Natural Killer Cells: From No Receptors to Too Many

Immunity, Vol. 6, 371–378, April, 1997, Copyright 1997 by Cell Press

Natural Killer Cells: From No Receptors to Too Many Lewis L. Lanier DNAX Research Institute Department of Immunobiology 901 California Avenue Palo Alto, California 94304

Described more than 20 years ago based on their functional ability to kill certain tumor cell targets (Herberman et al., 1975; Kiessling et al., 1975), natural killer (NK) cells have subsequently been implicated in innate immunity against viruses, intracellular bacteria, and parasites (reviewed by Scott and Trinchieri, 1995). Like T cells, NK cells mediate cell-mediated cytotoxicity and secrete a diverse array of cytokines and chemokines. The mechanism of NK cell recognition and the receptors involved in this process have proven elusive. However, recent studies are beginning to delineate this complex and sophisticated system. It is now clear that NK cells are regulated by a fine balance between positive and negative signals derived from membrane receptors that determine their ability to mediate cytotoxicity and secrete cytokines in response to immune challenge. Unlike the situation with B and T cells, NK cell development and function does not require gene rearrangements (reviewed by Spits et al., 1995). Furthermore, it seems unlikely that a single ‘‘NK receptor’’ will be responsible for all of the diverse biological properties attributed to these cells. Instead, NK cells likely use an array of different receptors to trigger their effector functions, depending upon the nature of the target and the cytokines available in the local environment (reviewed by Lanier et al., 1997). In in vitro model systems, many different ‘‘costimulatory’’ or ‘‘adhesion’’ molecules (e.g., LFA-1, CD2, CD16, CD26, CD27, CD28, CD44, CD69, NKR-P1, DNAM-1, Ly6, 2B4, and others) have been shown to induce NK cell–mediated cytotoxicity and cytokine secretion (reviewed by Lanier et al., 1997). However, the role of these receptors in NK cell responses in vivo against pathogens, transformed cells, or allogeneic grafts has not been defined. Based on the ability of NK cells to preferentially kill certain tumors lacking major histocompatibility complex (MHC) class I, the existence of an immune surveillance process for the elimination of host cells with abnormal or absent class I molecules was proposed (Karre et al., 1986). Immunity against cells lacking MHC class I would potentially prevent pathogens (or transformed cells) from escaping immune detection simply by disrupting class I synthesis or transport. The ability of MHC class I on target cells to prevent NK cell–mediated cytotoxicity has been validated in several experimental models, both in vivo and in vitro. Genes encoding the NK cell receptors for MHC class I have been cloned, the mechanisms of signal transduction are being analyzed, and insights into the physiological consequences of this process are emerging. The NK Cell Complex There is a cluster of genes on mouse chromosome 6, in a region designated the NK cell complex (NKC), that

Review

encode type II membrane glycoproteins of the C-type lectin superfamily (reviewed by Ryan and Seaman, 1997; Yokoyama and Seaman, 1993). Genes in the NKC are preferentially expressed by NK cells and these genes can be divided into two subfamilies: NKR-P1, which contains three homologous genes (NKR-P1A, B, and C) (Giorda and Trucco, 1991; Giorda et al., 1992; Ryan et al., 1992, 1995; Yokoyama et al., 1991), and Ly49, which includes at least nine closely related members (Ly49A– Ly49I) (Brennan et al., 1996a; Chan and Takei, 1989; Mason et al., 1995; Smith et al., 1994; Stoneman et al., 1995; Wong et al., 1991; Yokoyama et al., 1989, 1990) (Figure 1). Recently, a rat NKC has been found on chromosome 4, syntenic with mouse chromosome 6, and rat homologs of the NKR-P1 and Ly49 genes are located in this region (Dissen et al., 1996). Consistent with their C-type lectin structure, NKR-P1 proteins have been shown to bind synthetic carbohydrates (Bezouska et al., 1994); however, physiological ligands have not been identified. A NKR-P1 loss mutant generated from a rat NK cell line was unable to kill certain murine tumor cell targets, and this activity was restored by genetic transfection with NKR-P1A cDNA, implicating NKR-P1 in target cell recognition and NK cell activation (Ryan et al., 1995). Ly49A (Chan and Takei, 1989; Yokoyama et al., 1989), the first gene discovered in the Ly49 family and the best characterized, encodes a disulfide-bonded homodimer that binds H-2D d and Dk ligands (Daniels et al., 1994a; Kane, 1994). NK cells expressing the Ly49A receptor are unable to kill target cells expressing H-2D d and Dk, suggesting that interactions between the receptor and ligand transmit ‘‘negative signals’’ that inactivate NK cell function (Karlhofer et al., 1992). Similarly, Ly49G2 has been shown to recognize H-2D d and H-2Ld (Mason et al., 1995) and Ly49C appears to bind several different H-2 ligands, possibly including H-2 d, H-2k, H-2 b, and H-2s (Brennan et al., 1994, 1996a). There is evidence for allelic polymorphism of the Ly49 genes and alternative splicing of the transcripts (Silver et al., 1996; Smith et al., 1994; Sundback et al., 1996). Additionally, each allele at a given Ly49 locus can be regulated independently by an as yet undefined mechanism (Held et al., 1995). All of the Ly49 receptors analyzed have been found on overlapping subsets of NK cells in frequencies that vary subtly in different mouse strains (Karlhofer et al., 1992; Mason et al., 1995, 1996; Stoneman et al., 1995; Yu et al., 1994). Humans have an NKC located on chromosome 12 p12.3–p13.1, syntenic with mouse chromosome 6 (Figure 1). Within this region are several genes of the C-type lectin superfamily encoding type II proteins that are preferentially expressed by NK cells and some T cells. Presently only one human NKR-P1 gene (CD161) has been identified (Lanier et al., 1994). Like the rodent NKR-P1 molecules, CD161 is expressed as a disulfide-bonded homodimer on most NK cells and a subset of ‘‘memory’’ T cells (Lanier et al., 1994). Natural ligands of this receptor have not been identified, but interestingly crosslinking of human NKR-P1 with monoclonal antibodies

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Figure 1. The NK Complex Diagrammatic representation of the genes located in the human and mouse NK complex on chromosome 12 p12.3–p13.1 and chromosome 6, respectively. The relative positions of the genes shown are not yet determined.

can either inhibit or activate cytotoxicity mediated by certain NK cell clones (Lanier et al., 1994; Poggi et al., 1996). As yet, human homologs of the Ly49 genes have not been found. However, by using subtractive hybridization to isolate genes preferentially transcribed in human NK cells, a small family of five C-type lectin genes, designated NKG2A–NKG2E, have been identified in the human NKC (Adamkiewicz et al., 1994; Houchins et al., 1991; Yabe et al., 1993), and these molecules may provide immune functions similar to Ly49. The NKG2 genes differ in both the extracellular and cytoplasmic domains, suggesting possible heterogeneity in ligand binding and signal transduction. While the Ly49 receptors are disulfide-bonded homodimers, the NKG2 glycoproteins form disulfide-bonded heterodimers with another glycoprotein designated CD94 (Brooks et al., 1997; Carretero et al., 1997; Lazetic et al., 1996). CD94 is encoded by a single gene of the C-type lectin superfamily (Chang et al., 1995) located within the human NKC (Renedo et al., submitted). Monoclonal antibodies against CD94 have been shown to affect NK cell recognition of target cells transfected with several HLA-A, B, and C genes (Moretta et al., 1994; Phillips et al., 1996; Sivori et al., 1996). Therefore, while the Ly49 and CD94/NKG2 receptors are not strictly structural homologs, they may have evolved to serve a similar function in rodents and humans, respectively. KIR The existence of human inhibitory NK cell receptors for polymorphic MHC class I molecules was predicted based on the observation that NK cells killed HLA class I–deficient B lymphoblastoid cell lines, but did not lyse these target cells when transfected with certain HLA class I genes (Shimizu and DeMars, 1989; Storkus et al., 1989). Membrane glycoproteins on NK cells involved in the recognition of HLA-A (Dohring et al., 1996; Pende et al., 1996), HLA-B (Litwin et al., 1994), and HLA-C (Moretta et al., 1993) were subsequently identified by using monoclonal antibodies that disrupted interactions between the inhibitory receptors on the NK cells and

their class I ligands on targets. Cloning of the cDNAs encoding these receptors (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995a) revealed the existence of a family of genes, designated the killer cell inhibitory receptors (KIR) (Long et al., 1996), on human chromosome 19 q13.4 (Baker et al., 1995). Unlike the Ly49 or CD94/NKG2A receptors, KIR are type I glycoproteins related to the immunoglobulin superfamily (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995a). More than 25 cDNA have been identified that potentially encode KIR glycoproteins (reviewed by Lanier et al., 1997; Long et al., 1997). Presently, it is uncertain how much of this diversity is a result of allelic polymorphism, alternative splicing of transcripts, or distinct genes. However, results from genomic Southern blot analysis are consistent with the existence of a small KIR gene family (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995a). Of the KIR implicated as inhibitory receptors for MHC class I ligands, three distinct protein isoforms have been described (Figure 2). KIR involved in HLA-C recognition are usually monomeric glycoproteins of z58 kDa containing two immunoglobulin-like domains (KIR-2D) in the extracellular region (Figure 2) (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995a). By contrast, KIR reactive with HLA-B are z70 kDa monomeric glycoproteins with three immunoglobulin-like domains (KIR-3D) (Figure 2) (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995b). Recent studies suggest that certain KIR reactive with HLA-A ligands possess three immunoglobulin domains in the extracellular region (Dohring and Colonna, 1996; Pende et al., 1996) and are expressed on the cell surface as disulfide-linked homodimers composed of two z70 kDa subunits (Pende et al., 1996) (Figure 2). Although a murine KIR has not been identified, two mouse genes related to human KIR, designated gp49A and gp49B, have been cloned from murine mast cells (Arm et al., 1991; Castells et al., 1994). Like the KIR-2D molecules, the gp49 glycoproteins contain two immunoglobulin-like domains in the extracellular region and the gp49B1 receptor is able to inhibit mast cell degranulation when it is coligated together with the high affinity IgE receptor (Katz et al., 1996). Recent studies indicate that all mouse NK cells express gp49B1 (Wang et al., 1997), and transfection of mouse NK cells with chimeric receptors containing the extracellular domain of human KIR linked to the cytoplasmic domain of gp49B1 has shown that the gp49B1 cytoplasmic domain is capable of inhibiting NK cell–mediated cytotoxicity (Rojo et al., 1997a). However, gp49B1 is probably not the murine equivalent of human KIR because a human gene with greater homology to gp49 than KIR has recently been identified (Rojo et al., 1997a). Furthermore, it seems unlikely that MHC class I will serve as the ligand for the gp49 receptors (Rojo et al., 1997a; Wang et al., 1997).

NK Recognition of MHC Class I The Ly49 receptors recognize polymorphic determinants of H-2 that map to the a1/a2 domain of the class I heavy chain (Karlhofer et al., 1992; Sentman et al., 1994). The extracellular domain of H-2D d is sufficient to

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Figure 2. Inhibitory Class I

Receptors

for

MHC

Diagrammatic representation of the Ly49, CD94-NKG2A, and KIR isoforms implicated as inhibitory receptors for MHC class I. A common feature in all of these receptors is ITIM sequences in the cytoplasmic domains. Disulfide bonds between the receptor subunits are indicated (C-C). Ly49 and CD94/ NKG2A are type II proteins of the C-type lectin superfamily, whereas the KIR are type I proteins of the immunoglobulin superfamily.

confer target cell protection against NK cell lysis, as demonstrated by experiments using H-2Dd tethered to the membrane of target cells via a glycosylphosphatidylinositol anchor (Sentman et al., 1996). While the affinity of the Ly49 receptors may be influenced by the presence of carbohydrate on the class I molecule (Brennan et al., 1995; Daniels et al., 1994b), it seems very unlikely that carbohydrates alone can explain the H-2 allele specificity of these receptors. An intact H-2 class I trimer (composed of a heavy chain, b2-microglobulin, and a bound peptide) is required for functional interactions with Ly49A (Correa and Raulet, 1995; Orihuela et al., 1996). Peptides are required to generate a stable H-2 molecule with the proper conformation; however, Ly49 is unable to discriminate between different peptides within the MHC complex (Correa and Raulet, 1995; Orihuela et al., 1996). Preliminary findings indicate that both the carbohydrate recognition domain and the stalk region of the Ly49 receptor are necessary for ligand binding (Brennan et al., 1996b). Like Ly49, KIR recognize a region in the a1 domain of the HLA class I heavy chain (Biassoni et al., 1995; Cella et al., 1994; Colonna et al., 1993; Gumperz et al., 1995; Mandelboim et al., 1996b). The three-immunoglobulin domain KIR designated NKB1 recognizes the HLA-Bw4 motif, which is conferred by amino acids 77–83 in the a1 domain of certain HLA-B heavy chains (Gumperz et al., 1995) (Figure 3). Interaction between this three-immunoglobulin domain KIR and HLA-B*5101 (a Bw4 allele) has been confirmed by direct binding assays using a recombinant KIR fusion protein (Rojo et al., 1997b). Other three-immunoglobulin domain KIR, recognized by the 5.133 and Q66 monoclonal antibodies, recognize HLA-A3 (Figure 3), although the structural properties of this specificity have not been well characterized (Dohring et al., 1996; Pende et al., 1996). The two-immunoglobulin domain KIR recognize a polymorphism at positions 77 and 80 of the HLA-C heavy chain (Colonna et al., 1993). KIR reactive with the EB6 (Moretta et al., 1993) or HP-3E4 (Lanier et al., 1995) monoclonal antibodies recognize HLA-Cw4 and related alleles (possessing asparagine at residue 77 and lysine at residue 80), whereas KIR detected with the GL183 monoclonal antibody (Moretta et al., 1993) bind HLA-Cw3 and related alleles (serine at residue 77 and asparagine at residue 80) (Figure 3). Direct binding studies using soluble recombinant two-immunoglobulin domain KIR and HLA-C

ligands have confirmed the specificity of this interaction (Dohring and Colonna, 1996; Fan et al., 1996; Wagtmann et al., 1995b) and demonstrate that the receptor and ligand bind with a 1:1 stochiometry (Fan et al., 1996). Carbohydrates on the HLA class I molecules are not required for interaction with KIR (Fan et al., 1996; Gumperz et al., 1995). As yet, no consensus has emerged on the role of b2microglobulin and peptides in KIR recognition. Mandelboim et al. (1996b) have suggested that the two-immunoglobulin domain KIR can recognize HLA-C ligands in the absence of bound peptides, whereas studies of a three-immunoglobulin domain KIR demonstrate that a peptide bound to the HLA-B*2705 class I heavy chain is necessary (Malnati et al., 1995; Peruzzi et al., 1996b). In the latter case, residues at positions 7 and 8 in the peptide influence the interaction between the class I complex and the KIR (Peruzzi et al., 1996a). Although different peptides may affect the conformation of the

Figure 3. HLA Class I Specificity of KIR Diagrammatic representation of the KIR reactive with HLA-A, B, and C ligands. The two-immunoglobulin domain KIR identified using the EB6 or HP-3E4 monoclonal antibodies recognizes HLA-Cw4 and other HLA-C alleles possessing asparagine at residue 77 and lysine at residue 80 in the HLA-C heavy chain. The two-immunoglobulin domain KIR identified using the GL183 monoclonal antibody recognizes HLA-Cw3 and other HLA-C alleles possessing serine at residue 77 and asparagine at residue 80 in the HLA-C heavy chain. The three-immunoglobulin domain KIR NKB1, identified using monoclonal antibody DX9, binds HLA-B alleles possessing the Bw4 motif. The three-immunoglobulin domain KIR NKAT4, identified by the monoclonal antibody 5.133, recognizes HLA-A3.

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class I molecule near the binding site of KIR, present results indicate that KIR do not discriminate between ‘‘self’’ and ‘‘foreign’’ peptides, making the physiological role of peptides in NK function uncertain. The HLA class I specificity of the human CD94/NKG2 receptors has not as yet been examined in detail. However, it appears that these receptors are more promiscuous than the KIR (Lazetic et al., 1996; Phillips et al., 1996; Sivori et al., 1996). CD94/NKG2 receptors can affect recognition of many, but not all, HLA-A, B, and C alleles (Phillips et al., 1996; Sivori et al., 1996). Unlike KIR, these receptors do not recognize the polymorphism defined by residues 77 and 80 in the HLA-C molecules and they are unable to distinguish between HLA-Bw4 and HLABw6 alleles (Phillips et al., 1996). Moreover, there is as yet no direct evidence that CD94/NKG2 heterodimers bind to class I molecules. They could conceivably function as coreceptors involved in signaling, rather than directly binding class I molecules (Lazetic et al., 1996; Phillips et al., 1996). Signal Transduction and Inhibitory Receptors Cellular responses are often controlled by the opposing actions of tyrosine kinases that activate and tyrosine phosphatases that terminate signaling (Thomas, 1995). For example, coligation of the immunoglobulin receptors and FcgRIIB on B cells stimulate the tyrosine kinases that phosphorylate the intracytoplasmic portion of FcgRIIB, which in turn recruits the SHP-1 phosphatase that terminates immunoglobulin signal transduction (D’Ambrosio et al., 1995). Molecular analysis of several membrane receptors with inhibitory function revealed a common sequence I/VxYxxL/V (the immune receptor tyrosine-based inhibitory motif [ITIM]), which binds the SHP-1 tyrosine phosphatase and halts positive signals transduced via other receptors (Scharenberg and Kinet, 1996). The two-immunoglobulin domain and threeimmunoglobulin domain KIR isoforms with a long cytoplasmic tail possess two ITIMs, separated by 26–28 amino acids (Colonna and Samaridis, 1995; D’Andrea et al., 1995; Wagtmann et al., 1995a). Studies from several groups have recently demonstrated that activation of NK cells results in tyrosine phosphorylation of the KIR ITIMs, recruitment of SHP-1 (and possibly SHP-2), and inhibition of NK cell–mediated cytotoxicity (Binstadt et al., 1996; Burshtyn et al., 1996; Campbell et al., 1996; Olcese et al., 1996). Like their function in NK cells, KIR can negatively regulate signals initiated in T cells via the T cell receptor (TCR) by recruitment of SHP-1 (Fry et al., 1996). The NKG2A receptor also has two ITIMs in the cytoplasmic domain (Houchins et al., 1997; Lazetic et al., 1996), and studies using chimeric receptors show that NKG2A ITIMs can recruit SHP-1 and inhibit NK cell– mediated cytotoxicity (Houchins et al., 1997). Many of the Ly49 glycoproteins have a single ITIM (reviewed by Ryan and Seaman, 1997), but since Ly49 receptors are disulfide-linked homodimers this would provide for two ITIMs per receptor. Negative signaling by the Ly49 receptors and murine gp49B1, which has two ITIMs in its cytoplasmic domain, also probably involves SHP-1 (Katz et al., 1996; Nakamura et al., 1997; Olcese et al., 1996; Rojo et al., 1997a).

As yet, little is known about the tyrosine kinases that phosphorylate the ITIMs of the NKG2A, KIR, and Ly49 receptors and the critical substrates of the tyrosine phosphatases, although these may likely depend upon the particular positive signaling receptor that initially activates the NK cell. A potential role for the lck src family tyrosine kinase in KIR function is suggested by experiments showing that overexpression of lck in NK cells enhances KIR phosphorylation (Binstadt et al., 1996). Studies have also indicated that a critical substrate of the SHP-1 phosphatase recruited by KIR receptors is the p36 adapter protein (Valiante et al., 1996), previously implicated in TCR and FcR signal transduction (Galandrini et al., 1996; Motto et al., 1996; Sieh et al., 1994). Certain isoforms of the KIR, Ly49, and NKG2 family lack ITIMs in their cytoplasmic domains, and evidence is accumulating to indicate that these receptors may activate, rather than inhibit, T and NK cell proliferation and cytotoxicity (Biassoni et al., 1996; Bottino et al., 1996; Houchins et al., 1997; Mandelboim et al., 1996a; Mason et al., 1996). As yet, little is known about the biochemical basis of this activity. It is unclear whether these molecules are sufficient to stimulate effector function directly or whether they serve as costimulators working in cooperation with other, as yet unidentified, activating receptors.

Educating an NK Cell The KIR, CD94/NKG2A, and Ly49 receptors are expressed on overlapping subsets of NK cells. This is advantageous in that it allows the host immune system to detect cells that have lost the expression of a single allele at only one MHC class I locus. However, it requires a positive selection process during development to ensure that all NK cells will express at least one inhibitory receptor that can bind to a self class I molecule. Studies of this process are complicated by the fact that a single NK cell clone can simultaneously express multiple Ly49 receptors in mouse and several KIR and/or CD94/NKG2 receptors in man. Furthermore, certain KIR or Ly49 isoforms are expressed in hosts who do not possess relevant ligands for these receptors (e.g., Ly49A, an H-2 d receptor, is expressed in B6 mice [Dorfman and Raulet, 1996; Karlhofer et al., 1992] and NKB1 KIR, an HLABw4 receptor, is expressed in HLA-Bw6 homozygous individuals [Gumperz et al., 1996]). Although these observations suggest that the expression of Ly49 or KIR receptors is not strictly ligand induced or dependent, the MHC haplotype of the host can in fact influence the level of Ly49 receptor expression and the function of these molecules. Studies of B6 mice expressing an H-2Dd transgene indicate that the transgenic mice do not kill H-2Dd-bearing targets, whereas NK cells from normal B6 mice do lyse these cells (Olsson et al., 1995). Similarly, NK cells arising from bone marrow progenitors transplanted into an allogeneic recipient are unable to lyse the host (Sykes et al., 1993). Cell surface expression of Ly49A on the NK cells in these H-2Dd transgenic mice and bone marrow chimeras are present at much lower levels than in normal B6 mice, indicating that an active process is involved (Olsson et al., 1995; Sykes et al.,

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1993). Studies comparing Ly49 expression and function in b2-microglobulin-deficient and normal mice further support the concept that the repertoire and function of these receptors are affected by the H-2 haplotype of the host (Dorfman and Raulet, 1996; Held et al., 1996; Raulet et al., 1995).

Physiological Role of Inhibitory MHC Class I Receptors on NK and T Cells The existence of NK cell subsets expressing different inhibitory Ly49 receptors can now explain the ability of F1 recipients to reject parental bone marrow grafts in certain mouse strains (a phenomenon called hybrid histocompatibility resistance) (Bennett et al., 1995; Yu et al., 1996). Similarly, rejection of transplanted MHC class I–deficient tumor cell lines is consistent with immune surveillance by NK cells for cells with abnormal class I expression (Karre et al., 1986; Piontek et al., 1985). Although it is convincingly demonstrated in these model systems, further studies are needed to determine the role of these inhibitory MHC class I receptors in conventional immune responses. Expression of inhibitory MHC class I receptors on NK or T cells could lower the threshold required for activation when the antigen-presenting cell also displays abnormally low levels of class I. Because certain viruses are known to down-regulate host class I synthesis or transport (reviewed by Maudsley and Pound, 1991), the diminished levels of MHC class I on virus-infected cells might render these cells more susceptible to NK cell–mediated cytotoxicity or augment NK cell–derived cytokine production. However, this has not as yet been demonstrated in vivo. Similarly, diminished MHC class I on virus-infected antigen-presenting cells or transformed cells could lower the threshold for TCR-mediated activation in T cells bearing inhibitory KIR or Ly49 receptors. A potentially important example of KIR function on human T cells has recently been reported (Ikeda et al., 1997). In these studies, a CTL cell line established from a patient with metastatic melanoma was shown to recognize an HLA-A-restricted peptide encoded by a normal human gene that is generally silent in adult tissues, but expressed in melanoma tumors. Surprisingly, this CTL clone was unable to kill the autologous primary melanoma that expressed both the tumor antigen and normal levels of HLA class I on the tumor. However, the CTL did lyse a spontaneous variant of the melanoma that had lost expression of HLA-C. Further studies revealed that this CTL expressed a two-immunoglobulin domain inhibitory KIR that prevented lysis of primary melanoma cells, but permitted lysis of the HLA-C loss variant. Because this melanoma antigen is in fact a normal self-protein, it is intriguing to speculate that the physiological function of the KIR might be to prevent autoimmunity against normal tissues occasionally presenting this antigen, but permit elimination of abnormal cells expressing this protein. While a single example, these observations should prompt a more extensive study to explore the biological role of these inhibitory MHC class I receptors in autoimmunity, cancer, and infectious diseases.

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