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Inflammatory caspases, including caspase-11, are upregulated in CD8+ T cells after Ag-specific activation, but little is known about their function in T cells. We report that caspase-11–deficient (Casp11−/−) T cells proliferated more readily in response to low-affinity and low-abundance ligands both in vitro and in vivo due to an increased ability to signal through the TCR. In addition to increased numbers, Casp11−/− T cells had enhanced effector function compared with wild-type cells, including increased production of IL-2 and reduced expression of CD62L. Casp11−/− T cells specific for endogenous Ags were more readily deleted than wild-type cells. These data indicate that caspase-11 negatively regulates TCR signaling, possibly through its ability to regulate actin polymerization, and inhibiting its activity could enhance the expansion and function of low-affinity T cells.

The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the αβ TCR. However, T cells that express the γδ TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ T cell numbers increased 10-fold in the infected ear and resulted in a novel γδ T cell population not found in naive skin. Circulating γδ T cells were specifically recruited to the site of inflammation and differentially contributed to dermal populations based on their CD27 expression. Recruited γδ T cells, the majority of which were CD27+, were granzyme B+ and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident γδ T cell populations that made IL-17 were CD27−. Using a double-chimera model that can discriminate between the resident dermal and recruited γδ T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflammation. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and collateral damage in the tissue during viral infection. These findings have important implications for our understanding of immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune defense.

Tissue resident memory (Trm) CD8 T cells represent a newly described memory T cell population. We have previously characterized a population of Trm cells that persists within the brain after acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm cells do not undergo recall expansion after dissociation from the tissue. Furthermore, these Trm cells do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells, we compared the gene expression profiles of Trm cells isolated from the brain with those of circulating memory T cells isolated from the spleen after an acute virus infection. Trm cells displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors, and overexpressed several inhibitory receptors. Cumulatively, these data indicate that Trm cells are a distinct memory T cell population disconnected from the circulating memory T cell pool and display a unique molecular signature that likely results in optimal survival and function within their local environment.

The molecular mechanisms that regulate mature T cell fate and enable cells to differentiate into memory T cells are largely unknown. Memory T cells share certain key features with stem cells: they both have the ability to self-renew and are long-lived. The Wnt–β-catenin signaling pathway is a key player in regulating stem cell self-renewal and differentiation. We generated a conditional knockout mouse that specifically lacks β-catenin in mature T cells and report in this article that β-catenin is not involved in regulating effector versus memory T cell differentiation. β-catenin–deficient memory T cells were phenotypically and functionally indistinguishable from control cells and made normal recall responses. β-catenin deficiency does not affect T cell migration, T cell function in a model of chronic infection, or lymphopenia-induced proliferation. Together, our data suggest that self-renewal and differentiation are regulated differently in memory T cells compared with epithelial and hematopoietic stem cells.

In the real world, mice and men are not immunologically naive, having been exposed to numerous antigenic challenges. Prior infections sometimes negatively impact the response to a subsequent infection. This can occur in serial infections with pathogens sharing cross-reactive Ags. At the T cell level it has been proposed that preformed memory T cells, which cross-react with low avidity to epitopes presented in subsequent infections, dampen the response of high-avidity T cells. We investigated this with a series of related MHC class-I restricted Ags expressed by bacterial and viral pathogens. In all cases, we find that high-avidity CD8+ T cell precursors, either naive or memory, massively expand in secondary cross-reactive infections to dominate the response over low-avidity memory T cells. This holds true even when >10% of the CD8+ T cell compartment consists of memory T cells that cross-react weakly with the rechallenge ligand. Occasionally, memory cells generated by low-avidity stimulation in a primary infection recognize a cross-reactive epitope with high avidity and contribute positively to the response to a second infection. Taken together, our data show that the phenomenon of original antigenic sin does not occur in all heterologous infections.

In the thymus, the transcription factor ThPOK is essential for the development of the CD4 helper T cell lineage, whereas active repression of ThPOK is critical for the development of the CD8 cytotoxic T cell lineage. ThPOK gene silencing is thought to be irreversible in peripheral CD8 T cells. We noticed that ThPOK repression is readily abrogated upon in vitro TCR stimulation of peripheral CD8 T cells. This observation prompted us to investigate a role for ThPOK in the CD8 T cell response to an acute viral infection. We observed that a functional deficiency of ThPOK does not affect CD8 T cell differentiation into effector T cells and the long-term persistence of Ag-specific memory T cells. However, in the absence of functional ThPOK, clonal expansion is significantly less in both primary and secondary CD8 T cell responses. Long-lived, Ag-specific CD8 T cells with a functional deficiency in ThPOK fail to produce high amounts of IL-2 and also fail to express high levels of granzyme B upon rechallenge. Our data reveal an unexpected role for ThPOK in CD8 T cells in promoting expansion and boosting the response to antigenic challenge.

The transcription factor X-box-binding protein-1 (XBP-1) plays an essential role in activating the unfolded protein response in the endoplasmic reticulum (ER). Transcribed XBP-1 mRNA is converted to its active form by unconventional cytoplasmic splicing mediated by inositol-requiring enzyme-1 (IRE-1) upon ER stress. We report activation of the IRE-1/XBP-1 pathway in effector CD8+ T cells during the response to acute infection. Transcription of unspliced XBP-1 mRNA is up-regulated by IL-2 signals, while its splicing is induced after TCR ligation. Splicing of XBP-1 mRNA was evident during the expansion of Ag-specific CD8+ T cells in response to viral or bacterial infection. An XBP-1 splicing reporter revealed that splicing activity was enriched in terminal effector cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1). Overexpression of the spliced form of XBP-1 in CD8+ T cells enhanced KLRG1 expression during infection, whereas XBP-1−/− CD8+ T cells or cells expressing a dominant-negative form of XBP-1 showed a decreased proportion of KLRG1high effector cells. These results suggest that, in the response to pathogen, activation of ER stress sensors and XBP-1 splicing contribute to the differentiation of end-stage effector CD8+ T cells.

Ag-specific CD8+ T cells immunized in the absence of CD4+ T cell help, so-called “unhelped” CD8+ T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8+ T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4+ T cells. Secondly, we generated mice doubly deficient in CD4+ T cells and TRAIL and analyzed their CD8+ T cell response to LmOVA. Memory CD8+ T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4+ T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8+ T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8+ T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4+ T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4+ T cell help for the CD8+ T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.

IL-7 signals are crucial for the survival of naive and memory T cells, and the IL-7R is expressed on the surface of these cells. Following viral infection, the IL-7R is expressed on only a subset of effector CD8 T cells, and has been demonstrated to be important for the survival of these memory precursors. IL-7 message levels remain relatively constant during the T cell response to lymphocytic choriomeningitis virus, but a short-lived burst of GM-CSF is observed soon after infection. Retroviral expression of a chimeric GM-CSF/IL-7R, in which binding of GM-CSF by T cells leads to IL-7 signaling, allows for the delivery of an IL-7 signal in all effector T cells expressing the receptor. In mice infected with lymphocytic choriomeningitis virus, CD8 and CD4 T cells transduced with this chimeric receptor underwent an enhanced proliferative response compared with untransduced populations in the same host. Similarly, TCR transgenic CD8 cells expressing the chimeric receptor produced higher effector numbers during the peak of the T cell response to infection. Surprisingly, the enhanced proliferation did not lead to higher memory numbers, as the subsequent contraction phase was more pronounced in the transduced cell populations. These findings demonstrate that artificial IL-7 signaling during an infection leads to significantly increased Ag-specific effector T cell numbers, but does not result in increased numbers of memory progeny. The extent of contraction may be dictated by intrinsic factors related to the number of prior cell divisions.

Recent studies have suggested a role for MHC class Ib molecules in providing signals for memory T cell differentiation during the early phases of acute infection. To test this hypothesis, we assessed the development of effector and memory CD8 T cells in transgenic mice expressing a single chain H-2Dd/β2-microglobulin (β2M) fusion protein on a β2M-deficient background. These mice thus express a single MHC class Ia in the absence of all other β2M-dependent class Ia and Ib molecules. Following infection with a recombinant vaccinia virus expressing a known Dd-restricted epitope from HIV-1 gp160, the development of effector and memory cells CD8 T cells was comparable to control mice. Furthermore, these memory cells responded rapidly and robustly to antigenic restimulation. Therefore, we conclude that full CD8 memory differentiation requires only a single MHC class Ia chain, ruling out a requirement for MHC class Ib molecules in this process.

CD8α+ dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8α+ DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8α+ DCs are the principal DC subset that initiate CD8+ T cell immunity.

Following a primary immune response, a portion of effector T cells gives rise to long-lived memory cells. Although primary expansion and differentiation of effector CD8 T cells is dictated by a brief exposure to Ag, it is unclear whether full memory differentiation is also programmed within the same short window. By carefully modulating the kinetics of Listeria monocytogenes infection, we analyzed the requirements for the programming of effector and memory T cell development in vivo. We find that although limiting the infectious period to the first 24–48 h does not impact the size of the primary CD8 response, the ensuing memory population is significantly diminished. This effect is particularly pronounced in the development of tissue-homing memory cells and is inversely proportional to the initial infectious dose. In contrast to CD8 responses, the differentiation of primary CD4 responses was highly dependent on the continued presence of the infection. Shortening the duration of the infection greatly reduced the development of CD4 effector responses in the spleen and prevented their trafficking to peripheral sites of infection. We propose that the stimulus received by CD8 T cells during the early stages of infection largely contribute to the differentiation of CD8 effector cells, whereas continued or distinct signals received at later stages influence their ability to differentiate into memory cells.

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4+ and CD8+ T cells are markedly different. Gads−/− CD4+ T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads−/− CD4+ T cells continued to proliferate at a higher rate than wild-type CD4+ T cells, demonstrating a defect in homeostatic proliferation. Gads−/− CD8+ T cells had a memory-like phenotype, produced IFN-γ in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads−/− T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads−/− CD4+ T cells, but not CD8+ T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads−/− CD4+ T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4+ and CD8+ T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4+ T cells.

CD8 T cells need CD4 T cells to develop into long-lived, functional memory cells that provide protection against pathogen rechallenge. We investigated whether signaling via CD40 expressed on the CD8 cells themselves is involved in this cooperation. In murine responses to Listeria monocytogenes and lymphocytic choriomeningitis virus, we found no evidence of any requirement for CD40-CD40 ligand interaction at this level. No differences were observed between CD40−/− and CD40+/+ CD8 T cells that had matured in the same environment when comparing their expansion in a primary or secondary response, their contribution to memory, and their ability to enter nonlymphoid tissues such as the liver. Thus, we find no evidence that CD40 ligand-expressing CD4 T cells are required to activate CD40 on CD8 T cells directly for the full differentiation of the cytotoxic T cell response.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8+ T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8+ T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8+ T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8+ T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8+ T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

We have characterized the function of Notch-regulated ankyrin-repeat protein (Nrarp) in mouse cell lines and in hematopoietic stem cells (HSCs). Nrarp overexpression is able to block Notch-induced activation of CBF-1. In AKR1010 thymoma cells, Nrarp overexpression blocks CBF-1-dependent transcriptional activation of Notch-responsive genes and inhibits phenotypic changes associated with Notch activation. Enforced expression of Nrarp in mouse HSCs results in a profound block in T lineage commitment and progression through early stages of thymocyte maturation. In contrast, Deltex-1 overexpression in HSCs can also block T lineage commitment but not progression through the early double negative stages of thymocyte maturation. The different effects of Deltex-1 and Nrarp overexpression suggest that alternate Notch signaling pathways mediate T vs B lineage commitment and thymocyte maturation.

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (Kb−/−Db−/− mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (β2m−/− mice). The susceptibility of Kb−/−Db−/− mice cannot be explained by the failure of CD8+ T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8+ T cells were a relatively small population in uninfected Kb−/−Db−/− mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-γ following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of Kb−/−Db−/− mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8α−/− mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8+ effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2−/− double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.

Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor RORγt in mature T cells down-regulates their surface TCR expression. The RORγt transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of RORγt on c-Rel transcription. Furthermore, ectopic expression of RORγt inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that RORγt controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of RORγt expression in thymocytes is essential for their maturation.

Chemokines are likely to play an important role in regulating the trafficking of developing T cells within the thymus. By using anti-CD3ε treatment of recombinase-activating gene 2 (Rag2−/−) mice to mimic pre-TCR signaling and drive thymocyte development to the double positive stage, we have identified murine GPR-9-6 as a chemokine receptor whose expression is strongly induced following pre-TCR signaling. GPR-9-6 mRNA is present at high levels in the thymus, and by RT-PCR analysis its expression is induced as normal thymocytes undergo the double negative to double positive transition. Furthermore we show that TECK (thymus-expressed chemokine), a chemokine produced by thymic medullary dendritic cells, is a functional ligand for GPR-9-6. TECK specifically induces a calcium flux and chemotaxis of GPR-9-6-transfected cells. In addition, TECK stimulates the migration of normal double positive thymocytes, as well as Rag2−/− thymocytes following anti-CD3ε treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor 9 (CCR9). Our results suggest that TECK delivers signals through CCR9 important for the navigation of developing thymocytes.

Ag is presented to CTL as peptide associated with MHC class I molecules, which are present on most types of cells. We have investigated the presentation of Db-restricted lymphocytic choriomeningitis virus (LCMV) peptides by a fibroblast line (MC57) and a dendritic cell line (JawsII) to splenocytes from LCMV-immune C57BL/6 mice. We found that when LCMV-infected MC57 were used to restimulate the spleen cells, the resulting CTL line lost its ability to respond to the two dominant epitopes of the immune response to LCMV glycoprotein (gp)33 and nucleoprotein (np)396 but remained strongly lytic for targets coated with the subdominant gp276 epitope. In contrast, when LCMV-infected JawsII cells were used to restimulate the splenocytes, the resulting line continued to target gp33 and np396 but lost reactivity to gp276. When uninfected JawsII or MC57 cells were coated with peptides and used as stimulators, the resulting CTL lines continued to recognize all three epitopes, indicating that costimulatory or other potential innate differences in Ag presentation between the two cell lines are unlikely to account for the selective expansion of CTL specificities. When infected, both cell types produce similar levels of infectious LCMV, have similar levels of the NP and GP proteins from which np396 and gp33 are derived, and can be recognized by CTL specific for each of the three epitopes. These data indicate that in the generation of peptides for MHC-I binding and presentation to CTL, MC57 and JawsII process the same set of virus proteins in quantitatively different ways.

Targeted disruption of genes in mouse embryonic stem cells, incorporation of the mutation into the germline, and transmission to offspring was realized only in the late 1980s. Research in the laboratories of Mario Capecchi, Rudolf Jaenisch, Elizabeth Robertson, and Oliver Smithies pioneered this effort. It is difficult to believe, but reports of the first targeted deletion of a gene of immunological import and the generation and analysis of mice homozygous for the mutation were published only in 1990. Two papers independently reported the generation of mice in which both copies of the β2- microglobulin (β2M) gene had been disrupted (1, 2). So novel and powerful was this technology that both groups had previously published (in 1989) the derivation of the targeted embryonic stem cells, their injection into blastocysts, and the generation of mice heterozygous for the β2M mutation (3, 4). Disruption of the β2M gene and production of “β2M-knockout mice” confirmed in the whole animal the many previously suspected roles of the molecule. Thus, the mice lacked surface expression of the classical MHC class IH-2K and H-2D proteins because β2M is the L chain partner for the class I H chain. Similar results had been shown previously in β2M-deficient cell lines. The β2M-knockout mice lacked CD8+ T cells because MHC class I expression by thymic epithelial cells is required to “positively select” class I-restricted, TCR αβ+, CD8+ T cells. In addition, one of the papers from1990 showed that functional expression of the neonatal Fc receptor, also known to be β2M associated, was lacking in the knockout mice (2). Finally, both groups reported that T cells expressing a γδ TCR were present in normal numbers in the spleen and thymus of β2M-knockout mice. Perhaps more important than these expected phenotypes, the knockout mice showed what β2M did not do. The homozygous β2M-knockout mice were healthy, and both males and females were fertile. The knockout mice had apparently normal development, ruling out previous suggestions of an involvement of β2M or MHC molecules in a wide range of developmental and signaling functions. Whatever those functions may be, the role of β2M was dispensable. In addition, the normal breeding behavior of homozygous β2M-knockout mice lent no support to the suggestion that MHC class I was somehow involved in mating preferences of mice. More important still are the later discoveries that depended on or benefited from the use of β2M-knockout mice. The idea that NK cells would respond to “missing self ” to become activated to remove MHC-deficient cells had been proposed (5). The new knockout mice provided a clear demonstration of this with the finding that it was almost impossible to reconstitute a lethally irradiated wild-type recipient with bone marrow stem cells from a β2M-deficient donor (6). The knockout marrow was rapidly rejected. This was a novel incarnation of the MHC-linked barrier to transplantation known as the hybrid histocompatibility (Hh) phenomenon (7). Rejection of knockout marrow was shown to be due to the inability of β2M-deficient stem cells to deliver “OFF” signals to recipient NK cells via their MHC-specific inhibitory receptors. The β2M-knockout mice lacked NKT cells (8, 9). These TCR-positive cells develop in the thymus and express either the CD4 coreceptor or no coreceptor. A nonclassical, β2M-associated class I molecule, CD1, was later shown to be the ligand for this lineage (10). For NKT cells, their selection in the thymus came with a novel twist: in this case, the selecting MHC class I was expressed on double positive thymocytes themselves and not epithelial cells, as shown by the use of radiation chimeras with wild-type and β2M-knockout partners. The β2M-knockout mice also contributed to our understanding of the self-peptide dependence for positive selection of CD8+ T cells in the thymus. Fetal thymus lobes from mice lacking β2M (which do not develop mature CD8+ T cells) were used in an organ culture system to demonstrate that, with a polyclonal TCR repertoire, the addition of a diversity of class I-binding peptides plus exogenous β2M was required to positively select a significant population of mature CD8+ T cells (11). With stem cells bearing a single MHC class I-restricted TCR, a single peptide, related to the agonist peptide but with extremely low affinity for the TCR, was shown to be required for positive selection without inducing negative selection (12). In 1994, β2M-knockout mice were found to suffer from iron overload (13). Livers from the knockout mice had many times higher levels of iron than did heterozygous control animals. This mimicked the HLA-linked human condition known as hereditary hemochromatosis. Mutation in a novel class I-like gene within the HLA complex, called HLA-H, was later reported to be highly associated with the disease (14). The β2M-knockout mice have contributed greatly to the analysis of T cell, NKT cell, and NK cell function. Their generation in 1990 represents a landmark in biology as a whole. The knockouts ushered in the modern era of targeting genes of interest for deletion in the germline or for conditional deletion via the use of “floxed” alleles and tissue-specific or timed expression of the cre recombinase.

The pool of naïve T cells specific for any foreign peptide/MHC-I epitope is diverse and contains cells that differ in their functional avidity. Theoretical considerations imply that there are likely to be more low than high avidity T cells specific for any foreign antigen. We studied to what extent lower avidity T cells participate in an immune response and we investigated how the dominance of high avidity T cells evolves over the course of an infection. To control the strength of stimulation that CD8 T cells receive during an immune response, we used a model system in which OT-1 TCR transgenic CD8 T cells were stimulated during a Listeria monocytogenes infection either by their native ligand SIINFEKL or by different SIINFEKL-derived altered peptide ligands. Using this system we observed that even very weak TCR-ligand interactions are sufficient to induce rapid T cell proliferation and to generate functional effector and memory CD8 T cells. However, the strength of the TCR-ligand interaction impacts when expansion stops and contraction begins and when the cells exit lymphoid organs, i.e. strongly stimulated T cells contract and exit lymphoid organs later than do weakly stimulated cells. Our data challenges the prevailing view that strong TCR ligation is a prerequisite for CD8+ T cell activation. Instead, very weak interactions are sufficient for activation, but strong TCR ligation is required to sustain T cell expansion.

CD8+ T cells are critical for protection against viral and intracellular bacterial infections. They are characterized by their ability to acquire cytolytic activity to lyse infected target cells. This effector stage is associated with the expression of a variety of intracellular and cell surface markers. Here, we examined the role of direct signaling by the cytokine IL-2 on the acquisition of CD8+ T cell effector functions. IL-2 signals via its heterotrimeric high-affinity receptor composed of the IL-2Rα-chain (CD25), β-chain (CD122) and common γ-chain (CD132). Previous work from our lab and others has shown that direct IL-2 signaling on CD8+ T cells is imperative for their differentiation into memory cells capable of secondary expansion. Using a mixed bone marrow chimeric approach in which wildtype (WT) and CD25 knockout (KO) cells are responding in the same environment we now demonstrate that KO CD8+ T cells are defective in effector differentiation following primary acute infection. CD25 KO effectors show decreased lytic activity and expression of effector-associated proteins and transcription factors, such as Granzyme B and T-bet. In addition, these cells show increased expression of naïve/memory-associated factors, such as Bcl2, Eomesodermin and IL-2 production, despite their inability to expand upon rechallenge. Overall, we conclude that direct IL-2 signaling is required for complete CD8+ T cell effector differentiation following infection in vivo. This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health Grant AI-19335 (to M.J.B.)

A small fraction of the CD8 T cell effector population that responds to an infection progresses through the contraction phase to the memory stage. The factors regulating the extent of contraction are poorly understood. In previous experiments, parameters such as inflammation, antigen presentation, etc. were modified during the priming/expansion phase and the consequences of these changes were monitored in the contraction phase. A major caveat of these studies is that the experimental setup did not allow examination of the contraction phase without altering the priming phase first. We developed a system based on bim-/- CD8 T cells that overcomes this confounding problem. Competition for limited resources has been widely postulated to be the cause of cell death during the contraction phase, but our data show that competition does not affect contraction kinetics. All effector cells present at the peak of the response have the potential to become bona fide memory cells, thus excluding selection based on functionality. We propose that cell death during contraction is the default pathway for an effector cell and identify a set of genes that may be key regulators for surviving contraction. This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grant AI-19335 (to M.J.B.). M.P. is a fellow of The Leukemia & Lymphoma Society. The authors have no conflicting financial interests.

Anti-IL-2 antibodies have been used with the premise that they block IL-2 signaling in vitro and in vivo, but recent findings demonstrate that anti-IL-2 antibodies form a complex with IL-2 in vivo that actually enhances IL-2 activity. NK cells are one cell type affected by these IL-2 complexes. We asked at what stage IL-2 complexes act on NK cells and whether they could be utilized to generate functional NK cells with potential therapeutic value. We found that the administration of IL-2 complexes resulted in the rapid generation of NK cells in IL-15−/− animals. IL-15 is required for the development and survival of NK cells. Mature, transferred NK cells survived in IL-15−/− animals treated with IL-2 complexes, but proliferated only modestly. These data suggest that the IL-2 complex preferentially expands an immature subset of NK cells. To determine whether this expansion results in the generation of functional NK cells, we treated lethally irradiated, bone marrow reconstituted mice with IL-2 complexes and assessed the activity of NK cells by an in vivo killing assay. Bone marrow chimeras that were treated with IL-2 complexes displayed greatly enhanced NK lytic activity compared to control chimeras 4 days after initiation of treatment. We conclude that IL-2 complex has the potential to rapidly generate functional NK cells that may reduce the risk of viral infections and influence GVHD processes.

Cooperation of CD4+ and CD8+ T cells is required for the development of primary effector and memory CD8+ T cells following immunization with non-inflammatory immunogens. In this study, we characterized endogenous CD4+ and CD8+ T cell responses to male-specific minor histocompatibility (HY) antigens. We find that male cells are rejected 10–12 days after transfer into female mice of the same strain, and this rejection coincides with the expansion and effector function of CD8+ CTLs to two H-2Db-restricted epitopes. While anti-male CD4+ T cell responses are readily detectable day 5 post-transfer, CD8+ T cell response are not detectable until day 10. The early CD4+ T cell response is not dependent on direct presentation of antigen by donor male cells, but depends on processing and presentation of the male cells by female APC. The primary and recall CD8+ T cell responses and rejection of HY incompatible cells are dependent on a sustained CD4+ T cell response and the absence of this response is not replaced by inflammation or treatment with anti-CD40 agonist Ab. Unexpectedly, HY-specific CD4+ T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8 effector response can be largely abrogated by depleting T cells from the male inoculum, and we further demonstrate that donor CD8+ T cells in particular suppress or veto the host anti-HY CD8+ T cell responses.

The cellular basis of the cross-priming observed with minor histocompatibility antigens on H-2 different cells was investigated. Cytotoxic T cells induced against minor alloantigens show absolute H-2 restriction at the effector level ([51Cr]-release). That is, F1(BALB/c × BALB.B) (H-2d/b) cytotoxic cells induced by immunization with B10(H-2b) cells are not able to lyse B10.D2(H-2d) targets. But an injection of B10 cells does prime F1 mice for a secondary cytotoxic response to B10.D2. The technique of inducing cytotoxic effector function polyclonally with Con A in the absence of alloantigen was used here to establish that such cross-priming reflects what happens at the cytotoxic cell level. It is shown that an F1 animal previously injected with B10 cells has expanded pools of memory cytotoxic cells reactive with B10 and B10.D2. From this it is concluded that: a) minor H structures on B10.D2 and B10 do cross-react at the cytotoxic T cell level during in vivo priming, and b) because normal cells cross-prime whereas tumor cells do not, then the F1 cytotoxic precursors are probably committed to respond to antigen on cells bearing either the maternal or paternal H-2 haplotype before they encounter antigen. Cross-priming may be explained by foreign minor H antigens being presented to F1 host T cells on the surface of host macrophages. Therefore priming is not restricted to the H-2 type of the injected cells but to both H-2 types of the F1 host.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c × BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A.Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

Mitogen-induced cytotoxicity was studied by culturing mouse spleen cells with an optimum mitogenic dose of concanavalin A (Con A) for 2 days and, after washing, assessing their ability to lyse tumor targets. We show that rapid lysis occurs only when the correct concentration of an agglutinating mitogen (phytohemagglutinin P (PHA) or Con A) is present in the assay. PHA is more efficient than Con A in revealing the cytotoxic effect. The Con A-induced effector cytotoxic cell is shown to be sensitive to anti-ϑ serum and complement. Cytotoxic T cells were also induced by H-2 different allo-immunization. These effector cells specifically lyse targets which bear the immunizing H-2 antigens in the absence of PHA. When PHA is present in the assay, they will also lyse syngeneic tumor targets nonspecifically. Since not all dividing T cells (e.g., T lymphomas, and T blasts induced by M locus different, H-2 similar mixed lymphocyte culture) lyse PHA-P815, we propose that this assay measures only a subset of effector T cells, namely, cytotoxic T cells, regardless of their antigen specificity. The tumor cells, P815 and EL4, are sensitive both to antigen-specific T cell-mediated lysis in the absence of PHA and to nonspecific, PHA-revealed lysis. Small lymphocytes, T cell blasts, and B cell blasts are sensitive to lysis by T cells which are directed against H-2 antigens on their surface, but are not very susceptible to nonspecific T cell lysis in the presence of PHA. The reason for this difference in susceptibility of tumor and normal cells to PHA-revealed nonspecific T cell lysis is not known but may have some relevance to in vivo tumor rejection.

Murine T cells immunized with allogeneic cells exert a cytotoxic effect on appropriate target cells, as measured by 51Cr release. Lysis of the 51Cr-labeled target can be inhibited by addition to the assay of unlabeled cells which bear target antigens. With variants of lymphoid tumors which express different amounts of serologically defined H-2 antigens as competitors in the assay, a positive correlation is shown to exist between the amount of serologically defined antigen and the degree of inhibition of T cell-mediated lysis. The immunizing small lymphocytes are shown to be very poor inhibitors of 51Cr release from target cells which are more susceptible to T cell-mediated lysis. This is interpreted as showing that small lymphocytes are bound very poorly by cytotoxic T cells. With H-2 cross-over strains which carry only the H-2K or H-2D antigens which a cytotoxic population is directed against, it is shown that, 1) approximately equal amounts of cytotoxicity are directed against the K and D antigens, and 2) separate cytotoxic T cells recognize the K and D antigens. Thus, by this criterion, cytotoxic T cells are monospecific and therefore do not acquire their antigen-specific receptors cytophilically.

Spleen cells from normal mice do not give a detectable in vitro cytotoxic T cell (CTL) response to minor H antigens. Spleen cells from animals primed in vivo with minor H antigens give a strong CTL response when boosted in culture with the appropriate stimulating cells. Here I have studied the requirements for priming a CTL response to minor H antigens. It is shown that priming is just as antigen specific as is cytolytic effector function. That is, the priming cells have to carry the same minor antigens as the challenge cells. Inducing a graft-vs-host reaction in vivo does not nonspecifically allow spleen cells to respond to minor H antigens in vitro. Using minor H congenic mice (congenic for H-Y and/or H-7) I have also tried, and failed, to demonstrate a carrier effect in priming. Female mice primed to H-Y were challenged in culture with cells bearing H-Y and H-7 antigens in the hope that a helper response to H-Y would augment a CTL response to H-7. This did not happen, however. Such primed and boosted cells gave a strong secondary CTL response to H-Y but none to H-7. It is concluded that in order to prime for a detectable in vitro response to minor antigens it is necessary to expose the CTL precursors to antigen in vivo. This either expands the size of the pool of precursors by cell division or changes them in some qualitative way.