improved viral clearance, the de- cells, the T cells arising in these chimeras develop within the re- gree of T cell exhaustion was reduced, with larger numbers of cipient’s thymus (50). Thus, regardless of their genetic origin, the IFN-␥ producing gp276-specific T cells when compared with developing T cell precursors are selected exclusively on thymic the fully MHC-disparate mixed chimeras (see Fig. 2) or the epithelium of recipient origin (51). Therefore, we sought to deter- class II matched allogeneic mixed chimeras 98 days postinfec- mine whether the defective ability of fully MHC-disparate mixed tion (Fig. 6B). Finally, whereas a significant number of chime- chimeras to generate donor-restricted CD8ϩ antiviral responses The Journal of Immunology 2623

FIGURE 8. T cell adoptive immunotherapy improves viral control. BALB/c3B6 mixed chimeras were generated as above. Forty-eight hours before LCMV clone 13 challenge, chimeras were given 12 ϫ 106 naive ϩ ϩ ϫ 6 CB6.F1-sorted CD4 splenocytes, sorted CD8 splenocytes, or 12 10 of each subset. Viral titers were followed, and are shown for day 127 postinfection. Line indicates median.

cells primarily support positive selection, with perhaps a minor role for hemopoietically derived cells (19, 44, 51). Thus, in the Downloaded from setting of fully MHC-disparate mixed chimerism, the development of approaches that promote the selection of an adequate T cell repertoire may be an important consideration in providing robust immune protection against chronic viral infections residing in do- nor-derived cells. http://www.jimmunol.org/ Adoptive T cell immunotherapy restores the immune competence FIGURE 7. Thymic parenchyma is the dominant determinant of T cell repertoire restriction in mixed chimeras. BALB/c3B6 nude chimeras of fully MHC-disparate mixed chimeras were generated as above and given a B6, BALB/c, or CB6.F1 thymus under To test whether gaps in the T cell repertoire play a major role in the the kidney capsule at the time of induction. Ninety days after reconstitu- immune incompetence of fully MHC-disparate mixed chimeras, ϫ 5 tion, the resultant chimeras were infected with 2 10 PFU LCMV Arm- we evaluated the ability of adoptively transferred T cells to restore strong. A, Representative flow plots display CD8ϩ (x-axis), IFN-␥ϩ (y- b control of chronic viral infections. Naive T cells from CB6.F1 axis) splenocytes after restimulation with the indicated peptide (D /NP396, d donors were used for adoptive transfer because they are tolerant to B6 or L /NP118, BALB/c) at the peak of the response, 8 days postinfection. b d ϩ ϩ both host (H-2 ) and donor (H-2 ) Ags, and more importantly, B, The percentage of CD8 , IFN-␥ cells in the spleen is shown for each by guest on October 3, 2021 bϫd indicated epitope. Error bars represent SEM, n ϭ 4 for each group. have been positively selected over an H2 environment. Fully MHC-disparate mixed chimeras were generated as above, and 48 h before challenge with LCMV clone 13, the mice were infused with was the result of inefficient positive selection of an effective T cell either 1.2 ϫ 107 CD4 T cells, 1.2 ϫ 107 CD8 T cells, or a com- repertoire. bination of both populations from CB6.F1 donors. Viral load was To explore this, we generated fully MHC-disparate mixed chi- assessed over time, and as in our previous studies, fully MHC- meras in athymic B6 nude mice using T-depleted BALB/c bone disparate chimeras that did not receive T cells failed to control marrow. In conjunction with the donor bone marrow infusion, viral replication and maintained high-level viremia. Adoptive ϩ ϩ these recipient mice were reconstituted with a thymus transplant transfer of either CD4 or CD8 T cells alone led to trends toward from a syngeneic (B6), an allogeneic (BALB/c), or semiallogeneic improved viral clearance, but these did not achieve statistical sig-

(CB6.F1) donor. These fully MHC-mismatched chimeric mice nificance (Fig. 8). However, fully MHC-disparate mixed chimeras ϩ ϩ were challenged with 2 ϫ 105 PFU of LCMV Armstrong, which reconstituted with both CD4 and CD8 T cells showed signifi- shares all defined epitopes with clone 13, but offers the opportunity cantly improved viral control (Fig. 8, p Ͻ 0.05). This further sup- to clearly examine the restriction of the antiviral T cell response to ports our hypothesis that a primary mechanism underlying the im- multiple H-2b and H-2d dominant epitopes in an acute response. mune deficiency of fully MHC-disparate mixed hemopoietic Fully MHC-disparate mixed chimeras that received a B6 thymus chimeras is the failure to generate a T cell repertoire that provides b b mounted potent host-restricted responses to the D /NP396 and D / an adequate number of donor-restricted T cells. gp33 epitopes 8 days postinfection (Fig. 7), similar to the quality of responses in control nonchimeric B6 nude mice receiving a B6 Discussion thymus (data not shown). Furthermore, these same chimeras gen- In light of the increasing number of clinical trials using nonmy- erated poor, but detectable donor-restricted responses to the Ld/ eloablative approaches to establish therapeutic chimerism across a

NP118 epitope, indicating some degree of positive selection of a range of MHC disparities in bone marrow and solid organ trans- donor-restricted response (Fig. 7). In contrast, the anti-LCMV re- plantation (1, 34, 52), we undertook studies to evaluate the im- sponses in fully MHC-disparate mixed chimeras receiving a mune competence of allogeneic mixed chimeras and to define the BALB/c thymus transplant were dominated by the donor-restricted mechanisms underlying any observed immunodeficiencies. The re- d b L /NP118 epitope, and responses to the host-restricted D /NP396 sults presented in this study demonstrate that, whereas the estab- and Db/gp33 epitopes were greatly compromised. Finally, mixed lishment of a state of stable multilineage mixed chimerism con- chimeras that had received a CB6.F1 thymus showed a broad- sistently promotes robust donor-specific transplantation tolerance, based response, including both donor- and recipient-directed T depending on the degree of MHC disparity this state does not cells (Fig. 7B). These data support the notion that thymic epithelial necessarily confer immune competence. Rather, we observe that 2624 HEMOPOIETIC CHIMERAS AND CHRONIC INFECTION when the donor is fully MHC disparate, mixed chimeras have sig- may contribute to the profound defects in the generation and main- nificant defects in protective immunity that become manifest after tenance of donor-restricted CTL responses. challenge with a persistent pathogen requiring a coordinated and Evidence to support the conclusion that an insufficient pool of sustained immune response to achieve viral control. The observa- donor-restricted T cells is the primary defect in fully MHC-dis- tion that fully MHC-disparate mixed chimeras exhibit sustained parate mixed chimeras comes primarily from the observation that viremia coupled with a systemic inflammatory cytokine storm and the adoptive transfer of donor-derived T cells that had been se- progressive weight loss suggests that these recipients were indeed lected on donor MHC molecules during their thymic development mounting an immune response, albeit an ineffective and possibly can restore immune competence of fully MHC-disparate mixed immunopathologic one. chimeras. Furthermore, the observation that fully and semi-MHC- At first glance, the immune deficiencies of the fully MHC-dis- disparate mixed chimeras differ significantly in their immune com- d parate mixed chimeras seem surprising given that the peripheral petence despite presenting the same allo-Ags (H-2 ) on donor- immune system of these chimeras contains approximately equiv- derived hemopoietic cells to developing thymocytes in the alent numbers of donor- and recipient-derived APC and T cells recipient thymus suggests that the immune defects in the fully (data not shown). However, the donor or recipient genetic origin of disparate mixed allogeneic chimeras are not likely to be solely T cells in the peripheral pool provides only the germline TCR explained by excessive negative selection in this system. However, segments available for rearrangement, but this does not supersede further studies could be undertaken to determine whether exces- the epigenetic influences of the thymic selecting environment that sive negative selection also might contribute to defects in immune ultimately determines the restriction and composition of the pe- competence when a supraphysiologic number of MHC molecules are present during thymic selection, for example, if mixed chimer- ripheral T cell repertoire (8, 50). The observation that T cell chi- Downloaded from merism in these recipients develops over a period of several weeks ism were established using a donor bearing two different MHC dϫk suggests that the bulk of these donor-derived T cells emerged from haplotypes (e.g., H-2 ) into a recipient bearing yet another two bϫs the recipient’s thymus, where they will have undergone positive MHC haplotypes (e.g., H-2 ) as would occur in fully MHC- and negative selection on recipient thymic epithelium and on both disparate human transplants. recipient- and donor-derived dendritic cells. This observation is The observation that fully MHC-disparate chimeras have de- fects in generating donor-restricted responses is consistent with

consistent with earlier observations in cross-species rat-to-mouse http://www.jimmunol.org/ early studies suggesting that the nonhemopoietically derived thy- mixed chimeras, which have been shown to support host-restricted mic epithelial cells play a dominant role in positively selecting the T cells derived from xenogeneic donors (50). T cell repertoire (51, 54, 55). Similarly, our thymic reconstitution Conceptually, the immune deficiencies in fully MHC-disparate experiments demonstrated that the thymic environment can dra- mixed chimeras could involve inadequacies of the T cell repertoire matically alter the resulting T cell response to LCMV Armstrong induced by either of two nonmutually exclusive mechanisms, as in fully MHC-disparate mixed chimeras. In these experiments, the follows: 1) the introduction of donor hemopoietic cells to the re- use of LCMV Armstrong allowed us to reproducibly study the cipient’s thymus could result in more extensive negative selection, restriction of the antiviral T cell response to multiple H-2b and thereby narrowing the T cell repertoire and potentially eliminating H-2d dominant epitopes. Studies by several investigators in large important pathogen-specific T cells (53), or alternatively, 2) pos- by guest on October 3, 2021 animal models have shown that transplantation of the donor thy- itive selection in the recipient’s thymus may not support the de- mus can enhance tolerance induction (56, 57). It will also be in- velopment of an adequate pool of T cells capable of recognizing teresting to determine whether thymic transplantation also has sal- viral peptides presented on donor MHC molecules, thus compro- utary effects on immune competence in the setting of persistent mising the elimination of infected cells of donor origin. pathogenic challenges such as LCMV clone 13. The wide range of tools and assays that are available to assess The functional consequence of the defective repertoire is that immune responses to LCMV allowed us to directly assess quan- the fully MHC-disparate mixed chimeras are unable to detect and titative and qualitative aspects of the recipient- and donor-re- eliminate infected donor-derived cells upon challenge with a per- stricted anti-LCMV response in mixed chimeras. Our studies sug- sisting pathogen such as LCMV clone 13. This immunological gest that the principal defect in fully MHC-disparate mixed blind spot allows the donor-derived cells to act as a reservoir for chimeras is the lack of a sufficient population of T cells that are ongoing viral replication that appears to perpetuate host-restricted capable of recognizing viral peptides presented by donor-MHC T cell exhaustion as well. This was evident for subdominant Db/ rather than a hole in the repertoire of T cells that recognize viral gp276 responses, which in congenic and semi-MHC-disparate peptides presented on recipient-type MHC molecules. Although mixed chimeras retain function and correlate with clearance, ϳ we have shown previously that low-level ( 10-fold lower) donor- whereas in fully disparate chimeras this response loses IFN-␥ restricted responses are generated in fully MHC-disparate mixed function, coincident with higher viral titers and immune pathology chimeras and are adequate to clear an acute LCMV Armstrong (37, 58). This is somewhat unexpected given that persistent virus infection (18), the data presented in this study indicate that these was predominantly detected in donor-derived cells. However, it is low-level responses are neither sustained nor sufficient to control possible that virus replication in donor cells leads to low-level the persistent LCMV clone 13 variant. The defective generation of recipient cell infection or chronic cross-presentation of viral pep- a donor-restricted response was most clearly demonstrated by pro- tides by recipient APC, contributing to enhanced exhaustion of the found defects in the ability of fully MHC-disparate mixed chime- recipient-restricted T cells. ras to kill donor cells bearing viral peptides in an in vivo killing Our studies provide potential insights for the development of assay. This is consistent with earlier work showing a lack of donor- strategies to minimize immunodeficiency or restore immune com- restricted killing using ex vivo killing assays (8). Similarly, the petence in the setting of mixed hemopoietic chimerism. First, our defective production of Ab from donor-derived B cells in the studies emphasize the importance of MHC matching not only to MHC-disparate mixed chimeras implies that defects in donor-re- minimize the barrier to tolerance induction, but also for the pres- stricted T cell help may play a role in viral persistence. Although ervation of protective immunity. Our findings indicate that for the not crucial for the humoral response, which can be supplied by preservation of protective immunity, the degree of MHC incom- recipient-derived B cells, the defects in donor-restricted help also patibility is not as critical as the degree of MHC sharing between The Journal of Immunology 2625 donor and recipient. Fully incompatible chimeras, which do not restriction specificity of mixed allogeneic (P1 ϩ P2—P1) irradiation chimeras. share MHC, exhibit severe immunoincompetence, whereas an F Cell. Immunol. 121: 185–195. 1 9. Sharabi, Y., and D. H. Sachs. 1989. Mixed chimerism and permanent specific donor (shared MHC) appears to reconstitute viral control. Notably, transplantation tolerance induced by a nonlethal preparative regimen. J. Exp. sharing of MHC class I by donor and recipient appears to be the Med. 169: 493–502. more important factor for restoring competency. Chimeras without 10. Cobbold, S. P., G. Martin, S. Qin, and H. Waldmann. 1986. Monoclonal anti- bodies to promote marrow engraftment and tissue graft tolerance. Nature 323: a shared class I Ag exhibit deficient donor-restricted killing, main- 164–166. taining a viral reservoir exclusively in donor-derived cells. Studies 11. Wekerle, T., J. Kurtz, H. Ito, J. V. Ronquillo, V. Dong, G. Zhao, J. Shaffer, M. H. Sayegh, and M. Sykes. 2000. Allogeneic bone marrow transplantation with of mixed chimerism induction in human transplantation have most co-stimulatory blockade induces macrochimerism and tolerance without cytore- commonly been done across either one or two MHC haplotype ductive host treatment. Nat. Med. 6: 464–469. matches (1) (www.immunetolerance.org). However, in other pro- 12. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al. tocols in which chimerism induction has been attempted across 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 wider degrees of MHC disparity (34), chimerism has been tran- and CD28 pathways. Nature 381: 434–438. sient. Although caution should be used in extrapolating from mu- 13. Durham, M. M., A. W. Bingaman, A. B. Adams, J. Ha, S.-Y. Waitze, T. C. Pearson, and C. P. Larsen. 2000. Administration of anti-CD40 ligand and rine studies to the clinical setting, our studies nonetheless suggest donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance that attention to the evaluation of protective immunity during clin- without cytoreductive conditioning. J. Immunol. 165: 1–4. ical chimerism induction trials, particularly across greater degrees 14. Tomita, Y., A. Khan, and M. Sykes. 1994. Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplan- of MHC disparity, should be emphasized. tation in mice conditioned with a nonmyeloablative regimen. J. Immunol. 153: A second approach to restoring protective immunity, when chi- 1087–1098. merism is established in the absence of MHC matching, is adoptive 15. Wekerle, T., M. H. Sayegh, J. Hill, Y. Zhao, A. Chandraker, K. G. Swenson, G. Zhao, and M. Sykes. 1998. 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