CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells,respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene

Contact sensitivity (CS) is a form of delayed-type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed-type hypersensitivity responses, by CD4⁺ T helper-1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8⁺, CD4⁺, or both, T cells. MHC class I single-deficient and MHC class I/class II double-deficient mice, both of which have a drastic reduction in the number of CD8⁺ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II-deficient mice and normal mice treated with an anti-CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti-CD8 mAb depletion of class II-deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4⁺ T cells in class II-deficient mice by anti-CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I-restricted CD8⁺ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II-restricted CD4⁺ T cells down-regulate this inflammatory response.     

Expression of class II,but not class I,major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4⁺ T helper lymphocytes sensitized to parasite egg antigens. However, CD8⁺ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4⁺ and CD8⁺ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4⁺ and CD8⁺ T cells. The results clearly show that schistosome-infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild-type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon-gamma, interleukin (IL)-4, IL-10] responses; nor were they able to present egg antigens to specifically sensitized CD4⁺ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild-type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II-restricted CD4⁺ T helper cells. They also suggest that CD8⁺ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.     

The murine buccal mucosa is an inductive site for priming class I-restricted CD8+ effector T cells in vivo

The present study shows that Langerhans cells of the buccal mucosa and the skin share a similar phenotype, including in situ expression of MHC class II, the mannose receptor DEC-205 and CD11c, and absence of the costimulatory molecules B7.1, B7.2 and CD40 as well as Fas. Application of 2,4-dinitrofluorobenzene (DNFB) onto the buccal mucosa is associated with a rapid migration of dendritic cells (DC) to the epithelium and induction of B7.2 expression on some DC. Buccal sensitization with DNFB elicited a specific contact sensitivity (CS) in response to skin challenge, mediated by class I-restricted CD8⁺ effector T cells and down-regulated by class II-restricted CD4⁺ T cells, demonstrated by the lack of priming of class I-deficient mice and the enhanced response of class II-deficient mice, respectively. CS induced by buccal immunization is associated with priming of class I-restricted CD8⁺ effector T cells endowed with hapten-specific cytotoxic activity. Thus, the buccal epithelium is an inductive site, equivalent to the epidermis, for the generation of CS independent of CD4 help, and of cytotoxic T lymphocyte (CTL) responses mediated by class I-restricted CD8⁺ T cells. We propose that immunization through the buccal mucosa, which allows antigen presentation by epithelial DC efficient for priming systemic class I-restricted CD8⁺ CTL, may be a valuable approach for single-dose mucosal vaccination with subunit vaccines.     

A human CD4 transgene rescues CD4−CD8+ cells in β2-microglobulin-deficient mice

The specificity of the αβ T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4^?CD8⁺ or CD4⁺CD8^? phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4^?CD8⁺ T cells in β2-microglobulin-deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

Lymphocyte-activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4+ T lymphocytes

The activation requirements for antigen-dependent proliferation of CD4⁺ T cells are well documented, while the events leading to the inactivation phase are poorly understood. Here, we tested the hypothesis that the lymphocyte-activation gene 3 (LAG-3), a second major histocompatibility complex (MHC) class II ligand, plays a regulatory role in CD4⁺ T lymphocyte activation. CD4⁺ class II-restricted T cell clones were stimulated by their relevant antigen (hemagglutinin peptide or diphteria toxoid) and antigen-presenting cells with or without anti-LAG-3 monoclonal antibody (mAb). Kinetic studies were performed to monitor different activation parameters, including the measurement of thymidine incorporation, expression of activation antigens and cytokine secretion. Results showed that the time course from the initial time points up to the peak time point was not modified in the presence of anti-LAG-3 mAb. However, addition of these antibodies, either as whole IgG or as Fab fragments, led to increased thymidine incorporation values for late time points and, hence, to a shift in the decreasing proliferation curve. We also showed that expression of activation antigens, such as CD25, was higher in the presence of anti-LAG-3 mAb, and that cytokine concentrations, i.e. of interferon-γ or interleukin-4, were higher in the corresponding culture supernatants. In addition, we tested whether the effects of anti-LAG-3 mAb were limited to antigen-dependent. MHC class II-restricted responses. The proliferative responses of CD4⁺ T cell clones following stimulation with either interleukin-2, mitogens, a combination of anti-CD2 mAb, immobilized anti-CD3 or anti-T cell receptor mAb were not altered by anti-LAG-3 mAb. The allogeneic proliferative response of a CD8⁺ T cell clone was also not affected. Overall, the present analysis reveals a modulating effect of anti-LAG-3 mAb, mediated specifically on antigen-dependent, MHC class II-restricted responses of CD4⁺ T cell lines. These results support the view that LAG-3/MHC class II interaction down-regulates antigen-dependent stimulation of CD4⁺ T lymphocytes.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.     

Immunotargeting of insulin reactive CD8 T cells to prevent Diabetes

We have previously demonstrated, in the collagen-induced arthritis model (CIA), that repetitive injections of immature bone-marrow-derived dendritic cells (iDCs) induce the expansion of a population of CD4CD49b-expressing cells, and that their adoptive transfer results in protection against CIA in a prophylactic setting. However, the in vivo mechanism responsible for their expansion, as well as their therapeutic potential in established disease remains to be defined. In the present study, we show that expression of the MHC class II molecules on iDCs is required for their expansion thus identifying these cells as MHC class II-restricted T cells. Using adoptive transfer of Thy1.1 positive cells, it is shown that iDC-induced CD4⁺CD49b⁺ T cells home to the lymph nodes draining the inflamed tissue. The high immunomodulatory potential of these cells was underscored following their adoptive transfer in a model of contact hypersensitivity. Finally, we assessed and compared the therapeutic potential of iDC-inducible CD4⁺CD49b⁺ T cells with that of iDCs in established CIA. Repetitive injections of iDCs in arthritic mice failed to decrease the severity of established disease. In contrast however, a single injection of iDC-induced CD4⁺CD49b⁺ T cells reversed clinical symptoms of arthritis and provided long-lasting protection. Together, our data indicate that iDC-induced CD4⁺CD49b⁺ T cells are bona fide T regulatory cells with strong immunomodulatory properties that are not only able to prevent disease onset, but also to interfere with an ongoing inflammatory immune response.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

The lack of CD8α cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56^lck. A T cell-specific transgene encoding CD8α truncated at the cytoplasmic domain (“tailless CD8α”), was introduced into CD8α-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. “Tailless CD8α” was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4^? T cells (6% of T lymphocytes) was found to have cell surface expression of “tailless CD8α” and endogenous CD8β indicating that these cells may belong to the CD8⁺ T cell lineage. A consistent result was obtained from CD8α-deficient mice bearing the “tailless CD8α” and the MHC class I-restricted 2C TcR transgenes. A small population of CD4 T cells expressing CD8β the “tailless CD8α” and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When “tailless CD8α” mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8⁺ CD4^? T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8α is not absolutely required but dramatically enhances the eficiency of thymic maturation of CD8⁺ T cells. The lack of CD8α cytoplasmic domain in peripheral CD8⁺ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.     

CD8 T cells from major histocompatibility complex class II-deficient mice respond vigorously to class II molecules in a primary mixed lymphocyte reaction

Mature CD4⁺ and CD8⁺ T cells are restricted by major histocompatibility complex (MHC) class II and class I molecules, respectively. In a primary mixed lymphocyte reaction (MLR), CD8⁺ T cells from C57BL/6 (B6) mice can respond to allo-class I molecules, but not allo-class II molecules. However, a significant fraction of CD8⁺ T cells from C57BL/6 class II-deficient (B6Aα^?) mice violate this rule by responding vigorously in a MLR to class II molecules. The frequency of responding cells is ～ 50 % of that of B6 CD8⁺ T cells responding to B6bm1 allo-class I molecules. This response requires neither appropriate co-receptor, i.e. CD4, nor exogenous lymphokines, indicating that interactions between the T cell receptors (TCR) and class II molecules are remarkably efficient. Since these CD8⁺ T cells are positively selected by class I molecules in the thymus of class II-deficient mice, these CD8⁺ T cells should interact with both classes of MHC molecules. The absence of thymic negative selection by class II molecules may result in the production of these CD8⁺ T cells. The data imply that a substantial fraction of CD4⁺CD8⁺ double-positive thymocytes in wild-type mice interacts with both classes of MHC molecules prior to thymic selection.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

CD4 T cells can reject major histocompatibility complex class I-incompatible skin grafts

We have re-investigated the roles of CD4 and CD8 T cell subsets in skin graft rejection across a single class I MHC disparity. Recipient mice were transplanted with skin from donors transgenic for the class I MHC molecule K^b. As expected, CD8 T cells were sufficient for rapid injection; but surprisingly, CD4 T cells were also competent to do the same. Rejection was dependent on one or the other subset, since elimination of both resulted in indefinite graft survival. The possibility that alloantibody was the downstream effector of CD4 mediated rejection was excluded because CD8-depleted mice rendered B cell deficient still rejected rapidly, but T cell-depleted recipients with pre-existing high titers of alloantibody were unable to do so. In addition, if CD4 cells act to reject by recruiting and/or activating macrophages then this was not dependent on CR3, IFN-γ or TNF-α. Transplantation of skin grafts where the MHC class I disparity was at the level of passenger leukocytes only, demonstrated that transient bystander damage could occur, but that this was insufficient to result in full rejection. We surmise that for CD4 T cells to reject an MHC class I-incompatible graft it is necessary that an appropriate allogeneic peptide is processed and presented in the context of recipient MHC class II. CD4 T cells from B6 mice may fail to reject skin from MHC class I mutants because of the lack of such MHC class II-restricted presentation.     

Increased number of cytotoxic T cells within CD4+8− T cells in β2-microglobulin,major histocompatibility complex class I-deficient mice

Targeted disruption of β₂-microgobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4^?8⁺ T cells. Despite this, β₂M^?/? mice reject skin grafts and cope with most viral infections tested. We asked whether CD4⁺8^? cytotoxic T cells could play a role in compensating for the defect in CD4^?8⁺ cytotoxic T cell function. We found that the cytotoxic activity against class II⁺ targets is significantly higher among CD4⁺8^? T cells of β₂M^?/? than among those of β₂M^+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4⁺8^?, class II-specific T cells is at least fivefold higher in β₂M ^?/?than in β₂M^+/+ mice. These results suggest that CD4+8^? cytotoxic T cells could play a major role in carrying out cytotoxic function in β₂M^?/? mice.     

TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4⁺ T-cell help (CD4 help) plays a pivotal role in CD8⁺ T-cell responses against viral infections. However, the role in primary CD8⁺ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8⁺ T-cell responses to vaccinia virus (VACV) in MHC class II^−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8⁺ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8⁺ T cells in i.p. infected MHC II^−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II^−/− mice when a small viral dose was used. These data suggested that primary CD8⁺ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8⁺ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8⁺ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8⁺ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8⁺ T cells than i.p. infection.