CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice,but provide minimal protection

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 (K(b-/-)D(b-/-) 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 (beta(2)m(-/-) mice). The susceptibility of K(b-/-)D(b-/-) 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 K(b-/-)D(b-/-) 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-gamma following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of K(b-/-)D(b-/-) 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 CD8alpha(-/-) 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.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice

B6.K(b-)D(b-) mice are devoid of class Ia but express normal levels of class Ib molecules. They have low levels of CD8 T cells in both the thymus as well as peripheral T cell compartments. Although the percentage of splenic CD8 alpha alpha T cells is increased in these animals, approximately 90% of CD8 T cells are CD8 alpha beta. In contrast to B6 animals, most of the CD8 T cells from these mice have a memory phenotype (CD44(high)CD122(high) CD62L(low)) including both CD8 alpha beta and CD8 alpha alpha subsets. In the thymus of B6.K(b-)D(b-) animals, there is a decrease in the percentage of SP CD8 T cells, although most are CD44(low), similar to that seen in B6 mice. The spleens from day 1-old B6 and B6.K(b-)D(b-) mice have a relatively high proportion of CD44(high)CD62L(low) CD8 T cells. However, by day 28 most CD8 T cells in B6 mice have a naive phenotype while in B6.K(b-)D(b-) mice the memory phenotype remains. Unlike CD44(high) cells that are found in B6 animals, most CD44(high) cells from B6.K(b-)D(b-) mice do not secrete IFN-gamma rapidly upon activation. The paucity of CD8 T cells in B6.K(b-)D(b-) mice might be due in part to their inability to undergo homeostatic expansion. Consistent with this, we found that CD8 T cells from these animals expand poorly in X-irradiated syngeneic hosts compared with B6 CD8 T cells that respond to class Ia Ags. We examined homeostatic expansion of B6 CD8 T cells in single as well as double class Ia knockout mice and were able to estimate the fraction of cells reactive against class Ia vs class Ib molecules.     

Effective control of chronic gamma-herpesvirus infection by unconventional MHC Class Ia-independent CD8 T cells

Control of virus infection is mediated in part by major histocompatibility complex (MHC) Class Ia presentation of viral peptides to conventional CD8 T cells. Although important, the absolute requirement for MHC Class Ia-dependent CD8 T cells for control of chronic virus infection has not been formally demonstrated. We show here that mice lacking MHC Class Ia molecules (K(b-/-)xD(b-/-) mice) effectively control chronic gamma-herpesvirus 68 (gammaHV68) infection via a robust expansion of beta2-microglobulin (beta2-m)-dependent, but CD1d-independent, unconventional CD8 T cells. These unconventional CD8 T cells expressed: (1) CD8alphabeta and CD3, (2) cell surface molecules associated with conventional effector/memory CD8 T cells, (3) TCRalphabeta with a significant Vbeta4, Vbeta3, and Vbeta10 bias, and (4) the key effector cytokine interferon-gamma (IFNgamma). Unconventional CD8 T cells utilized a diverse TCR repertoire, and CDR3 analysis suggests that some of that repertoire may be utilized even in the presence of conventional CD8 T cells. This is the first demonstration to our knowledge that beta2-m-dependent, but Class Ia-independent, unconventional CD8 T cells can efficiently control chronic virus infection, implicating a role for beta2-n-dependent non-classical MHC molecules in control of chronic viral infection. We speculate that similar unconventional CD8 T cells may be able to control of other chronic viral infections, especially when viruses evade immunity by inhibiting generation of Class Ia-restricted T cells.     

Distinct regulation of H2-M3-restricted memory T cell responses in lymph node and spleen

CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

Response to Listeria monocytogenes in mice lacking MHC class Ia molecules

MHC class Ia-deficient mice (H2 Kb-/- Db-/-) inoculated with the intracellular pathogen Listeria monocytogenes (LM) displayed a three- to fourfold expansion of splenic CD8+ T cells 6 days following infection. Culture of these spleen cells in vitro gave rise to CTL that recognized LM-infected target cells and were restricted by the class Ib molecules, Qa1b and M3. Exposure of target cells to heat-killed LM (HKLM) rather than live bacteria did not result in CTL-mediated lysis. Target cells pulsed with three LM peptides known to bind M3, f-MIGWII, f-MIVTLF, and f-MIVIL, were recognized by effector cells from both B6 and Kb-/- Db-/- animals. In vivo analysis showed that B6 and Kb-/- Db-/- mice clear LM from the spleen and liver rapidly with similar kinetics, whereas TAP.1-/- mice, which are deficient in class Ia and Ib molecules, clear LM slowly upon infection. To establish the in vivo role of CD8+ T cells in Kb-/- Db-/- animals, we showed that depletion of such cells from the spleens of immune mice prevented the adoptive transfer of protective immunity to syngeneic recipients. Spleen cells from Kb-/- Db-/- mice were also capable of generating responses directed against syngeneic as well as allogeneic class Ia molecules in vitro. Thus, class Ia-deficient animals have a CD8+ T cell repertoire capable of recognizing both class Ia and class Ib molecules and can generate protective immunity to LM.     

V beta T cell repertoire of CD8+ splenocytes selected on nonpolymorphic MHC class I molecules

In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8(+) T splenocytes. We have compared the CD8(+) T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8(+) repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V alpha (AV) and V beta (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8(+) splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8(+) cells, even when the number of CD8(+) splenocytes is dramatically reduced (90% in the double knockout mice).     

MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection

Infection of B6 mice with the intracellular pathogen Listeria monocytogenes (LM) results in the activation of CD8(+) T cells that respond to Ag presented by both MHC class Ia and class Ib molecules. Enzyme-linked immunospot analysis reveals that these CTL populations expand and contract at different times following a primary sublethal LM infection. Between days 4 and 6 postinfection, class Ib-restricted CTL exhibit a rapid proliferative response that is primarily H2-M3 restricted. The peak response of class Ia-restricted CD8(+) T cells occurs a few days later, after the majority of bacteria have been cleared. Although class Ia-restricted CTL exhibit a vigorous recall response to secondary LM infection, we observe limited expansion of class Ib-restricted memory CTL, even in MHC class Ia-deficient mice (B6.K(b-/-)D(b-/-)). Despite this lack of enhanced expansion in vivo, class Ib-restricted memory CTL retain the ability to proliferate and expand when provided with Ag in vitro. Furthermore, we demonstrate that in vivo depletion of CD8(+) T cells in LM-immune B6.K(b-/-)D(b-/-) mice severely impairs memory protection. Together, these data demonstrate that class Ib-restricted CTL play an important role in clearing a primary LM infection and generate a memory population capable of providing significant protection against subsequent infection.     

The source of early IFN-gamma that plays a role in Th1 priming

When naive CD4 T cells are primed, they rapidly differentiate into polarized Th1 and/or Th2 phenotypes. A major factor in producing such polarization is the early production of cytokines (IL-12 and IFN-gamma in the case of Th1 cells and IL-4 in the case of Th2 cells). One issue that remains unresolved is the source of the early IFN-gamma that synergizes with IL-12 to fully polarize CD4 T cells into Th1 cells. We have examined this question by injecting mice with anti-CD3 and examining cells from normal and various MHC-knockout mice. We found that IFN-gamma is induced rapidly in a small subset of CD8 T cells. This subset is absent in mice that lack beta2-microglobulin, but not in K(b)D(b)-double-knockout mice, indicating that these CD8 T cells are dependent on nonclassical MHC class Ib molecules. The early burst of IFN-gamma polarizes CD4 T cells toward Th1 cells, in part by stimulating the release of IL-12 from APC. We also use TAP- and CD1-knockout mice to show that such cells are not CD1-restricted NK T cells, nor are they dependent on TAP-1 transport for surface expression of the relevant MHC class Ib molecule. Therefore, they arise on MHC class Ib molecules that do not depend on TAP-1 transporters.     

Selective dependence of H2-M3-restricted CD8 responses on IL-15

We studied whether CD8 T cell responses that are mediated by unconventional MHC class Ib molecules are IL-15 dependent in mice. CD8(+) T cell responses to Listeria monocytogenes infection that are restricted by the MHC class Ib molecule H2-M3 decreased in the absence of IL-15, whereas other primary MHC class Ib- and MHC class Ia-restricted responses were IL-15 independent. This result was confirmed in MHC class Ia-deficient mice in which IL-15 deficiency also reduced H2-M3-restricted but not all CD8 T cell responses to L. monocytogenes. IL-15 deficiency did not affect proliferation or survival of responding H2-M3-restricted CD8(+) T cells, but IL-15 was necessary to detect H2-M3-restricted CD8(+) T cells in naive mice. This finding suggests that these CD8(+) T cells require IL-15 during development, but become IL-15 independent after activation. IL-15 was necessary for the survival of most class Ib-restricted CD8(+) T cells, starting at the mature thymocyte stage in naive mice, but does not affect a distinct CD44(low)/CD122(low) subpopulation. These data suggest that the nature of the selecting MHC class Ib molecule determines whether CD8(+) T cells acquire IL-15 dependence during thymic development.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

An MHC class Ib-restricted TCR that cross-reacts with an MHC class Ia molecule

TCR transgenic 6C5 T cells recognize an insulin B chain epitope presented by the nonclassical class I MHC molecule, Qa-1(b). Positive selection of these T cells was shown previously to require Qa-1(b). Despite dedicated specificity for Qa-1(b), evidence presented in the current study indicates that 6C5 T cells can cross-recognize a classical class I molecule. Clonal deletion was observed unexpectedly in 6C5.H-2(bxq) mice, which do not express I-E MHC class II molecules and thus should not be subject to superantigen-mediated negative selection. 6C5 T cells were observed to respond in vivo and in vitro to spleen cells from allogeneic H-2(q) mice, and specificity was mapped to D(q). Evidence was obtained for direct recognition of D(q), rather than indirect presentation of a D(q)-derived peptide presented by Qa-1(b). Polyclonal CD8(+) T cells from class Ia-deficient K(b)D(b-/-) mice reacted in vitro to allogeneic spleen cells with an apparent frequency comparable to conventional class Ia-restricted T cells. Our results provide a clear example of a Qa-1-specific TCR that can cross-react with a class Ia molecule and evidence supporting the idea that this may be a common property of T cells selected by class Ib molecules.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

Nonconventional CD8+ T cell responses to Listeria infection in mice lacking MHC class Ia and H2-M3

CD8(+) T cells restricted to MHC class Ib molecules other than H2-M3 have been shown to recognize bacterial Ags. However, the contribution of these T cells to immune responses against bacterial infection is not well defined. To investigate the immune potential of MHC class Ib-restricted CD8(+) T cells, we have generated mice that lack both MHC class Ia and H2-M3 molecules (K(b-/-)D (b-/-)M3(-/-)). The CD8(+) T cells present in K(b-/-)D (b-/-)M3(-/-) mice display an activated surface phenotype and are able to secrete IFN-γ rapidly upon anti-CD3 and anti-CD28 stimulation. Although the CD8(+) T cell population is reduced in K(b-/-)D (b-/-)M3(-/-) mice compared with that in K(b-/-)D (b-/-) mice, this population retains the capacity to expand significantly in response to primary infection with the bacteria Listeria monocytogenes. However, K(b-/-)D (b-/-)M3(-/-) CD8(+) T cells do not expand upon secondary infection, similar to what has been observed for H2-M3-restricted T cells. CD8(+) T cells isolated from Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit cytotoxicity and secrete proinflammatory cytokines in response to Listeria-infected APCs. These T cells are protective against primary Listeria infection, as Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit reduced bacterial burden compared with that of infected β(2)-microglobulin-deficient mice that lack MHC class Ib-restricted CD8(+) T cells altogether. In addition, adoptive transfer of Listeria-experienced K(b-/-)D (b-/-)M3(-/-) splenocytes protects recipient mice against subsequent Listeria infection in a CD8(+) T cell-dependent manner. These data demonstrate that other MHC class Ib-restricted CD8(+) T cells, in addition to H2-M3-restricted T cells, contribute to antilisterial immunity and may contribute to immune responses against other intracellular bacteria.     

T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules

Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved. Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed. Class I-deficient beta2-microglobulin-/- mice show increased susceptibility to infection with S. typhimurium. Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected. The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules. Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1. Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element. The Qa-1-restricted CTL recognition depends on TAP and proteasome functions. Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria. Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.     

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.     

Coreceptor function of CD4 in response to MHC class I molecule

Specificity of T cell receptor (TCR) and its interaction with coreceptor molecules play decisive role in successful passing of T lymphocytes via check-points during their development and finally determine the efficiency of adaptive immunity. Genes encoding alpha- and beta-chains of TCR hybridoma 1D1 have been cloned. The hybridoma 1D1 was established by the fusion of BWZ.36CD8alpha cell line with CD8+ memory cells specific to MHC class I H-2Kb molecule. Exploiting retroviral transduction of thymoma 4G4 cells with TCR genes and coreceptors CD4 and CD8, variants of this cell line expressing on the surface CD3/TCR complex and coreceptors, separately or simultaneously have been obtained. The main function of CD4 is stabilization of interaction between TCR and MHC class II molecule. Nevertheless, we have found that CD4 could successfully participate in the activation of transfectants via TCR specific to MHC class I molecule H-2Kb. Moreover, coreceptor CD4 dominates CDS, because the response of transfectants CD4+CD8+ is blocked by antibodies to CD4 and MHC Class II Ab molecule but not to coreceptor CD8. The response of CD4+ cells was not due to cross-reaction between TCR 1D1 with MHC class II molecules, because transfectants do not respond to splenocytes of H-2b knockouted mice with impaired assembly of TCR/beta2-microglobulin/peptide complexes resulting in their absence on the cell surphace. The effect of domination was not due to sequestration of kinase p56lck, because truncated CD4 with the loss of binding motif for p56lck remained functional in 4G4 cells. Results obtained can explain the number of features of intrathymic selection and represent experimental basis for development of new methods of cancer gene therapy.