In vivo antigen delivery by a Salmonella typhimurium type III secretion system for therapeutic cancer vaccines

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratumoral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.     

Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 +/- 12.7% versus 15 +/- 1.4%). CD4(+) and CD8(+) T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1 alpha) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.     

A Safe Bacterial Microsyringe for In Vivo Antigen Delivery and Immunotherapy

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the “killed but metabolically active” (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.     

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.     

High-efficiency lentiviral vector-mediated gene transfer into murine macrophages and activated splenic B lymphocytes

The goal of the present report was to determine if lentiviral vectors could mediate gene transfer into murine terminally differentiated macrophages and mature B lymphocytes as a new strategy of gene delivery into professional antigen-presenting cells (APC). We demonstrated that nondividing tissue resident macrophages were efficiently transduced in vitro by lentiviral vectors. Gene transfer efficiencies of up to 90% were demonstrated using a green fluorescent protein (GFP) reporter gene-containing vector and expression was stable for the length of cell culture. Transduced macrophages were functionally competent, preserving their phagocytic activity, accessory cell function, interleukin (IL)-12 secretion, and nitric oxide (NO) production similar to control untransduced macrophages. Lentiviral vector mediated transduction of CD19(+) B cell blasts was demonstrated to be in the range of 60%-70% GFP-positive cells. These transduced cells retain the ability to upregulate CD80 and CD86 similar to control B cell cultures. In addition, we show that the human immunodeficiency virus type 1 (HIV-1) accessory proteins Nef, Vpr, Vif, and Vpu are not required for the transduction of both resident macrophages and activated B lymphoblasts. We conclude that HIV-1-based lentiviral vectors can mediate efficient gene transfer into primary murine macrophages and mature B lymphocytes.     

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.     

Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.     

Viral vectors for inducing CD8+ T cell responses

CD8(+) T cells (T(CD8+)) can mediate protective immunity to intracellular pathogens and tumours. Viruses generate strong T(CD8+) responses and, therefore, represent attractive vectors for generating vaccines aimed at producing T(CD8+)-mediated protective immunity. This review will examine the immunological properties of viruses that make them good candidates as vaccine vectors, as well as the manipulations of both vector and antigen that may be required to produce an effective vaccine. The areas addressed include virus infection of dendritic cells in vivo, stimulation of the innate immune response via intracellular and extracellular pattern recognition receptors, the effect of antigenic form on the pathways of antigen presentation and the requirement for elimination of viral genes that target various aspects of the innate and adaptive immune response.     

Incomplete CD8(+) T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen

CD8(+) cytotoxic T lymphocytes (CTLs) recognize antigen in the context of major histocompatibility complex (MHC) class I molecules. Class I epitopes have been classified as dominant or subdominant depending on the magnitude of the CTL response to the epitope. In this report, we have examined the in vitro memory CTL response of H-2(d) haplotype murine CD8(+) T lymphocytes specific for a dominant and subdominant epitope of influenza hemagglutinin using activation marker expression and staining with soluble tetrameric MHC-peptide complexes. Immune CD8(+) T lymphocytes specific for the dominant HA204-210 epitope give rise to CTL effectors that display activation markers, stain with the HA204 tetramer, and exhibit effector functions (i.e., cytolytic activity and cytokine synthesis). In contrast, stimulation of memory CD8(+) T lymphocytes directed to the subdominant HA210-219 epitope results in the generation of a large population of activated CD8(+) T cells that exhibit weak cytolytic activity and fail to stain with the HA210 tetramer. After additional rounds of restimulation with antigen, the HA210-219-specific subdominant CD8(+) T lymphocytes give rise to daughter cells that acquire antigen-specific CTL effector activity and transition from a HA210 tetramer-negative to a tetramer-positive phenotype. These results suggest a novel mechanism to account for weak CD8(+) CTL responses to subdominant epitopes at the level of CD8(+) T lymphocyte differentiation into effector CTL. The implications of these findings for CD8(+) T lymphocyte activation are discussed.     

Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and na?ve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting na?ve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.     

Recombinant adeno-associated virus vectors induce functionally impaired transgene product-specific CD8+ T cells in mice

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8(+) T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product-specific CD8(+) T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector-induced CD8(+) T cells proliferated poorly, produced low levels of IFN-gamma in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8(+) T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.     

The amount of surface HLA-I on T lymphocytes decreases in breast infiltrating ductal carcinoma patients

Human leucocyte antigen class I (HLA-I), which includes HLA-A, -B and -C, is an essential immune factor participating in the antitumour immune response. The changes in HLA-I expression in peripheral blood T lymphocytes in cancer patients have yet to be defined. This study examined the expression of HLA-I on CD4(+) and CD8(+) T lymphocytes in female patients with stage I - IV breast infiltrating ductal carcinoma, benign breast tumour diseases (mammary intraductal papilloma or breast fibroadenoma), and in healthy controls. HLA-I was down-regulated on CD4(+) T lymphocytes from patients with stage III and IV cancer, and on CD8(+) T lymphocytes in patients with stage I - IV cancer compared with healthy controls. HLA-I expression in T lymphocytes may contribute towards immune-balance disorders in tumour patients.     

miRNA-mediated Silencing in Hepatocytes Can Increase Adaptive Immune Responses to Adenovirus Vector-delivered Transgenic Antigens

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K^b/S_190–197-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.     

Adenoviral transgene ubiquitination enhances mouse immunization and class I presentation by human dendritic cells

Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors (Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A*0201 humanized HHD mice and (2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells (DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a (51)Cr-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.     

Liver Damage Preferentially Results from CD8+ T Cells Triggered by High Affinity Peptide Antigens

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)–restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4⁻CD8⁻B220⁺ T cells occurs more frequently from a CD8⁺ precursor than from CD4⁺ T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8⁺ cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC–restricted TCR. The findings show that CD4⁻CD8⁻B220⁺ T cells preferentially derive from a CD8⁺ precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.     

Chemokine receptor CXCR3 facilitates CD8(+) T cell differentiation into short-lived effector cells leading to memory degeneration

Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8(+) T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8(+) T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3(-/-) antigen-specific CD8(+) T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8(+) T cells. Early after infection, CXCR3(-/-) antigen-specific CD8(+) T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-α are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3(-/-) CD8(+) T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8(+) T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8(+) T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.     

Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery

Dendritic cells (DCs) are being evaluated in immunization protocols to enhance immunity against infectious diseases and cancer. Interaction of T-helper cells expressing CD40 ligand (CD40L) with its cognate CD40 receptor on DCs leads to a mature DC phenotype, characterized by increased capacity of antigen presentation to cytotoxic T cells. The authors examined the ability of third-generation self-inactivating lentiviral vectors expressing CD40L to induce autonomous maturation of ex vivo expanded human monocyte-derived dendritic cells. Transduction with lentiviral vectors achieved a highly efficient gene transfer of CD40L to DCs, which correlated with phenotypic maturation as shown by the expression of immunologic relevant markers (CD83, CD80, MHCI) and secretion of IL-12, whereas DC phenotype was not affected by a control vector expressing only the green fluorescent protein marker. Addition of recombinant IFN-gamma to DCs at the time of CD40L transduction further enhanced IL-12 production, and when co-cultured with allogeneic and autologous CD8+ and CD4+ T cells, a potent activation was observed. Autologous responses against an HLA-A2-restricted influenza peptide (Flu-M1) and a tumor-associated antigenic peptide (gp100 210M) were significantly enhanced when CD40L transduced DCs were used as antigen-presenting cells for in vitro stimulation of CD8+ cytotoxic T lymphocytes. These results demonstrate that endogenous expression of CD40L by lentivirally transduced DCs induced their autonomous maturation to a phenotype comparable to that induced by optimal concentrations of soluble CD40L, providing a novel tool for genetic manipulation of DCs.     

Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen

Interferon (IFN)-gamma plays an important role in the innate immune response against intracellular bacterial pathogens. It is commonly thought that natural killer cells are the primary source of this cytokine that is involved in activating antibacterial effects in infected cells and polarizing CD4+ T cells toward the Th1 subset. However, here we show that both effector and memory CD8+ T cells have the potential to secrete IFN-gamma in response to interleukin (IL)-12 and IL-18 in the absence of cognate antigen. We demonstrate that memory CD8+ T cells specific for the ovalbumin protein secrete IFN-gamma rapidly after infection with wild-type Listeria monocytogenes (LM). Furthermore, small numbers of ovalbumin-specific, memory CD8+ T cells can reduce spleen and liver bacterial counts in IFN-gamma-deficient mice 3 d after LM infection. Up-regulation of the receptors for IL-12 and IL-18 provides a mechanism for the ability of memory CD8+ T cells to respond in this antigen nonspecific manner. Thus, CD8+ T cells play an important role in the innate immune response against intracellular pathogens by rapidly secreting IFN-gamma in response to IL-12 and IL-18.     

Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.