Effects of recombinant human interleukin 7 on T-cell recovery and thymic output in HIV-infected patients receiving antiretroviral therapy: results of a phase I/IIa randomized, placebo-controlled, multicenter study.

BACKGROUND: The immune deficiency of human immunodeficiency virus (HIV) infection is not fully corrected with ARV therapy. Interleukin-7 (IL-7) can boost CD4 T-cell counts, but optimal dosing and mechanisms of cellular increases need to be defined. METHODS: We performed a randomized placebo-controlled dose escalation (10, 20 and 30 µg/kg) trial of 3 weekly doses of recombinant human IL-7 (rhIL-7) in ARV-treated HIV-infected persons with CD4 T-cell counts between 101 and 400 cells/µL and plasma HIV levels <50 copies/mL. Toxicity, activity and the impact of rhIL-7 on immune reconstitution were monitored. RESULTS: Doses of rhIL-7 up to 20 µg/kg were well tolerated. CD4 increases of predominantly naive and central memory T cells were brisk (averaging 323 cells/µL at 12 weeks) and durable (up to 1 year). Increased cell cycling and transient increased bcl-2 expression were noted. Expanded cells did not have the characteristics of regulatory or activated T cells. Transient low-level HIV viremia was seen in 6 of 26 treated patients; modest increases in total levels of intracellular HIV DNA were proportional to CD4 T-cell expansions. IL-7 seemed to increase thymic output and tended to improve the T-cell receptor (TCR) repertoire in persons with low TCR diversity. CONCLUSIONS: Three weekly doses of rhIL-7 at 20 µg/kg are well tolerated and lead to a dose-dependent CD4 T-cell increase and the broadening of TCR diversity in some subjects. These data suggest that this rhIL-7 dose could be advanced in future rhIL-7 clinical studies. CLINICAL TRIALS REGISTRATION: NCT0047732.

Valproic acid in association with highly active antiretroviral therapy for reducing systemic HIV-1 reservoirs: results from a multicentre randomized clinical study.

OBJECTIVES: Conflicting results have been reported regarding the ability of valproic acid (VPA) to reduce the size of HIV reservoirs in patients receiving suppressive highly active antiretroviral therapy (HAART). In a randomized multicentre, cross-over study, we assessed whether adding VPA to stable HAART could potentially reduce the size of the latent viral reservoir in CD4 T cells of chronically infected patients. METHODS: A total of 56 virologically suppressed patients were randomly assigned either to receive VPA plus HAART for 16 weeks followed by HAART alone for 32 weeks (arm 1; n = 27) or to receive HAART alone for 16 weeks and then VPA plus HAART for 32 weeks (arm 2; n = 29). VPA was administered at a dose of 500 mg twice a day (bid) and was adjusted to the therapeutic range. A quantitative culture assay was used to assess HIV reservoirs in CD4 T cells at baseline and at weeks 16 and 48. RESULTS: No significant reductions in the frequency of CD4 T cells harbouring replication-competent HIV after 16 and 32 weeks of VPA therapy were observed. In arm 1, median (range) values of IU per log(10) billion (IUPB) cells were 2.55 (range 1.20-4.20), 1.80 (range 1.0-4.70) and 2.70 (range 1.0-3.90; P = 0.87) for baseline, week 16 and week 48, respectively. In arm 2, median values of IUPB were 2.55 (range 1.20-4.65), 1.64 (range 1.0-3.94) and 2.51 (range 1.0-4.48; P = 0.50) for baseline, week 16 and week 48, respectively. CONCLUSIONS: Our study demonstrates that adding VPA to stable HAART does not reduce the latent HIV reservoir in virally suppressed patients.

Design and implementation of a randomized crossover study of valproic acid and antiretroviral therapy to reduce the HIV reservoir.

BACKGROUND: HIV reservoirs represent the major obstacles for eradication and are defined as a cell type that allows persistence of replication-competent HIV in patients on optimal long-term antiretroviral therapy (HAART). Several pilot clinical trials have been implemented to assess the value of experimental therapy to reduce reservoir size or eradicate HIV. In order to eradicate HIV, valproic acid was used as a new strategy to increase viral gene expression in the nucleus of infected cells with the expectation of generating a direct cell death or destruction by nearby cytotoxic cells. Previous pilot studies using VPA have showed conflicting results on the ability of VPA to reduce the size of HIV reservoirs. PURPOSE: As the role of VPA on HIV reservoirs remains unclear, we conducted a multicenter clinical trial with a specific study design to obtain optimal information on reservoir changes while exposing the smallest number of individuals to the experimental medication. METHOD: To this aim, a randomized, crossover design with 2 different treatment durations was implemented. By doubling the therapeutic period in one study arm, we were in a position to assess the impact of an extended duration of VPA on the size of the HIV reservoir and to evaluate the duration of treatment effects upon VPA withdrawal in the other arm. However, limitations for this type of study design included the logistical complexity of 2 uneven study arms and longer study duration. CONCLUSION: Despite the absence of demonstrable impact of VPA on reservoir size, such crossover study design should be considered in the early stage testing of novel HIV therapeutics targeted to reduce reservoir size or eradicate HIV.

Paracrine transfer of mouse mammary tumor virus superantigen.

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive V beta s. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V beta s led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II+ cells in mice led to expansion of V beta 6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

Complement receptor 2 mediates enhancement of human immunodeficiency virus 1 infection in Epstein-Barr virus-carrying B cells.

Although the CD4 glycoprotein is the primary receptor for HIV-1, recent reports have suggested that other molecules might be involved in the enhancement of HIV-1 infection. We investigated the possible role of the complement receptor 2 in enhancement of HIV-1 infection in CD4+ EBV-containing B cells by infecting such cells in the presence of sera from HIV sero-positive donors, with or without added human complement. A marked increase in production of viral p24 and infectious progeny virus was observed only when infection had been carried out in the presence of human complement. The addition of mAb to the human complement receptor 2 completely inhibited this enhancement. This mechanism was CD4 dependent, suggesting a cooperative effect between these two ligands in the potentiation of viral entry.

T cell receptor-major histocompatibility complex class II interaction is required for the T cell response to bacterial superantigens.

Bacterial and retroviral superantigens (SAGs) stimulate a high proportion of T cells expressing specific variable regions of the T cell receptor (TCR) beta chain. Although most alleles and isotypes bind SAGs, polymorphisms of major histocompatibility complex (MHC) class II molecules affect their presentation to T cells. This observation has raised the possibility that a TCR-MHC class II interaction can occur during this recognition process. To address the importance of such interactions during SAG presentation, we have used a panel of murine T cell hybridomas that respond to the bacterial SAG Staphylococcal enterotoxin B (SEB) and to the retroviral SAG Mtv-7 when presented by antigen-presenting cells (APCs) expressing HLA-DR1. Amino acid substitutions of the putative TCR contact residues 59, 64, 66, 77, and 81 on the DR1 beta chain showed that these amino acids are critical for recognition of the SAG SEB by T cells. TCR-MHC class II interactions are thus required for T cell recognition of SAG. Moreover, Mtv-7 SAG recognition by the same T cell hybridomas was not affected by these mutations, suggesting that the topology of the TCR-MHC class II-SAG trimolecular complex could be different from one TCR to another and from one SAG to another.

Highly biased CDR3 usage in restricted sets of beta chain variable regions during viral superantigen 9 response.

Superantigens encoded by the mouse mammary tumor virus can stimulate a large proportion of T cells through interaction with germline-encoded regions of the T cell receptor beta chain like the hypervariable region 4 (HV4) loop. However, several lines of evidence suggest that somatically generated determinants in the CDR3 region might influence superantigen responses. We stimulated T cells from donors differing at the BV6S7 allele with vSAG9 to assess the nature and structure of the T cell receptor in amplified T cells and to evaluate the contribution of non-HV4 elements in vSAG recognition. This report demonstrates that vSAG9 stimulation caused the expansion of TCR BV6-expressing T cells, although to varying degrees depending on the BV6 subfamily. The BV6S7 subfamily was preferentially expanded in all donors, but in donors homozygous for the BV6S7*2 allele, a significant number of BV6S5 T cells were amplified and showed a highly biased beta chain junctional region (BJ) and CDR3 usage. As CDR3 regions are involved in major histocompatibility complex (MHC)-peptide interaction, such a selection is highly suggestive of an intimate MHC-TCR interaction and would imply that the topology of the MHC-vSAG-TCR complex is similar to the one occurring during conventional antigen recognition.

Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

HLA-DR alleles differ in their ability to present staphylococcal enterotoxins to T cells.

Staphylococcal enterotoxins (SEs) have been shown to bind to major histocompatibility complex (MHC) class II proteins and stimulate T cells in a V beta-specific manner, and these V beta specificities for various SEs have been well documented in mice and humans. This study was undertaken in order to examine the ability of human class II molecules to present SEs to human and murine T cell hybridomas. Using a panel of transfectants expressing individual HLA class II antigens, we have shown that HLA-DR alleles differ in their ability to bind and present SEs. Since the HLA-DR proteins share a common alpha chain, these results indicate that the polymorphic beta chain plays an important role in SE binding and presentation to T cells. In addition, we have shown that human class II isotypes markedly differ in their ability to present SEs. The results of this study should provide information on the region of MHC class II molecules that interacts with foreign, and perhaps self, super-antigens.

Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4.

The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4.

Cytolytic T lymphocyte function is independent of growth phase and position in the mitotic cycle.

We have investigated mitotic cell cycle and growth phase regulation of homogeneous cytolytic T lymphocytes (CTL). Two independently derived CTL clones were stained with the DNA-binding dye Hoechst 33342, sorted in a fluorescence-activated cell sorter according to their position in the cell cycle, and then assayed for specific lytic activity using a short-term (30 min) (51)Cr release assay. Results show that lytic activity remained unchanged throughout the cell cycle. Furthermore, there was no significant difference in the lytic activity of CTL clones growing exponentially or arrested in a plateau phase. These results demonstrate that T cell-mediated cytolysis is independent of growth phase and position in the cell cycle.

CD4-mediated enhancement or inhibition of T cell activation does not require the CD4:p56lck association.

CD4 is the coreceptor molecule expressed on the surface of T cells specific for or restricted by class II molecules of the major histocompatibility complex (MHC). Its expression on T cells is required for an optimal response to antigen (Ag). Three mechanisms have been invoked for the involvement of CD4 in T cell activation. First, it was shown that CD4 binds to MHC class II molecules on antigen presenting cells (APCs) thereby favoring an adhesion between effector cells and APCs. Association of CD4 to the T cell receptor and to the tyrosine kinase p56lck have also been shown to be critically involved in the positive function of CD4. Here, we demonstrate that the interaction of CD4 with p56lck is not required to enhance the response of two CD4-dependent, Ag-specific T cell hybridomas. Mutant forms of CD4 (TCD4), which lose association to p56lck, were expressed in these T cells and were shown to enhance the Ag-specific response as efficiently as the wild-type CD4. Moreover both CD4-dependent and independent T cell responses were inhibited by CD4-specific mAbs even when CD4 was not associated with p56lck. These results indicate that mechanisms distinct from sequestration of p56lck and/or negative signaling operate in these inhibitions. Results demonstrating enhancement of TCR-mediated signaling by the coaggregation of TCD4 mutant to the TCR further confirm that the association of p56lck to CD4 is not absolutely required for the regulatory functions of CD4. Our results suggest that the mechanisms implicated in the enhancement of T cell stimulation via CD4 depend solely on the extracellular and transmembrane domains of CD4.

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery.

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.

The CD4 molecule is not always required for the T cell response to bacterial enterotoxins.

T cells respond in a V beta-restricted fashion to bacterial enterotoxins bound to major histocompatibility complex (MHC) class II molecules. The requirement for CD4 in MHC class II-restricted T cell responses is very well established. We have assessed the role of CD4 in the T cell response to the bacterial enterotoxins Staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1. Three CD4- murine T cell hybridomas were transfected with the human CD4 molecule and assayed for interleukin 2 production in the presence of accessory cells bearing human MHC class II molecules and of the appropriate enterotoxin. The results clearly indicate that CD4- cells responded even to suboptimal concentrations of enterotoxin(s) equally well as CD4+ cells. Furthermore, expression of CD4 did not result in the acquisition of previously undetectable reactivity to enterotoxins. These results suggest that unlike the case with antigen-specific responses, formation of a T cell receptor-CD3/CD4 supramolecular complex is not always essential for T cell activation by bacterial enterotoxins.

Cell surface expression of class II histocompatibility antigens occurs in the absence of the invariant chain.

The invariant chain is a glycoprotein transiently associated with the alpha and beta subunits of class II antigens of the major histocompatibility complex during their transport to the cell surface. An expression assay with cDNA clones transfected into simian COS cells was used to test whether the invariant chain is required for assembly and transport of human class II antigens. COS cells do not express detectable levels of RNA from the endogenous invariant chain gene. Cell surface expression of the DP, DQ, and DR antigens was observed in COS cells transfected with the respective alpha and beta chain cDNA clones. Analysis of RNA from the transfected cells showed that the human genes were transcribed in COS cells and that the endogenous simian class II and invariant chain genes were not induced. Cotransfections with an invariant chain cDNA clone did not alter the levels of class II antigens at the cell surface. Biosynthetic labeling and immunoprecipitation demonstrated that the invariant chain cDNA was expressed into a protein which associated with DR alpha and beta chains. Efficient expression of DR antigen in absence of invariant chain was also observed at the surface of a human fibroblast line stably transfected with DR alpha and beta cDNA. This study demonstrates that expression of all three human class II antigens can be achieved with cDNAs cloned in expression vectors. Furthermore, cell surface expression of class II major histocompatibility complex antigens can occur in absence of invariant chain. The postulated role of the invariant chain in class II antigen transport to the cell surface must be reevaluated. The invariant chain may rather be involved in functional properties of class II molecules such as antigen presentation.

Binding sites for bacterial and endogenous retroviral superantigens can be dissociated on major histocompatibility complex class II molecules.

Bacterial and retroviral superantigens (SAGs) interact with major histocompatibility complex (MHC) class II molecules and stimulate T cells upon binding to the V beta portion of the T cell receptor. Whereas both types of molecules exert similar effects on T cells, they have very different primary structures. Amino acids critical for the binding of bacterial toxins to class II molecules have been identified but little is known of the molecular interactions between class II and retroviral SAGs. To determine whether both types of superantigens interact with the same regions of MHC class II molecules, we have generated mutant HLA-DR molecules which have lost the capacity to bind three bacterial toxins (Staphylococcus aureus enterotoxin A [SEA], S. aureus enterotoxin B [SEB], and toxic shock syndrome toxin 1 [TSST-1]). Cells expressing these mutated class II molecules efficiently presented two retroviral SAGs (Mtv-9 and Mtv-7) to T cells while they were unable to present the bacterial SAGs. These results demonstrate that the binding sites for both types of SAGs can be dissociated.

Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression.

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is critical for its binding to the DR alpha chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF47A) or at position 227 (SEAD227A) failed to induce interleukin 1 beta and tumor necrosis factor-alpha messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAF47A or SEAD227A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.

Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features.

Mouse mammary tumor virus (MMTV)-encoded superantigens (SAGs) influence the murine T cell repertoire and stimulate a strong mixed lymphocyte response in vitro. These SAGs are encoded by the open reading frame of the 3' long terminal repeat of MMTV, termed MMTV SAGs. The T cell response to MMTV SAGs is V beta restricted and requires expression of the class II molecules of the major histocompatibility complex (MHC) on the presenting cells. While human T cells respond to bacterial SAGs, it is not known if human T cells or human MHC class II molecules can interact with MMTV SAGs. A fibroblastic cell line expressing the human MHC class II molecule HLA-DR1 and the Mtv-7 sag gene encoding Mls-1 was used to stimulate human T cells. We show here that human T cells efficiently proliferate in response to Mls-1 presented by HLA-DR1. This T cell response was inhibited by mAbs directed against CD4 or MHC class II molecules but not by mAbs specific for CD8 or MHC class I molecules. Moreover, the response to Mls-1 was limited to human T cells expressing a restricted set of T cell receptor V beta chains. Human T cells expressing V beta 12, 13, 14, 15, and 23 were selectively amplified after Mtv-7 sag stimulation. Interestingly, these human V beta s share the highest degree of homology with the mouse V beta s interacting with Mls-1. These results show a strong evolutionary conservation of the structures required for the presentation and the response to retrovirally encoded endogenous SAGs, raising the possibility that similar elements operate in humans to shape the T cell repertoire.

Correlated expression of T cell growth factor dependence, sensitivity to Vicia villosa lectin, and cytolytic activity in hybrids between cytolytic T cells and T lymphomas.

Somatic cell fusion between cytolytically active, T cell growth factor- (TCGF) dependent murine T cell lines (CTL lines) and noncytolytic, TCGF-independent murine T lymphoma lines has yielded two types of somatic cell hybrids (5): cytolytic hybrids, growth of which is dependent on TCGF, and hybrids with very weak or undetectable cytolytic activity which grow at the same rate with or without TCGF. Here we report that the former can produce stable variants that resemble the latter type. Some of these TCGF-independent variants still have TCGF receptors. High susceptibility to the cytotoxic effects of Vicia villosa lectin, a marker distinguishing the parental CTL lines from T lymphomas, is expressed by the TCGF-dependent hybrids but not by the TCGF-independent variants. The two types of hybrids also differ in the expression of surface glycoproteins. We propose that there exists a genetic element in the CTL line that represses the TCGF-independent replication mechanism of the T lymphoma parent in the TCGF-dependent hybrids and that this genetic element is lost or switched off in the TCGF-independent variants.

HLA-DR polymorphism affects the interaction with CD4.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic and bind peptides for presentation to CD4+ T cells. Functional and adhesion assays have shown that CD4 interacts with MHC class II molecules, leading to enhanced responses of CD4+ T cells after the activation of the CD4-associated tyrosine kinase p56lck. We have addressed the possible contribution of allelic polymorphism in the interaction between CD4 and MHC class II molecules. Using mouse DAP-3-transfected cells expressing different isotypes and allelic forms of the HLA-DR molecule, we have shown in a functional assay that a hierarchy exists in the ability of class II molecules to interact with CD4. Also, the study of DR4 subtypes minimized the potential contribution of polymorphic residues of the peptide-binding groove in the interaction with CD4. Chimeras between the DR4 or DR1 molecules, which interact efficiently with CD4, and DRw53, which interacts poorly, allowed the mapping of polymorphic residues between positions beta 180 and 189 that can exert a dramatic influence on the interaction with CD4.