Human immunology and immunotherapy: main achievements and challenges

Cell subsets

The characterization of new immune cell subsets has been a constant feature in the immunology field. This evolution is clearly reflected in the discovery of an innate counterpart of T lymphocytes, collectively named innate lymphoid cells (ILCs)⁶, and in the identification of different types of effector CD4 and regulatory T cells⁷.

Trained and adaptive immune memory

Other classical concepts in fundamental immunology, such as immune memory, are also changing. The specificity and the capacity to generate long-lived memory cells are two properties that have been classically used to distinguish innate immunity from adaptive immunity. Adaptive immunity is clearly based on the specific recognition of antigenic determinants by somatically diversified receptors (B cell and T cell receptors (BCR and TCRs, respectively)) and on its capacity to respond more effectively to restimulation with the same antigen. In contrast, innate immune responses have traditionally been considered nonspecific and without the capacity to adapt⁸¹. However, the discovery of germline-encoded pattern recognition receptors (PRRs) and the “trained innate” immunity (or innate immune memory) have provoked a shift in our understanding of the immune response. In 1997, Medzhitov et al. demonstrated that pattern recognition receptors (PRRs) expressed on innate cells recognize invariant molecular structures expressed by invading pathogens⁸². After the interaction, PRRs trigger the expression of costimulatory molecules and activate important signaling pathways to induce the activation of innate and adaptive immune cells. PRRs mainly belong to four families: Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and peptidoglycan recognition proteins (PGRPs)^83,84. The profiles of PRRs expressed by innate cells can lead to partially specific recognition of a type of microorganism; e.g., innate cells can distinguish between gram-negative and gram-positive bacteria and modulate the immune response based on this recognition, although they cannot differentiate between bacterial species⁸⁵.

The idea that only jawed vertebrates developed immunological memory has also been challenged by the observation of resistance to reinfection in organisms that lack an adaptive immune response, such as plants⁸⁶ and invertebrates^87,88. Recent studies have shown that monocytes and macrophages exposed to Candida albicans or β-glucans exhibited an enhanced secondary response⁸⁹. In addition, immunization of mice with bacillus Calmette-Guérin (BCG, the tuberculosis vaccine) induces T cell-independent protection against secondary infections by Candida albicans, Schistosoma mansoni or influenza virus^90–93. Thus, organisms are protected not only against the original microorganism but also to unrelated pathogens.

The mechanisms underlying the establishment of this innate immune memory differ from those involved in adaptive immune memory⁸¹. After infection or vaccination, innate immune cells (such as monocytes and macrophages) display long-term functional changes through epigenetic and metabolic reprogramming, including histone acetylation, methylation and modulation of noncoding RNAs^94–96. In turn, the faster and more pronounced reactivity of adaptive immune cells (T and B lymphocytes) upon reinfection is characterized by permanent changes in the genome of cells, such as mutations, gene rearrangement, clonal expansions, as well as epigenetic modifications, all of which ensure a more persistent effect than is endowed by trained immunity^81,94,95.

Other cells for which immunological memory has been described include Tγ/δ cells⁹⁷ and innate lymphoid cells⁹⁸. Recently, some authors have proposed that NK cells are also capable of immunological memory^99–102. Antigen-specific recall responses by human NK cells were observed by Nikzad et al.¹⁰³ in humanized mice and in varicella zoster virus (VZV)-exposed adult human volunteers, in which cytotoxic NK cells were recruited to sites of an VZV test antigen challenge on the skin. Sensitization with haptens using mice lacking T cells and B cells led to the generation of hapten-specific memory NK cells⁹⁹. The recall response persisted for more than four months after priming, and was adoptively transferred to naïve mice¹⁰⁰. Interestingly, NK cells exhibit memory that is not only specific to a given virus, such as cytomegalovirus^101,102, but that is also induced in the absence of a defined antigen^104,105.

Furthermore, new studies suggest that trained immunity is not a phenomenon that is restricted to immune cells, because epithelial stem cells also retain memory of previous inflammatory challenges by displaying an enhanced wound-healing capacity upon skin damage¹⁰⁶. Given the data outlined above, immunological memory is now recognized to be highly diverse and not restricted to B cell- or T cell-mediated adaptive immunity. Much remains to be learned in this field, but the different manifestations of immunological memory described above offer an important basis for clinical applications, such as the development of novel vaccination strategies¹⁰⁷ or new therapies for pathological situations in which immunological memory can be detrimental, such as allergies or autoimmune diseases^94,108,109.

Interaction of the immune system and the microbiome

The immune system has evolved in the presence of commensal microorganisms that colonize barrier surfaces of vertebrates and invertebrates^1,110. The cross talk between the natural host microbiome and immune system is particularly interesting in the gastrointestinal tract, where the density and diversity of indigenous bacteria, viruses and fungi are greatest compared to those of other anatomical sites¹¹¹. In the literature, reports of observed changes in microbial community composition during diseases are diverse and include those in inflammatory bowel disease (IBD), obesity, metabolic syndrome, and multiple sclerosis^112–116. However, the microbiome can be influenced by different factors, such as the specific niche that it occupies, diet, stress, environmental factors, and host genetics, and a specific correlation does not necessarily infer causation. The presence of these commensals in mucosal tissues has been known since before Metchnikoff, but the current knowledge on the role of the microbiome in shaping the immune system throughout life came mostly from the development of next-generation sequencing (in particular, the reduction in the cost of 16S ribosomal RNA gene sequencing) and the use of germ-free animal models, which can be colonized even with human microbiota¹¹⁷.

Germ-free mice are characterized by atrophy of Peyer’s patches with few germinal centers and isolated lymphoid follicles, a lower number of B, T, and dendritic cells and a decreased level of immunoglobulins, particularly IgA and IgG¹¹⁸. These effects are observed at the mucosal and systemic levels, and they can be reversed within weeks after the colonization of germ-free mice with commensal bacteria¹¹⁹. Moreover, colonization with commensal Bacteroides fragilis revealed the immunomodulatory effect of bacterial polysaccharides in restoring systemic cells and the differentiation of CD4+ T cells into regulatory T cells (Foxp3+ Tregs), which in turn favor mucosal immunomodulation¹²⁰. The induction of Th17 cell maturation by segmented filamentous bacteria has also been reported¹²¹. These important examples emphasize the major roles of the commensal microbiome in the maturation of mucus-associated lymphoid tissue and the systemic immune system. The development of new technologies to better track the locations and activities of distinct microbial populations is essential to elucidate host-microbe interactions, through which other systems, such as the nervous system, seem to play important roles^2,122–125.

The better characterization of some immune cell subsets, trained immunity, and host-microbiome interactions provides a few very good examples that prove the maturation of immunology in the last few decades. In this sense, studies with mouse models have significantly contributed to the increase in our fundamental knowledge; however, the differences between murine and human immunology are notable, and conclusions drawn from mouse studies are sometimes not fully translated to humans³¹. If we want to fully exploit the power of the immune system for human health, greater effort is required for understanding human immunology. Immunologists, in cooperation with experts from other fields, have developed a variety of protocols and tools to achieve greater selectivity in the identification and analysis of human cell subsets, types of cytokines and receptors, chemokines, etc. These tools range from biological approaches that rely on next-generation sequencing, mass spectrometry, and bioinformatics to immune monitoring technologies based on multiparameter flow cytometry and single-cell gene expression analysis. Although not without limitations, these techniques provide a much better picture of the whole immune system than individual and independent approaches.

Infectious diseases

Infectious diseases are caused by pathogens (viruses, bacteria, fungi or parasites that infect the host body), and they remain a leading cause of mortality worldwide. Prominent examples include illnesses produced by Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Plasmodium falciparum or the current coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has already infected millions of people and produced thousands of deaths in many countries.

For a number of years, many people believed Koch’s postulates, which implied that virulence traits reside solely in the pathogen. However, recent advances in molecular biology have shown that host genes play major roles in infection, together with a wide range of environmental variables¹²⁷.

To date, six gene products endowing infectious disease susceptibility have been validated in the literature: (1) hemoglobin subunit beta; (2) band 3-anion transport protein; (3) Duffy antigen/receptor, which is associated with Plasmodium spp. infections; (4) the prion protein associated with Creutzfeldt–Jakob disease; (5) fucosyltransferase 2 and 3, which is associated with Norwalk virus infections; and (6) C-C motif chemokine receptor 5 (CCR5) coreceptor, encoded by an immune-related gene and leads to the impairment of the entry of the human immunodeficiency virus (HIV) into helper T cells, thus avoiding/decreasing the progression to acquired immunodeficiency syndrome¹²⁸.

Another gene associated with infectious disease and the immune system is the natural-resistance-associated macrophage protein (NRAMP1), which encodes an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. It is a susceptibility locus for increased ratios of infection with Leishmania spp. parasites and certain strains of Salmonella spp., Mycobacterium bovis and Mycobacterium tuberculosis^129,130. In addition, it has been suggested that functional variants of immunoglobulin Fc gamma RIIa (CD32) are related to the development of invasive encapsulated bacterial infections¹³¹.

Moreover, because of recently acquired genomic data, new human polymorphisms have been discovered, some of which play roles in changing immunoglobulin levels, seroconversion rates or the intensity of antigen-specific immune responses. In addition, they also contribute to human susceptibility to infection by viruses such as influenza, rhinovirus and respiratory syncytial virus¹³². These polymorphisms are mapped within the MHC (HLA-DQB1*03, HLA-DRβ1, or HLA-DPβ1), natural killer cell immunoglobulin-like receptors 1 and 4 (KIR3DL1 and KIR2DS4) and natural killer lectin-like receptor D1 (KLDR-1)¹³³.

Several recent studies available as preprints have analyzed certain genes that may explain the differences in the variable expression of and susceptibility to COVID-19 by patients, either by affecting the host receptor for the virus (angiotensin I converting enzyme 2 (ACE-2))¹³⁴, immune genes (TLR7 and others) or blood groups (group O seems to be the most protective)¹³⁵, and more extensive omics studies are now underway with larger numbers of patients.

Autoimmune diseases

In 1901, the physician Paul Ehrlich first used the term “Horror autotoxicus” to describe the way autoimmunity contradicts the natural aversion to self-injury (“Living with the Enemy”, reviewed in¹³⁶). Currently, according to the American Autoimmune Related Disorders Association, more than 100 autoimmune diseases have been identified. Historically, these diseases were considered to be rare, but current epidemiological data have shown that they affect approximately 3–5% of the population worldwide. Some of the most common autoimmune diseases include type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (https://www.aarda.org/diseaselist/). Although significant progress has been made in understanding the mechanisms of autoimmune diseases and the nature of self-tolerance, these disease remain major burdens on health systems around the world.

Autoimmune diseases arise when the immune system attacks normal components of the body¹³⁷. The concept of immune tolerance is defined as the ability of the immune system to prevent the _targeting of self-molecules, self-cells or self-tissues. On the other hand, the failure to distinguish self from nonself is often termed a break of tolerance, and it is the basis for an autoimmune disease¹³⁸.

What are the mechanisms that lead to a break in tolerance? Autoimmune diseases are complex disorders that are believed to arise from a combination of genetic (mutations and higher inheritance frequency of some types of major histocompatibility complex alleles), epidemiological (age and sex) and environmental (infections, microbiota, tobacco, chemicals and pharmaceutical drugs). factors These factors trigger a break in self-tolerance with the activation of self-reactive lymphocytes through several mechanisms, such as molecular mimicry, the overexpression and abnormal expression of MHC class II molecules in peripheral tissues, thymic aging, and immunodeficiencies (discussed below) and many others. Some lymphocytes escape control due to polymorphisms in several genes that affect the routes of lymphocyte activation. Other causes may include defective antigen presentation by some MHC variants with specific polymorphisms. Therefore, the self-reactive lymphocytes that have escaped control and react against self-constituents initiate the autoimmune process¹³⁹.

Although a large number of genome-wide association studies (GWAS) have led to the identification of hundreds of polymorphisms associated with the development of different autoimmune diseases, it has proven difficult to define the role of most of these polymorphisms in the breakdown of tolerance to a self-antigen^139–145. It is worth highlighting, however, that the MHC remains the main genetic factor associated with human autoimmunity^138,139.

Other gene variants identified are common to many autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type I diabetes, ulcerative colitis, autoimmune hepatitis and numerous other autoimmune diseases. For example, the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene encodes a protein that inhibits T-cell activation in the adaptive immune system, whereas it promotes myeloid cell activation; interferon regulatory factor 5–transportin 3 (IRF5–TNPO3) is involved in the accumulation of lymphocytes within lymphoid organs and failed elimination of autoreactive naïve T cells; BTB domain and CNC homolog 2 (BACH2) has a critical role in immunoglobulin class-switching recombination, somatic hypermutation of immunoglobulin encoding genes and the activation of tissue macrophages. A more complete list of genes associated with autoimmunity can be found in the review by Wang et al.¹³⁸

Researchers are currently looking for the missing heritability in autoimmune diseases by focusing on the study of methylome profiles, genetic cargos in extracellular vesicles, genetic alterations, and ways in which the microbiome may affect these diseases.

Rejection of transplants

Immune-mediated rejection of tissue allografts was first described in 1945 by the British immunologist Peter Medawar^146,147. Only three years later, George Snell described the MHC, which carries the histocompatibility genes, and one decade later, Jean Dausset described the human leukocyte antigen (HLA); each of these scientists was recognized with the Nobel Prize in Physiology and Medicine¹⁴⁸. Since its discovery, MHC has emerged as the most polymorphic gene locus in eukaryotes with 24093 HLA and related alleles, more than 362709 nucleotide variants reported in the Individual-Participant Data–International ImMunoGeneTics/Human Leukocyte Antigen (IPD–IMGT/HLA) work group database (https://www.ebi.ac.uk/ipd/imgt/hla/), release 3.39.0, 2020/01/20¹⁴⁹.

Although the main barrier for long-term organ and tissue grafting is driven by HLA incompatibilities, other important players play roles in transplant rejection. In particular, minor histocompatibility antigens, which are peptides derived from allelic variants of normal cellular proteins, presented by class I or II MHC antigens induce cellular immune responses in HLA-matched individuals who lack the same allelic variant¹⁵⁰.

Natural killer (NK) cells also play important roles in transplantation through their killer cell immunoglobulin-like receptors (KIRs), which are receptors for HLA class I molecules. NK cells expressing an inhibitory KIR-binding self-HLA can be activated when exposed to allografts that lack a ligand for the inhibitory receptor¹⁵¹. The locus that codifies these receptors displays a considerable degree of polymorphism, with 1110 alleles reported in the Individual-Participant Data–International/Killer Cell Immunoglobulin-Like Receptors (IPD/KIR) work group database, release 2.9.0, 2019/12/11¹⁴⁹.

More recently, we have begun to appreciate the importance of non-HLA genetic factors in the development of transplant rejection; examples include polymorphisms in the genes encoding cytokines, such as tumor necrosis factors (TNF), interleukins (IL-1, IL-6 and IL-10), interferon gamma (IFN-γ), and transforming growth factor-β3 (TGF-β3). Other genes encode pathogen recognition receptors, with nucleotide-binding oligomerization domain-containing 2 (NOD2 (CARD15)) being the most widely studied, although conclusive data have not been obtained to date¹⁴⁸.

Immunodeficiencies

Primary immunodeficiencies (PIDs) comprise a heterogeneous group of more than 400 genetic disorders that result in defects in the immune response¹⁵². PIDs are considered Mendelian disorders because they are mainly autosomal recessive disorders that often display incomplete penetrance, which affects the severity and onset of the disease. With the exception of immunoglobulin A (IgA) deficiency, PIDs are considered to be rare disorders, as their prevalence worldwide ranges from 1 to 9 among 100,000 people¹⁵³. Unsurprisingly, these types of diseases are not uncommon in highly consanguineous populations such as those in the Middle East/Northern Africa (MENA) region. The incidence of consanguinity marriage in these areas ranges between 20 and 56%, which leads to a unique population in which autosomal recessive diseases arise, with the prevalence of PID in these countries as high as 30 in 100,000 people¹⁵⁴.

Although more than 400 genes have been described for PIDs, approximately 60% of the causal genes remain unknown, and next-generation sequencing studies performed in MENA populations are contributing to the search for currently unknown genes that cause PIDs¹⁵⁵. A complete and updated list of PID-causing genes and diseases can be found at the European Society for Immunodeficiencies (ESID) webpage (https://esid.org)¹⁵⁶.

Clinical manifestations of PIDs are highly variable; many disorders involve an increased susceptibility to several types of infections, but some patients develop autoimmune diseases. Patients usually present recurrent sinus or ear infections or pneumonia within a one-year period; other indicators are failure to thrive, poor response to prolonged use of antibiotics, and persistent thrush or skin abscesses¹⁵³.

Depending on the affected pathway, PIDs are associated with varying levels of severity, times of onset, and risks of infection by certain groups of microorganisms. According to the International Union of Immunological Societies (IUIS) (https://iuis.org/committees/iei/), 430 inborn errors of immunity can be classified as follows: (a) immunodeficiencies that affect cellular and humoral immunity; (b) combined immunodeficiency (CID) with associated or syndromic features; (c) predominant antibody deficiencies; (d) diseases of immune dysregulation; (e) congenital defects of phagocyte number, function, or both; (f) defects in intrinsic and innate immunity; (g) autoinflammatory disorders; (h) complement deficiencies; and (i) phenocopies of a PID^156,157.

However, PIDs are broadly classified as follows according to the component of the immune system affected:

• T-cell immunodeficiency, e.g., defects in the IFN-γ/IL-12 pathway and mutations in the autoimmune regulator (AIRE) gene.

• B-cell (antibody-mediated) immunodeficiency: gamma-globulinemia, X-linked common variable immunodeficiency (CVID), selective IgA deficiency, specific antibody deficiency, and IgG subclass deficiency.

• Combined immunodeficiency: Wiskott–Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome and severe combined immunodeficiency (SCID).

• Phagocyte defects: chronic granulomatous disease, hyperimmunoglobulin E (IgE) syndrome and leukocyte adhesion deficiency.

• Complement defects (deficiency in early, late or regulatory complement components)¹⁵⁸.

Autoinflammatory diseases

Systemic autoinflammatory diseases (AIDs) are characterized by recurrent acute inflammatory episodes secondary to a dysregulated inflammatory process that typically develops during childhood, with recurrent episodes of fever, rashes, and disease-specific patterns of organ inflammation. Genetically speaking, these are hereditary disorders, andto date, more than 40 genes (Table 1) have been identified as causes of AIDs, which can be grouped according to the pathway that is altered¹⁵⁹.

1. Inflammasome. The inflammasome is a multiprotein intracellular complex that detects pathogenic microorganisms and stressors and activates the highly pro-inflammatory cytokines IL-1β and IL-18. Genes affected in this group are MEFV (Mediterranean fever pyrin innate immunity regulator), which is related to familial Mediterranean fever (FMF); NLRC4 (NLR family CARD domain-containing 4); NLRP1 (NLR family pyrin domain-containing 1) and WDR1 (WD repeat domain 1)¹⁵⁹.

2. Type-I interferon (IFN)-mediated disorders. These disorders are characterized by the upregulated expression of genes induced by IFN. The gain of function by variants of TMEM173 (transmembrane protein 173) is the core manifestation of this disorder group, but other genes have been identified, including DDX58 (DExD/H-box helicase 58), DNASE2 (lysosomal deoxyribonuclease 2), POLA1 (DNA polymerase alpha 1 subunit) and USP18 (ubiquitin-specific peptidase 18)^159,160.

3. Ubiquitination disorders. Ubiquitination is a process that marks proteins for degradation via the proteasome, which is required for the processing of intracellular antigens (such as virus proteins or mutated tumor proteins) and their presentation by class I HLA molecules. Ubiquitination involves three main steps: activation, conjugation and ligation, which are performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). Ubiquitination disorders are caused by variants of the PSMB8, PSMB9, PSMA3 and PSM4 genes (proteasome 20S subunit beta 8, subunit beta 9, subunit alpha 3 and subunit alpha 4, respectively), affecting the proteasome subunits, proteasome maturation protein gene (POMP) and/or proteasome assembly chaperone 2 (PSMG2), by encoding proteasome assembly molecules¹⁶¹. In addition, other genes in this group, such as OTULIN (OTU deubiquitinase with linear linkage specificity), encode ubiquitin peptidases, i.e., proteins involved in ubiquitination assembly complexes, such as HOIL-1 (heme-oxidized IRP2 ubiquitin ligase 1) and HOIP (NHP2-like protein 1 homolog). Finally, the loss of function due to variants of the TNFAIP3 (TNF-alpha-induced protein 3, also known as A20) gene, which encodes a protein with ubiquitin ligase and ubiquitinase activity, has also been described¹⁵⁹.

4. Inflammatory or innate immune regulators. A large number of genes have been found to affect the pathways/mechanisms involved in macrophage and B-cell differentiation and lymph node development, among many functions. Genes in this group include ADA2 (adenosine deaminase 2), TNFRSF11A (TNF receptor superfamily member 11a), ADGRE2 (adhesion G protein-coupled receptor E2), TRNT1 (tRNA nucleotidyltransferase 1), LACC1 (laccase domain-containing 1) and AP1S3 (adaptor related protein complex 1 subunit sigma 3)¹⁵⁹.

Allergy

Allergic diseases can be termed complex diseases that involve both genetic and environmental factors, and they influence not only the development of IgE-mediated sensitivity in the case of hypersensitivity type I allergies but also the subsequent development of clinical symptoms in a range of tissues, including skin, nose, and lung tissue¹⁶².

Since the first report of a link between chromosome 11q12 and atopy in 1989^163, knowledge about the common risk variants for allergic diseases has increased exponentially, mainly because of GWAS. Most allergic diseases have allergy-related traits such as asthma, with the strongest association mapped to chromosome 17q21. However, the disease-associated gene at this locus remains unclear; one of the candidate genes is ORMDL3 (sphingolipid biosynthesis regulator 3) due to its role in sphingolipid synthesis and the regulation of eosinophils. Other genes associated with asthma are interleukin 33 (IL33) and its receptor, IL1RL1 (interleukin 1 receptor-like 1), HLA region, SMAD3 (SMA- and MAD-related protein 3) and IL2RB (interleukin 2 receptor subunit beta)¹⁶⁴.

As asthma and other allergic-associated traits could be present in patients without allergies, some researchers performed GWAS analysis on cohorts of patients who had high levels of allergen-specific immunoglobulin E (IgE) or a positive skin prick test. As a result, 18 loci were identified, and the strongest association was on chromosome 11q13. This locus has been associated with two genes: C11orf30 (EMSY transcriptional repressor, BRCA2 interacting), a potential regulator of interferon-stimulated gene, and LRRC32 (leucine rich repeat-containing 32), which is involved in Transforming Growth Factor Beta (TGFβ)-signaling in T regulatory cells.

The rest of the associated loci involved in the pathogenesis of allergy highlight the importance of the Th2 responses (STAT6 (signal transducer and activator of transcription 6), TSLP (thymic stromal lymphopoietin), BCL6 (B-cell lymphoma 6 protein), IL1RL1 (interleukin 1 receptor-like 1), IL33 (interleukin 33), GATA3 (trans-acting t-cell-specific transcription factor binding protein 3)); innate immunity (TLR1/6/10 (Toll-like receptor 1/6/10)); TGFβ-signaling (LRRC32 (leucine rich repeat-containing 32), SMAD3 (mothers against decapentaplegic homolog 3)); T-cell (IL2 (interleukin 2), PTGER4 (Prostaglandin E Receptor 4)) and T regulatory box (LRRC32 (leucine rich repeat-containing 32), IL-2, NFATC2 (nuclear factor of activated T cells 2), FOXA1 (forkhead box A1))¹⁶⁴.

In the last two years, researchers have focused on epigenome-wide association study (EWAS) of allergy processes. The epigenetic landscape is specific for a given cell; thus, EWAS requires careful selection of the relevant cell type for a given biomedical condition. For allergies, EWAS has mainly been performed on nasal mucosal cells and whole blood (although the result was later normalized by the number of circulating eosinophils). Nasal mucosal cells comprise CD8⁺ T cells, CD4⁺ T cells, myeloid cells, innate lymphoid cells, B cells, double-negative T cells, granulocytes, CD117⁺ cells, and plasma cell populations¹⁶⁵. In all of these studies, 36 CpG-associated regions were identified, from which the SMAD3 gene, coding for an important regulator of T-cell differentiation, was replicated in three independent cohorts¹⁶⁶. Of all of the genes in whole blood identified using EWAS, only the ACOT7 (acyl-CoA thioesterase 7), EPX (eosinophil peroxidase), GJA4 (gap junction protein alpha 4) and METTL1 (methyltransferase-like 1) genes were confirmed in the nasal cell populations¹⁶⁷.

Cancer immunology

In 1909, Ehrlich proposed the idea that mutant cells arise continuously and that the immune system scans for and eradicates these mutant cells before they manifest clinically¹⁶⁸. However, immune surveillance remained a controversial topic until its acceptance in the 1990s¹⁶⁹.

Immune surveillance is the recognition and elimination of cancerous cells by lymphocytes, which act as sentinels that recognize transformed cells. Ultimately, during tumor progression, cancer cells show low immunogenicity and resistance to immune effector cells, thus expanding and escaping immune control. The way in which cancer cells modify the immune system has been called immune editing¹⁶⁹.

The key of immunosurveillance is cancerous cell expression of tumor antigens that can activate various immune cell phenotypes; for simplicity, any overexpressed, mutated, dysregulated, or rearranged gene product expressed by a cancerous cell may be considered a tumor antigen. It is critical to consider that most of these proteins, except those derived from virus-infected cancer cells, are primarily self-proteins, but they are expressed with mutation(s) or minor changes in their antigenic structure¹⁷⁰.

One mechanism by which cancer cells escape from immune recognition is antigenic modulation. For example, the loss of MHC class I molecule expression leads to aberrant antigen masking, which is one of the mechanisms described for tumor cells that escape specific antitumor T-cell immune responses¹⁷¹. In addition, the MHC-peptide-T cell receptor complex elicited by a tumor antigen shows weak stability, since high-affinity T-cells tend to be rendered tolerant to these antigens¹⁷².

Another mechanism is the direct inhibition induced by cancer cells due to their interaction with surface regulatory molecules, also called checkpoint molecules. These molecules include programmed cell death-1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which induce the inhibition of host T cells. Although these checkpoints usually help conventional immune responses control immune activation, they can also be used by tumor cells to inhibit antitumoral T-cell responses¹⁷³.

PD1) is a transmembrane protein expressed on T, B, and NK cells, and it binds to PD1 ligands (PD-L1 and PD-L2) on _target cells. When it binds to its ligand on tumor cells, PD1 inhibits tumor cell apoptosis, causes peripheral effector T-cell exhaustion, and promotes the conversion of effector T cells into regulatory T cells^172,174.

CTLA4 is also a physiological negative regulator of T-cell activation. The interaction with CD80/CD86 in the tumor leads to the inhibition of T-cell function and suppressed effector activity¹⁷⁵. Knowledge of these two checkpoint inhibitors has opened the door to new antitumoral therapeutic approaches, such as the use of monoclonal antibodies that block the aforementioned interactions (anti-PD1, anti-PD-L1, or anti-CTLA-4), which are called checkpoint inhibitors¹⁷⁶.

In addition, tumor cells create an inhibitory microenvironment around them. Malignant cells can recruit other cells, such as immune cells and fibroblasts, which can be corrupted by tumor cells. The interaction between tumor and nontumor cells creates the tumor microenvironment, which is mostly driven by the dynamics of the tumor promoting the proliferation/expansion of cancer cells. For example, tumor and stromal cells release multiple factors, such as the chemokine CCL28 (C-C motif chemokine ligand 28), which inhibits effector T-cell functions and attracts Tregs to the microenvironment¹⁷².

Tumor cells use different mechanisms to promote cancer progression and further metastasis. The complete immunological eradication of cancer is the goal of antitumoral immunotherapy and is discussed later in this review.

Immunosenescence and inflammaging

Aging is accompanied by the decline and dysregulation of immune efficacy, which results in an increased vulnerability to infectious diseases, diminished responses to vaccination, and reduced tumor clearance. Immune alterations mainly manifest as a reduction in the number of naïve peripheral blood cells and a relative increase in some types of memory cells¹⁷⁷.

Natural aging causes progressive atrophy of the thymus, which is called thymic involution. The endpoint is a significant decrease in naïve T cells, which reduces the diversity of the T-cell antigen receptor (TCR) repertoire and culminates in disrupted T-cell homeostasis¹⁷⁸. The cellular and molecular hallmarks of aging have been described as genomic instability, telomere attrition, epigenetic alterations, sarcopenia, changes in intracellular communications, cellular senescence, immunosenescence and mitochondrial dysfunction¹⁷⁹.

The process of aging alters the innate and adaptive immune systems. In terms of innate immunity, aging results in a decreased number of circulating monocytes and dendritic cells, reduced phagocytic properties of macrophages and neutrophils, and impaired antigen presentation by dendritic cells¹⁷⁹. As mentioned above, aging also generates a reduction in the T-cell and B-cell receptor repertoire due to the accumulation of senescent or exhausted lymphocytes, together with a decrease in the number of circulating naïve T and B cells^178,179. On the other hand, NK cell cytotoxicity is maintained in centenarians, and an increase in the number of these cells is observed in healthy aging people¹⁷⁷. Moreover, CD4+ T cells exhibit cytotoxic features in centenarians; this is an acquired characteristic for CD4+ T cells that usually have helper, but not cytotoxic functions under physiological conditions¹⁸⁰.

In addition to these features, chronic inflammation is considered the key that underlies the phenomenon called ‘inflammaging’, which is related to elevated self-reactivity and results in the typical chronic low-grade, systemic inflammatory phenotype observed in the elderly in the absence of acute infection. Currently, it is believed that self-reactive T cells are the main contributors to this process. It has been proposed that this basal inflammatory state contributes to the development of some diseases, such as Type II diabetes, Alzheimer’s disease and atherosclerosis¹⁷⁸. Understanding the mechanisms of age-related disorders in immune regulation is important for identifying more efficient strategies of immune rejuvenation and for the effective induction of vaccination-mediated immunity in older individuals¹⁷⁷.

Pathogens and vaccines for infectious diseases

Immunotherapy associated with pathogens was first linked to the prevention of infectious diseases, starting from variolization (in the X century), followed by Edward Jenner’s vaccination against smallpox (in the XVIII century) and subsequently many other preventive vaccines for infectious diseases. The great advances in the knowledge about infectious diseases took place in the nineteenth century, but the XX and XXI centuries are clearly the vaccination centuries, as many new successful vaccines (with attenuated or dead pathogens, subunits, recombinant proteins, carbohydrates or DNA) introduced against a variety of pathogens. Currently, vaccines are among the factors that, together with hygiene, antibiotics and surgery, save the most lives¹⁸¹. Vaccination enabled the eradication of smallpox infection worldwide in 1980, and we are quite close to eradicating polio¹⁸². However, new and better vaccines are urgently needed; e.g., a vaccine against the new coronavirus 2019, SARS-Cov-2; prevalent pathogens, such as human immunodeficiency virus (HIV); parasites, such as Plasmodium spp., which produce malaria; and bacteria, such as Mycobacterium tuberculosis. However, anti-vaccine groups in more affluent countries are putting society at risk for a return of the serious illnesses that had almost been forgotten, such as diphtheria and tetanus¹⁸³, with an increase in measles in unvaccinated people at epidemic levels, thus negating many of the advances made over many years.

Therapy with microorganisms

Other vaccines

Polyclonal antibodies (pAbs)—serotherapy

The discovery of antibodies by Dr. E. von Behring and Kitasato²¹³ at the end of the XIX century highlighted the potential of antibodies to neutralize tetanus and diphtheria toxins. This discovery opened the way to exploring the potential clinical applications of conventional antiserum-containing polyclonal antibodies from immunized animals/humans²¹⁴. This “serotherapy” was initiated by Dr. Roux and Dr. Yersin, who used anti-diphtheria serum to treat several children²¹⁵. After this initial success, the use of serotherapy was increased for use against diphtheria and other diseases but also led to the identification of problems, such as immunogenicity with the formation of immune complexes (Arthus reaction), the variability and limitation of the antibody batches, the content of a mixture of classes and subclasses of antibodies with different biological activities, and their temporal effects. For all of these reasons, therapy with polyclonal antibodies was very much restricted to special cases, such as the use of gamma-globulins for the prevention of Rhesus (RH) maternal-fetal incompatibility and tetanus or snake venom toxicity²¹⁶.

With the identification of gamma-globulin-deficient patients by Dr. Bruton in 1952²¹⁷, the use of immunoglobulins as therapeutic molecules for the treatment of humoral immunodeficiencies was initiated. However, some problems were encountered in the initial phases, mostly related to the serum preparation and aggregation/fragmentation of antibodies. Since their initial use, several efforts have been made to avoid impurities and to improve the purification process, and several commercial products are now available (as intravenous or subcutaneous preparations). Currently, many patients with humoral immunodeficiencies are successfully being treated to prevent them from catching infectious diseases. More recently, the therapeutic applications of immunoglobulins have expanded to other diseases, such as against COVID-19 caused by SARS-Cov-2 infection (see below), autoimmune disorders and Kawasaki syndrome in children²¹⁸. The beneficial effects seem to be mediated by several immunological mechanisms, including viral neutralization, inhibition of inflammatory cells and activation of immune regulators²¹⁴.

Monoclonal antibodies (mAbs)

The development of monoclonal antibodies (mAbs) by C. Milstein and G. Köhler in 1975²¹⁹ (Nobel Prize winners for Physiology/Medicine in 1984) changed medicine and immunology completely, along with many other disciplines. Monoclonal antibodies are produced from the fusion of two cells to generate a hybrid cell or hybridoma with two characteristics, i.e., the production of one specific antibody and immortality. Dr. Milstein is considered to be the father of modern immunology for his crucial contribution²²⁰. The development of many different mAbs has enabled the identification of new molecules and the development of more accurate diagnostic approaches; specific, fast and inexpensive technologies; processes for the purification/concentration of compounds; and better and more specific therapy. mAbs can now be used against specific _targets according to the concept of the “magic bullet”, a term coined by Dr. Paul Ehrlich at the beginning of the XX century (reviewed in ref. ²²¹).

Numerous different mouse and rat mAbs were produced against several molecules, but due to their murine origin, patients treated with these mAbs suffered from hypersensitivity and immune responses^222,223. Thus, most mAbs currently used in clinical applications are linked to radioactive elements and used for diagnostic purposes (Table 2).

In an effort to avoid immunogenicity, mAbs were subsequently modified by genetic engineering approaches to carry mostly sequences of human origin. Several research groups and companies developed chimeric and humanized mAbs (Table 2), and these mAbs included additional modifications, such as changes in the carbohydrates (glycosylation) and/or antibody regions, with the aim of improving their therapeutic action^224–228. Moreover, fragments of recombinant antibodies (Fabs, single-chain Fvs, different V regions, fusion proteins, smaller antibodies, etc.) increased the variability of these potential therapeutic agents.

The generation of fully human mAbs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or P1 vectors. The generation of knockout mice (in which mice lack Ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mAbs^229,230. Other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies (Table 2). Sir Greg Winter, Nobel Prize winner in Chemistry in 2018^231,232, became the pioneer of mAb humanization through the genetic engineering of an antibody (Campath 1), later developing a fully human antibody (antitumor necrosis factor alfa, TNF-a) using recombinant phage technology^225,233,234. Several companies are currently developing human antibodies using these and new technologies (reviewed in^{225,227,233,234}).

Since 1975, the list of approved mAbs for human therapy has continued to increase (Table 2), and many more mAbs are in clinical trials^235–237. The versatility of mAbs is based on a different mechanism of action²³⁸:

• Neutralization/blocking of soluble elements. For example, the neutralization of cytokines (TNF-α) and growth factors (vascular endothelium growth factor) prevents the exhibition of their effects, i.e., inflammatory and angiogenic effects, respectively^239,240.

• Complement activation. IgG/IgM antibodies activate the complement cascade by the classical route, which leads to the death of the _target cell^241,242.

• Cytotoxicity mediated by NK cells. NK cells can facilitate mAb killing of _target cells. The mAb, after binding to a _target cell, can attach to Fc receptors on the surface of NK cells to trigger the release of granzymes and perforin, thus inducing cell _target death^243,244.

• Induction of cell death by apoptosis. Certain mAbs directed against some membrane molecules can directly activate apoptosis²⁴³.

• Blocking activation signals. Antibodies can block some membrane receptors and avoid cell activation/proliferation activation/proliferation^243,245.

• Carriers of toxins, pro-drugs, enzymes, and radioactive elements. mAbs are able to concentrate select compounds around _target cells, providing a much more selective therapy than conventional chemo- or radiotherapy²⁴⁴.

• Check point inhibitors. Leading to a recent revolution in cancer therapy, the identification of several inhibitory molecules can be blocked by mAbs, thus leading to the activation and proliferation of antitumoral T cells. Molecules such as CTLA-4 and PD1 and its ligand PDL-1, maintain immune cells under controlled conditions. However, it is possible to reactivate the antitumoral immune responses by blocking some of these molecules with mAbs, either directed to only one of them or by using various antibodies in combination (for example, against CTLA-4 and PD1)²⁴⁶.

The results obtained with these therapeutic mAbs against checkpoint inhibitors in some types of cancer have been amazing. For their contribution to the understanding of the roles of CTLA-4²⁴⁷ and PD-1²⁴⁸, the Swedish academy gave the Nobel Prize in 2018 to Dr. J.P. Allison and Dr. T. Honjo, respectively²⁴⁹. However, this therapy is not efficacious in all types of cancers for several reasons, such as the expression of these and other checkpoint inhibitors in immune cells, the number of antitumoral cells in each patient, an immunosuppressant microenvironment, the rate of cancer mutations, and the expression of histocompatibility molecules.

Recombinant proteins

There is a large list of recombinant proteins that are currently being used for human therapy, including interleukin 2 (IL-2), interferons (IFNs) and GM-CSF.

IL-2 was identified in 1976 as a growth factor for T lymphocytes, and soon after Dr. Rosenberg started to use it in antitumoral therapy^250,251. Years later, in 1991, IL-2 was approved by the FDA for patients with metastatic renal cancer and in 1998 for the treatment of metastatic melanoma²⁵¹.

Interferon (IFN) was described in 1957 by Isaacs and Lindenmann²⁵². The interferon family is the largest family of cytokines and is classified into three different types (I, II, and III). Type I IFNs (including IFN-α and IFN-β) exhibit several molecular actions that may be very relevant for use in therapy for a range of pathologies (such as autoimmune diseases and cancers)²⁵³. In 1986, the FDA approved human IFN-α2a and IFN-α2b for patients with hairy cell leukemia and later on for patients with multiple sclerosis. Since their initial use, these interferon species have been approved for many other diseases, including chronic hepatitis B and C, lymphoma, advanced melanoma, and as adjuvants together with other therapies for several types of cancers^254,255.

Another cytokine is GM-CSF, which activates the production of granulocytes and monocytes from bone marrow myeloid progenitors and has shown adjuvant antitumoral effects^256,257. Other cytokines, such as IL-5, IL-7, IL-12, IL-15, IL-18, and IL-21^258,259, are being tested in several clinical trials, and it is expected that some of them, either alone or in combination, can be used in future antitumoral therapy.

Other recombinant proteins are already on the market, some of which are derived from antibodies, with some advantages such as small size, low immunogenicity and general ease of production. Examples are etanercept and abatacept (CTLA-4 Ig), which were approved by the European Medicines Agency in 2000 and 2007, respectively. The former is a chimeric protein that carries the external portion of the tumor necrosis factor (TNF) receptor linked to the IgG Fc region, which captures soluble TNF to block its inflammatory effects²⁶⁰. The latter example is a fusion protein that combines the extracellular portion of human CTLA-4 and IgG1 Fc. Abatacept is a competitive inhibitor that blocks T-cell activation and can be used in the treatment of inflammatory illnesses such as rheumatoid arthritis²⁶¹.

Cellular immunotherapy

Immunotherapy for COVID-19 patients

Coronavirus disease 2019 (COVID-19), which is produced by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), affects millions of people in many countries. Most of the infected patients (80–85%) are asymptomatic or have mild symptoms, but the disease in some patients progresses to a moderate or severe illness that requires hospitalization in intensive care units because of respiratory distress, multiorgan failure, and/or other pathologies, and more than one-half million fatal cases have been reported worldwide. The most vulnerable population includes aging patients and those with comorbidities such as hypertension, diabetes and cardiovascular diseases.

There are several aspects of the COVID-19 pathogeny that suggest an overreaction of the immune system in severely ill patients, with increased levels of inflammatory cytokines such as IL-6, IL-1 and others (creating the so-called “cytokine storm”), together with blood lymphopenia and CD8 T cell and NK cell exhaustion. Special therapies have not yet been identified to cure these patients, and preventive vaccines are not yet available, but some immunotherapies have been proposed as adjunct therapies, and some of these are currently in different phases of clinical trials³¹².

The immunotherapeutic strategies include the following:

1. _targeting inflammatory molecules. To attenuate the cytokine storm (IL-6 receptor, IL-6, IL-1, GM-CSF, VEGF, etc.), monoclonal antibodies against receptors and/or cytokines, receptor antagonists and/or inhibitors are proposed.

2. Passive immunotherapy. Patients who were infected and recovered, but developed neutralizing antibodies against the SARS-Cov-2 virus, can donate plasma to treat severe/critical patients. Some reports have indicated promising results in a low number of patients who received convalescent plasma^313,314, but conclusions cannot be drawn until several randomized studies and more patients are analyzed. In addition to the use of convalescence plasma, hyperimmune globulin therapy or monoclonal antibodies directed against the virus have also been proposed, and clinical assays are ongoing.

3. Immunomodulation therapy. Intravenous immunoglobulins are aimed at blocking inflammation and preventing secondary infections³¹². This approach is being used with success in cases of Kawasaki syndrome in children.

4. Cellular immunotherapy. To date, very little attention has been paid to the cellular immunotherapy approach in treatments of COVID-19, but several attempts may include the use of SARS-Cov-2-specific T and NK cells to trigger antiviral responses and autologous or allogenic Tregs to modulate inflammatory processes.

Future challenges in immunotherapy

Immunotherapy has been used for centuries, but only in recent years has this area expanded rapidly in several respects, mostly by the use of soluble elements (monoclonal antibodies and cytokines) and, more recently, with immune cells (cellular immunotherapy). There are many fields in which immunotherapy faces a range of challenges: