Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Host and donor immune responses contribute to antiviral effects of amotosalen-treated donor lymphocytes following early posttransplant cytomegalovirus infection

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6-->CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 x 10(6) untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 x 10(6) treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8(+) T cells, amotosalen-treated CD8(+) T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8(+) T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4(-)CD8(-) (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-gamma and TNF-alpha in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.     

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (～50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.     

Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 x 10(6) pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-gamma production by MCMV-primed CD8(+) T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log(10) and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44(high)) CD8(+) T cells persisted to day +100 following infusion, and expressed IFN-gamma when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.     

Acute primary infection with cytomegalovirus (CMV) in kidney transplant recipients results in the appearance of a phenotypically aberrant CD8+ T cell population

Human cytomegalovirus (CMV) is a beta-herpesvirus that causes a chronic subclinical infection in healthy man. The immune system is unable to eliminate the virus completely, allowing virus to persist in a latent state. In the immunocompromised host, this equilibrium is disturbed, resulting in a clinical infection. In immunocompromised rats, clinical CMV infection is associated with an increase in NK cells and CD8+ T cells, including a phenotypically aberrant CD8+ T cell population. Using flow cytometry, we examined the effect of acute CMV infection on the composition of leukocyte subsets in immunocompromised patients. Therefore, we used peripheral blood of CMV seronegative patients receiving a kidney from a seronegative (control group) or a seropositive donor. Of the patients receiving a seropositive kidney, only the patients undergoing acute CMV infection were included (experimental group). Special attention was paid to the phenotype of the cytotoxic T cells. The development of acute CMV infection resulted in an increased NK cell number and an activation of both CD4+ and CD8+ T cells, as determined by HLA-DR expression. An aberrant CD8+ T cell subset with decreased expression of CD8 and TCR alphabeta appeared in the infected patients. Furthermore, the size of this subpopulation of CD8+ T cells is positively correlated with the viral load.     

Dysfunction of Bone Marrow Vascular Niche in Acute Graft-Versus-Host Disease after MHC-Haploidentical Bone Marrow Transplantation

Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4⁺, but not CD8⁺ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4⁺ donor T cells through the Fas/FasL pathway.     

Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection in lung transplant recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D(+)R(-)) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D(+)R(-) LTRs (59.1%) seroconverted (CMV IgG Ab(+)). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8(+)IFN-gamma(+) T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4(+)IFN-gamma(+) T cells during viral latency. However, the majority of IgG(+) LTRs demonstrated CMV-specific CD4(+) and CD8(+) T cell proliferative responses from PBMC, with CD4(+)IFN-gamma(+) T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4(+) and CD8(+)IFN-gamma(+) T cells, including patients from whom CD4(+)IFN-gamma(+) T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p < or = 0.009) and preservation of allograft function (p < or = 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D(+)R(-) LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.     

CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity.

We have shown in a murine model system for acute, lethal cytomegalovirus (CMV) disease in the immunocompromised natural host that control of virus multiplication in tissues, protection from virus-caused tissue destruction, and survival are mediated by virus-specific CD8+ CD4-T lymphocytes. Protection from a lethal course of disease did not result in a rapid establishment of virus latency, but led to a long-lasting, persistent state of infection. The CD8- CD4+ subset of T lymphocytes was not effective by itself in controlling murine CMV (MCMV) multiplication in tissue or essential for the protective function of the CD8+ CD4- effector cells. The antiviral efficacy of the purified CD8+ CD4- subset was not impaired by preincubation with fibroblasts that presented viral structural antigens, but was significantly reduced after depletion of effector cells specific for the nonstructural immediate-early antigens of MCMV, which are specified by the first among a multitude of viral genes expressed during MCMV replication in permissive cells. Thus, MCMV disease provides the first example of a role for nonstructural herpesvirus immediate-early antigens in protective immunity.     

Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule L^d were transplanted into the L^d gene deletion mutant BALB/c-H-2^dm2, an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of L^d-positive donor-derived and L^d-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against L^d. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by L^d within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.Cytomegaloviruses (CMV) are kept under tight immune control (for reviews, see references 22 and 23). As a consequence, acute CMV infection is resolved rapidly and does not result in disease unless the host is immunologically immature or immunocompromised. Bone marrow (BM) transplantation (BMT) as a therapy of hematological malignancies is associated with a transient immunodeficiency. Accordingly, during the period of immunocompromise, transmission of donor-type CMV with the transplant as well as recurrence of CMV from latency established within the organs of the transplantation recipient both entail a risk for destructive virus replication in tissues resulting in multiple-organ CMV disease (16). In BMT recipients, CMV-induced interstitial pneumonia is a frequent and endangering manifestation of CMV disease (11, 27). However, CMV infection does not inevitably result in fatal disease. It appears that CD8 T-cell reconstitution is the decisive parameter in the control of CMV after BMT. Clinical data have shown that both efficient reconstitution of CD8 T cells (41) and supplementation of antiviral CD8 T cells by preemptive cytoimmunotherapy with T-cell lines (42, 50) correlate with a reduced risk of human CMV disease, whereas combined in vivo-ex vivo T-cell depletion, intended as a prophylaxis against graft-versus-host (GvH) disease, accidentally resulted in an increased incidence of CMV infections in BMT patients (14). Aspects of these clinical problems can be approached experimentally in a murine model of BMT and concurrent infection with murine CMV (for an overview, see reference 35). Specifically, depletion of CD8 T cells, but not of CD4 T cells, performed in vivo during the phase of reconstitution after BMT abolished the development of protective antiviral immunity, with an inevitably lethal outcome (34, 47) resulting from multiple-organ pathology (34), including BM aplasia (29, 30). Likewise, an insufficient endogenous reconstitution was successfully supplemented by experimental adoptive cytoimmunotherapy with antiviral CD8 T cells. Again, CD4 T cells were not effective (36, 37, 39, 47). Altogether, clinical data on human CMV infection and experimental data from the murine model have so far been concordant and have identified CD8 T cells as the principal effectors controlling CMV infections after BMT.These findings imply that all conditions which lower the efficacy of CD8 T-cell reconstitution will increase the risk for progression of asymptomatic CMV infection to fatal CMV disease. Histoincompatibility between graft and recipient is a factor likely to negatively influence the restoration of antiviral immunity. Accordingly, even though cases of severe human CMV disease have been reported also after autologous BMT (27, 40), the incidence of CMV-related complications is generally higher after histoincompatible BMT (51). In clinical BMT, donor and recipient are usually matched in major histocompatibility complex (MHC) class II molecules, whereas differences in minor histocompatibility loci and in MHC class I loci are tolerated if unavoidable. Complications caused in the CMV-infected recipient by histoincompatibility may include (i) an impaired engraftment of transplanted cells in the recipient BM stroma, (ii) an immunological GvH response as well as a host-versus-graft (HvG) response directed against the foreign minor or major histocompatibility molecules, and (iii) a lack of antiviral T-cell control resulting from an inappropriate repertoire of viral antigenic peptides presented by infected tissue cells of the transplantation recipient.In a first attempt to dissect these possibilities, we have established a murine model of experimental BMT performed across a single MHC class I disparity, namely, the presence and absence of the L^d molecule in BALB/c mice (MHC class I molecules K^d, D^d, and L^d) and the L^d gene deletion mutant BALB/c-H-2^dm2 (44), respectively. Depending on the choice of donor and recipient for the BMT, immunogenetical GvH and HvG conditions can be studied separately (35). Work presented herein focuses on the HvG setting with BALB/c as the donor strain and the mutant as the recipient. Hence, after incomplete depletion of hematopoietic cells of the recipients, this model entails a risk for graft rejection caused by a recipient response directed against the donor MHC class I molecule L^d. In addition, presentation of viral peptides by L^d, including the immunodominant IE1 nonapeptide of murine CMV (18, 38), is confined to donor-derived hematopoietic cells and their progeny, whereas the parenchymal and stromal sites of cytocidal infection (34) lack L^d as the prevailing peptide presenter. The aim of the study was to investigate the influence of this particular MHC class I disparity on the control of murine CMV after BMT.     

CD8+ T cells are a biologically relevant source of macrophage inflammatory protein-1 alpha in vivo

Chemokines are small proteins that direct the migration of leukocytes to inflammatory foci. Many cell types, including macrophages, fibroblasts, endothelial cells, and lymphocytes, produce chemokines in vitro, but biologically relevant sources of chemokines in vivo have not been well characterized. To investigate the pertinent sources of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in vivo, we used MIP-1 alpha-deficient (MIP-1 alpha-/-) mice as donors and as recipients in adoptive transfer experiments after a lethal infection with Listeria monocytogenes (LM). Unexpectedly, we found that the production of MIP-1 alpha by CD8+ T cells was critical in this system, as the cells from MIP-1 alpha-/- mice primed with LM were significantly less effective in protecting naive mice against a lethal infection by LM than were the CD8+ T cells from wild-type (wt) mice. This requirement for donor T cell production of MIP-1 alpha was confirmed by the observation that wt donor T cells do not mediate protection when coadministered with an anti-MIP-1 alpha polyclonal antiserum. Production of MIP-1 alpha by the recipient mice was not required for protection, because wt and MIP-1 alpha-/- recipients were equally well protected by wt T cells. A 2- to 3-fold decrease in the number of transferred lymphocytes was seen in the spleens of mice receiving T cells from MIP-1 alpha-/- mice compared with those receiving wt T cells. In addition, CD8+ T cells from MIP-1 alpha-/- mice had a reduced ability to kill LM-infected target cells in vitro. These findings demonstrate that T cell production of MIP-1 alpha is required for clearance of an intracellular pathogen in vivo.     

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.     

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.     

The male minor transplantation antigen preferentially activates recipient CD4+ T cells through the indirect presentation pathway in vivo

To evaluate the priming and trafficking of male Ag-reactive CD4(+) T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-A(b). By manipulating donor and recipient strain combinations, we permitted the Mar CD4(+) T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4(+) T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2-3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4(+) T cells as relevant participants in both the priming and effector phases of acute graft rejection.     

CD4+ T-cell reconstitution reduces cytomegalovirus in the immunocompromised brain

Cytomegalovirus (CMV) infection is the most common opportunistic infection of the central nervous system in patients with human immunodeficiency virus or AIDS or on immunosuppressive drug therapy. Despite medical management, infection may be refractory to treatment and continues to cause significant morbidity and mortality. We investigated adoptive transfer as an approach to treat and prevent neurotropic CMV infection in an adult immunodeficient mouse model. SCID mice were challenged with intracranial murine CMV (MCMV) and reconstituted with MCMV- or vesicular stomatitis virus (VSV)-sensitized splenocytes, T cells, or T-cell subsets. T cells labeled with vital dye or that constitutively generated green fluorescent protein (GFP) were identified in the brain as early as 3 days following peripheral transfer. Regardless of specificity, activated T cells localized to regions of the brain containing CMV, however, only those specific for CMV were effective at clearing virus. Reconstitution with unsorted MCMV-immune splenocytes, enriched T-cell fractions, or CD4(+) cells significantly reduced virus levels in the brain within 7 days and also prevented clinical disease, in significant contrast with mice given VSV-immune unsorted splenocytes, MCMV-immune CD8(+) T cells, and SCID control mice. Results suggest CMV-immune T cells (particularly CD4(+)) rapidly cross the blood-brain barrier, congregate at sites of specific CMV infection, and functionally eliminate acute CMV within the brain. In addition, when CMV-immune splenocytes were administered prior to a peripheral CMV challenge, CMV entry into the immunocompromised brain was prevented. Systemic adoptive transfer may be a rapid and effective approach to preventing CMV entrance into the brain and for reducing neurotropic infection.     

MHC-unrestricted transfer of antilisterial immunity by freshly isolated immune CD8 spleen cells

Immune CD8 cells, which play an essential role in the adoptive transfer of antilisterial immunity, can specifically lyse Listeria-bearing macrophages in vitro in an MHC-unrestricted manner. In contrast, the adoptive transfer of immunity by unseparated immune lymphocytes has been reported to be MHC-restricted. To address the restriction properties of CD8 effectors in vivo, we assessed their efficacy in protecting syngeneic and allogeneic recipients. Protection was determined by comparing the number of viable splenic Listeria in naive mice and in recipients of 60 million CD8-enriched, L3T4-depleted, Listeria-immune spleen cells, 2 days after the infusion of 10,000 Listeria. Donor cells from B6 (H-2b) mice transferred about 4 logs of protection in syngeneic recipients and more than 2 logs in allogeneic B10.A (H-2a) or B10.BR (H-2k) mice. Immune B10.A CD8 cells transferred equivalent protection to B6 mice. Protection was almost completely abrogated by the lysis or lethal irradiation of CD8 cells before transfer in vivo. On the other hand, the depletion of macrophages or NK cells did not impair adoptive transfer. By comparison, nonimmune CD8 cells from normal mice or from mice stimulated with an irrelevant Ag in vivo did not transfer substantial immunity to allogeneic recipients. We have noted previously that protective CD8 cells inhibit phagocyte accumulation in the spleen of Listeria-infected syngeneic recipients. In the present studies, we observed similar changes in adoptively immunized allogeneic mice. Reduced phagocyte accumulation may reflect Listeria-dependent lysis of infected phagocytes by immune CD8 cells. In support of this, we showed that Listeria-immune donor cells rapidly acquired the capacity to mediate Listeria-dependent, MHC-unrestricted lysis of macrophages after incubation with small amounts of IL-2 in vitro. In sum, our data establish that Listeria-immune CD8 cells can function in vivo in MHC incompatible hosts, and indirectly support the hypothesis that the destruction of infected phagocytes may be important in T cell-mediated immunity against Listeria and perhaps other intracellular pathogens.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.